CN1800373A - Hybridoma cell strain and its produced human VEGFR-3 resistant monoclonal antibody - Google Patents

Abstract

The invention discloses a hybridomas cell strain BDD073 CGMCC No.1537 and its produced monoclonal antibody of antihuman VEGFR-3. The heavy chain variable area of the monoclonal antibody has amino acid residue sequence of SEQ ID No.1 or dose displacement, deletion or adding to one to ten amino acid residues of the amino acid residue sequence of SEQ ID No.1 with antihuman VEGFR-3 inactivating active polypeptide; the light chain variable area has amino acid residue sequence of SEQ ID No.2 or dose displacement, deletion or adding to one to ten amino acid residues of the amino acid residue sequence of SEQ ID No.2 with antihuman VEGFR-3 inactivating active polypeptide.

Description

The monoclonal antibody of the human VEGFR-3 resistant of hybridoma cell strain and generation thereof-3

Technical field

The present invention relates to the monoclonal antibody of the human VEGFR-3 resistant-3 of hybridoma cell strain and generation thereof.

Background technology

Metastases is the lethal one of the main reasons of human cancer normally.Think that at present the path of metastases is mainly by following three kinds of modes: the first, the tumor cell invasion blood vessel directly enters the blood road and forms metastasis; The second, enter the lymphokinesis system by invading already present lymphatic vessel, and then transfer in the new tissue by lymph-circulation of blood and to form metastasis; The 3rd, tumour cell comes off from solid tumor, directly plants perienchyma and forms transfer.Vascular endothelial growth factor (vascular endothelial growth factor wherein, VEGFs) (vascular endothelial growth factor receptor, VEGFRs) signalling system has been brought into play vital role in tumor vessel generation and hyperplasia and lymphatic vessel hyperplasia process for family and acceptor vascular endothelial growth factor receptor thereof.Have been found that 5 kinds of VEGFs:VEGF-A at present, B, C, D and placenta growth factor (placenta growth factor, PIGF), these VEGFs can be directly optionally with film on three kinds of acceptors (VEGFR-1 (Flt1), VEGFR-2 (KDR) and VEGFR-3 (Flt4)) in one or both effects, promote vascular endothelial cell proliferation or mediate vasculolymphatic hyperplasia (Akagi K, Ikeda Y, Miyazaki M, et al.Vascular endothelial growth factor-C (VEGF-C) expression in human colorectal cancer tissues[J] .Br J Cancer, 2000,83:887-891.).VEGF-A and VEGF-B energy and VEGFR-1, the VEGFR-2 combination, stimulate the blood vessel endothelium hyperplasia and increase vascular permeability, (the Niki T that plays an important role in (vasculogenesis) and vasculogenesis (angiogenesis) process takes place at blood vessel, Iba S, Tokunou M, et al.Expression of vascularendothelial growth factors A, B, C, and D and their relationships to lymphnode status in lung adenocarcinoma[J] .Clin Cancer Res, 2000,6:243l-2439.); VEGF-C is the factor of finding in recent years relevant with lymphocytic hyperplasia with VEGF-D, VEGF-C and VEGFR-2 and VEGFR-3 effect promote tumor lympha to take place and shift (Salven P, Lymboussaki A, Heikkila P, et al.Vascular endothelial growth factors VEGF-B and VEGF-C are expressed in humantumors[.J] .Am j Pathol, 1998,153 (1): 103-108.); Different with VEGF-C, VEGF-D combines with VEGFR-3 and VEGFR-2, not only promote hyperplasia (the Kurahara H. of vasculolymphatic generation but also promotion blood vessel, Takao S., Maemura K., et al.Impact of Vascular Endothelial Growth Factor-Cand-D Expression in human pancreatic cancer[J] .Clinical Cancer Res, 2004,10:8413-8420.).

Lymphatic metastasis is the particularly common route of metastasis of cancer of tumour, follows the tumour of lymphatic metastasis and sees with cancer more, especially the cancer at positions such as stomach, pancreas, lung, mammary gland, colon, nasopharynx.The metastases multiform of lymphoglandula becomes the station of pursuing from the near to the remote to develop, and promptly " waterfall type " shifts, and can be " great-jump-forward " or " back runing type " and shift, and hazardness is very big.In recent years people to VEGF-C/D in people's tumour expression and and metastases between relation carried out a large amount of research.Clinical detection shows, (Stearns M.E. is closed in the phase shift of carrying down of many tumor lymphatic such as the up-regulated of VEGF-C/D and prostate cancer, colorectal carcinoma, cancer of the stomach, lung cancer in the primary tumor, Wang M., Hu Y., et al.Expression of a Flt-4 (VEGFR3) Splicing Variant in Primary Human ProstateTumors.VEGF D and Flt-4t (Delta773-1081) overexpression is Diagnostic forsentinel lymph node metastasis[J] .Lab Invest, 2004,84:785-795.).Salven etc. are in the 35 routine people's tumor specimens that detect, 19 examples detect VEGF-C, wherein lymphoma all has the expression of VEGF-C, head and neck scale carcinoma, malignant melanoma, sarcoma, mammary cancer primary tumor also have expression, 1 example has expression (the Salven P of VEGF-C in the metastasis 3 routine samples, Lymboussaki A, Heikkila P, et al.Vascularendothelial growth factors VEGF-B and VEGF-C are expressed in human tumors[J] .Am j Pathol, 1998,153 (1): 103-108.).In research, find the various human tumor tissues, VEGF-C expresses with lymphatic vessel and shifts being proportionate property (Kitadai Y, Amioka T, Haruma K, et al.Clinicopathological Significance of vascular endothelial growth factor (VEGF)-C in human esophageal squamous cel carcinomas[J] .Int J Cancer, 2001,93 (5): 662-666.).Tumor cells expression VEGF-C and VEGF-D at human pancreas cancer, and with closely related (the Kurahara H. of lymphatic metastasis and patient's prognosis, Takao S., Maemura K., et al.Impact ofVascular Endothelial Growth Factor-C and-D Expression in human pancreaticcancer[J] .Clinical Cancer Res, 2004,10:8413-8420.).Stearns etc. have detected the expression conditions of the corresponding sentinel lymph nodes of 52 routine T2a-T2b/T3 phase tumours, the result in the primary tumor that has sentinel lymph node to participate in, VEGF-D and Flt-4t Δ _773-1081Expression level significantly improves, and shows at VEGF-D or Flt-4t Δ _773-1081The inhibition of acceptor may be carried down to move and be had important effect (Stearns M.E. the blocking-up tumor lympha, Wang M., Hu Y., et al.Expression of a Flt-4 (VEGFR3) Splicing Variant inPrimary Human Prostate Tumors.VEGF D and Flt-4t (Delta773-1081) overexpression is Diagnostic for sentinel lymph node metastasis[J] .LabInvest, 2004,84:785-795.).

The importance that lymphatic vessel generated when The Animal Model Study then shifted for tumor lympha provides strong support.Mandriota etc. utilize Rip-VEGF-C (Rip is rat insulin promoter) transgenic mice to study influence (the Mandriota SJ that VEGF-C generates the tumor lympha pipe, Jussila L, Jeltsch M, et al.Vascularendothelial growth factor-c-mediated lymphangiogenesis promotes tumormetastasis[J] .EMBOJ, 2001,20 (4), 672-682.).Be in Rip regulation and control VEGF-C orientation expression down in the beta cell of endocrine pancreas, the result forms lymphatic vessel network (is specific marker with LYVE-1) widely on every side at the Langerhans knot of transgenic mice.And under same condition, the pancreas beta cell cancer that Ripl Tag2 mouse (transgenic mice that has the carcinoma of the pancreas gene) forms there is no lymphatic vessel and generates, and does not also shift.The double transgenic mouse of breeding after Rip-VEGF-C and the hybridization of RiplTag2 mouse then forms abundant lymphatic vessel around tumour, the frequency of pancreas nodus lymphoideus transferring rate also increases thereupon.The more important thing is, in same model, the overexpression of VEGF-A can promote the formation of blood vessel and tumour, but there is no vasculolymphatic generation, nodus lymphoideus transferring rate (Gannon G does not take place yet, Mandriota SJ, Cui L, et al.Overexpression of vascular endothelialgrowth factor-A165 enhances tumor angiogenesis but not metastasis duringbeta-cell carcinogenesis[J] .Cancer Res, 2002,62:603-608.).The MDA-MB-435 tumour cell that Skobe etc. cross expression by inoculation VEGF-C in the mouse body is studied the vasculogenesis and lymphatic vessel generation in the solid tumor, found that VEGF-C can induced tumor tissue lymphangiectasis and inside tumor on every side form newborn lymphatic vessel (Skobe M, Hawighorst T, Jackson D, et al.Velasco P, et al.Induction of tumour lymphangiogenesis by VEGF-C promotes breast cancermetastasis[J] .Nat Med, 2001b, 7:192-198.).Karpanen etc. are implanted to mammary cancer MCF-7 cell in the SCID mouse, the result shows that VEGF-C can promote tumour vasculolymphatic generation (the Karpanen T that is correlated with, Egeblad M, Marika I, et al.Vascular endothelial growth factor C promotes tumorlymphangiogenesis and intralymphatic tumor growth[J] .Cancer Res, 2001,61 (5): 1786-1790.).And use soluble VEGFR-3/Ig can suppress vasculolymphatic new life, show that blocking VEGF R-3 signal path may be one of approach that suppresses the tumor lympha transfer.Usefulness 293-EBNA heteroplastic transplantation models such as Stacker have also confirmed VEGF-D (the Stacker S that plays an important role in tumor lymphatic metastasis, CaesarC, Baldwin M, et al.VEGF-D promotes the metastatic spread of tumour cellsvia the lymphatics[J] .Nat Med, 2001,7:186-191.).Similar to the research of Gannon, the overexpression of VEGF-A has promoted the formation of blood vessel and tumour, but vasculolymphatic generation and nodus lymphoideus transferring rate are not had influence.

Up to now, both at home and abroad the research that suppresses the malignant tumour lymphatic metastasis is mainly concentrated on combining of blocking VEGF-C/D and VEGFR-3 and then suppress its tumor lymphatic and carry down and move.Blocking-up approach commonly used mainly contains two kinds: 1. the VEGFR-3 of using soluble achieves the goal with emulative combination of its part.Use LNM35 (human lung cancer cell line, have high shift phenotype and the high-caliber endogenous VEGF-C expresses) cell of overexpression VEGFR-3/Ig, can suppress in the immunodeficient mouse model that the tumor lymphatic pipe generates and tumor lympha is carried down moves.At sound homogenic of immunologic function is the identical method of use in the breast tumor mouse model, equally also obtained to suppress effect (the He Y of nodus lymphoideus transferring rate, Kozaki K, Karpanen T, et al.Suppression of tumor lymphangiogenesisand lymph node metastasis by blocking vascular endothelial growth factorreceptor 3signaling[J] .J Natl Cancer Inst, 2002,94:819-825.).2. use the signal path that the active antibody of neutralization suppresses VEGFR-3.But Shimizu etc. discover in the tumour cell excessive secretion VEGF-C/VEGF-D induced tumor tissue or the newborn vasculolymphatic formation in edge, the antibody of using the VEGF-C/VEGF-D barrier then can suppress tumor lymphatic metastasis (Shimizu, K., Hajime Kubo, Koji Yamaguchi, et al.Suppression of VEGFR-3 signaling inhibits lymph node metastasis ingastric cancer[J] .Cancer Sci, 2004,95:328-333.).Pytowski etc. use anti-mouse VEGFR-3 neutrality antibody vasculolymphatic hyperplasia capable of blocking in the mammary cancer of crossing VEGF expression-C.Simultaneously long blocking VEGF R-3 signal path is to established lymph vessels did not influence (Pytowski B on form, GoldmanJ, Persaud K, et al.Complete and specific inhibition of adult lymphaticregeneration by a novel VEGFR-3neutralizing antibody[J] .J Natl Cancer Inst, 2005,97:14-21.).This provides strong support for the method that is used as a kind of antitumor lymphatic metastasis with blocking VEGF R-3.

The research of being done at present all is confined to use anti-mouse VEGFR-3 monoclonal antibody and suppresses the aspect that tumor lympha shifts.Not seeing has human VEGFR-3 resistant in-3 and the report of the animal model of active antibody.The domestic report of also not seeing relevant human VEGFR-3 resistant-3 antibody of while.

Summary of the invention

The purpose of this invention is to provide the hybridoma cell strain that a strain can produce human VEGFR-3 resistant-3 monoclonal antibody.

The hybridoma cell strain that produces human VEGFR-3 resistant-3 monoclonal antibody provided by the present invention, name is called BDD073, this cell strain is preserved in Chinese common micro-organisms culture presevation management committee common micro-organisms center on November 25th, 2005, and deposit number is CGMCC No.1537.

The monoclonal antibody of the human VEGFR-3 resistant-3 that produces by above-mentioned hybridoma cell strain BDD073, name is called ABDD073, derive from Mus mouse (Mus musculus), its variable region of heavy chain has the SEQ ID № in the sequence table: 1 amino acid residue sequence or with SEQ ID № in the sequence table: 1 amino acid residue sequence is through replacement, disappearance or the interpolation of one to ten amino-acid residue and have in the human VEGFR-3 resistant-3 and active polypeptide; Variable region of light chain has the SEQ ID № in the sequence table: 2 amino acid residue sequence or with SEQ ID № in the sequence table: 2 amino acid residue sequence is through replacement, disappearance or the interpolation of one to ten amino-acid residue and have in the human VEGFR-3 resistant-3 and active polypeptide.

SEQ ID № in the sequence table: 1 forms the SEQ ID № in the sequence table by 117 amino-acid residues: 2 are made up of 132 amino-acid residues.

The gene (ABDD073) of coding human VEGFR-3 resistant-3 monoclonal antibody ABDD073, its variable region of heavy chain encoding gene has SEQ ID № in the sequence table: SEQ ID № in 3 dna sequence dna or the code sequence tabulation: 1 dna sequence dna or under the rigorous condition of height can with SEQ ID № in the sequence table: the nucleotide sequence of the 3 dna sequence dnas hybridization that limit; The variable region of light chain encoding gene has SEQ ID № in the sequence table: SEQ ID № in the tabulation of 4 dna sequence dna or code sequence: 2 dna sequence dna or under the rigorous condition of height can with SEQ ID № in the sequence table: the nucleotide sequence of the 4 dna sequence dnas hybridization that limit.

The rigorous condition of described height be 0.1 * SSPE (or 0.1 * SSC), in the solution of 0.1%SDS, hybridization and wash film under 65 ℃ of conditions.

SEQ ID № in the sequence table: 3 by 351 based compositions, and its encoding sequence is that coding has SEQ ID № in the sequence table: the protein of 1 amino acid residue sequence from 5 ' end the 1st to 351 bit base.SEQ ID № in the sequence table: 4 by 396 based compositions, and its encoding sequence is that coding has SEQ ID № in the sequence table: the protein of 2 amino acid residue sequence from 5 ' end the 1st to 396 bit base.

The expression vector, transgenic cell line and the host bacterium that contain gene A BDD073 of the present invention all belong to protection scope of the present invention.

Arbitrary segmental primer is to also within protection scope of the present invention among the amplification ABDD073.

The present invention also provides the single-chain antibody that is derived from human VEGFR-3 resistant-3 monoclonal antibody ABDD073, Fab antibody and human mouse chimeric antibody, and the polynucleotide sequence of these antibody of deriving of encoding.

The invention provides the monoclonal antibody ABDD073 of a kind of human VEGFR-3 resistant-3.VEGFR-3 is one of lymphatic vessel specificity marker thing, and therefore monoclonal anti antibody of the present invention can be applicable in the diagnosis of tumor lympha transfer.In addition, ABDD073 can suppress VEGF-D and combine with VEGFR-3, suppresses the propagation of people's red and white blood cell HEL, and suppresses the generation by people VEGF-D inductive chick chorioallantoic membrane blood vessel.ABDD073 reaches the effect that suppresses metastases by suppressing tumor lympha pipe new life, compare with traditional therapeutic monoclonal antibody, anti-VEGFR-3 monoclonal antibody have wide spectrum, low toxicity or nontoxic, a low chemical sproof advantage, its reason is: 1) sudden change of tumor vessel or lymphatic endothelial cells is few, is difficult for producing resistance; 2) though different types of tumors specific target protein difference, therefore its blood vessel and lymphatic vessel structure are roughly the same, have broad spectrum; 3) directly act on blood vessel or lymph endothelium, need not be penetrated into inside tumor, avoided the problem of inside tumor high pressure and drug distribution.Based on above-mentioned advantage, can ABDD073 be that activeconstituents is prepared into medicine for anti transfer of tumor.This preparation method for antibody is simple, can be produced by hybridoma cell strain BDD073CGMCC No1537 direct secretion.The present invention not only is that the biological function and the mechanism of action thereof of further research VEGFR-3 is laid a good foundation, and has the potential application prospect in the curative drug of preparation inhibition tumor lympha transfer and angiogenesis inhibitor.

The invention will be further described below in conjunction with specific embodiment.

Description of drawings

Fig. 1 for reorganization GST-VEGF-D and VEGFR-3/Fc in conjunction with the activation analysis result

Fig. 2 is the experimental result of various dose GST-VEGF-D to the influence of hel cell propagation

Fig. 3 is the The selection result with the active anti-VEGFR-3 monoclonal antibody of neutralization

Fig. 4 is immunoblotting (Western blot) qualification result of anti-VEGFR-3 monoclonal antibody ABDD073

Fig. 5 analyzes the tissue positioned result of VEGFR-3 monoclonal antibody ABDD073 in fetal rhythm, tire kidney, tire lung with immunohistochemistry technology

Fig. 6 identifies that for flow cytometry ABDD073 combines active result with the VEGFR-3 of hel cell surface expression

Fig. 7 is the interactional experiment in vitro result of ABDD073 blocking VEGF R-3 and VEGF-D

Fig. 8 is the confirmatory experiment result that the monoclonal antibody ABDD073 of anti-VEGFR-3 suppresses hel cell propagation

Fig. 9 is the experimental result that the GST-VEGF-D albumen of the solubility of detection various dose influences the chick chorioallantoic membrane vasculogenesis

Figure 10 is the monoclonal antibody ABDD073 of the anti-VEGFR-3 of the various dose inhibition experimental result to the chick chorioallantoic membrane blood vessel of being induced generation by GST-VEGF-D

Figure 11 is the agarose gel electrophoresis detected result of light, variable region of heavy chain encoding gene of the ABDD073 of pcr amplification

Figure 12 is the agarose gel electrophoresis detected result of the anti-VEGFR-3 single-chain antibody VL-linker-VH encoding gene of pcr amplification

Figure 13 is the SDS-PAGE detected result of the anti-VEGFR-3 single-chain antibody VL-linker-VH of abduction delivering

Embodiment

Method therefor is ordinary method if no special instructions among the following embodiment, and the primer synthesizes and examining order is finished by Shanghai Bo Ya Bioisystech Co., Ltd.

Embodiment 1, anti-VEGFR-3 MONOCLONAL ANTIBODIES SPECIFIC FOR and evaluation thereof

Use the monoclonal antibody that hybridoma technology prepares anti-VEGFR-3, concrete grammar may further comprise the steps:

One, the preparation of hybridoma cell strain and primary dcreening operation

With VEGFR-3/Ig recombinant protein (R﹠amp; D research system corp.) as immunogen BALB/C mice is carried out immunization 3 times.Inoculation position is peritoneal injection or subcutaneous injection, per injection dosage 50 μ g/mL.Immunization is after three days the last time, get spleen, shred, with cell suspension in the RPMI1640 substratum (GIBCO, USA) in, then in the presence of 50% polyoxyethylene glycol (PEG), splenocyte and SP2/0 murine myeloma cell are merged, and hybridoma is carried out preliminary screening with HAT selective medium (RPMI 1640197.5mL, HAT concentrated solution (100 *) 2.5mL, foetal calf serum 50mL).

Two, the screening of the monoclonal antibody of the VEGFR-3 positive and normal human serum feminine gender

The preparation method of monoclonal antibody: to BALB/C mice abdominal injection paraffin oil 0.5mL/ of six all age.After 10 days, the hybridoma suspension inoculation that step 1 is obtained through primary dcreening operation is in BALB/C mice abdominal cavity, 1 * 10 ⁷Individual cell/only.After about 10 days, collect ascites, the centrifuging and taking supernatant.By the albumin A affinity chromatography, monoclonal antibody purification from culture supernatant or ascites.The monoclonal antibody of purifying is carried out sterile filtration, refrigeration or freezing preservation.Identify the hypotype of antibody with the antibody subclass detection kit of HBT company.

The monoclonal antibody of normal human serum feminine gender with the sandwich enzyme-linked immunoabsorption screening VEGFR-3 positive, concrete grammar is:

1) the reorganization VEGFR-3/Ig of purifying, normal human serum, normal human IgG (company limited of China fir Golden Bridge in Beijing) wrap respectively by microtitre cylindrical void (strip wells), be diluted to 0.5 μ g/mL with coating buffer (0.1M carbonate buffer solution (pH 9.6)), each microtitre cylindrical void adds 100 μ l, hatches 12-24 hour (or at room temperature hatching 2 hours) for 4 ℃.

2) above-mentioned bag is added 10% calf serum (200 μ l/ hole) after the hole washing three times sealed opening up on the GARMENT WASH plate machine with washings (0.2M phosphate buffered saline buffer, 0.02% tween), hatched 12-24 hour (or at room temperature hatching 2 hours) for 4 ℃.After opening up on the GARMENT WASH plate machine washing three times, pat dry standby.

3) add the culture supernatant of the hybridoma that 100 μ l obtain through primary dcreening operation in each the microtitre cylindrical void after sealing, with the serum of splenectomy mouse as positive control (dilution in 1: 1000).Hatch after 30 minutes with washings for 37 ℃ and give a baby a bath on the third day after its birth time.The effect of normal human serum (1: 4000) and normal human IgG (0.5 μ g/mL) is a hybridoma of removing secretion and normal people Ig binding antibody.

4) will join in the microtitre cylindrical void by the sheep anti mouse Ig-HRP antibody (Jackson corp.) of 1: 5000 dilution proportion, hatch after 20 minutes for 37 ℃ and wash described cylindrical void as stated above.

5) add HRP enzyme substrates reaction solution A and B (each 80 μ l/ hole, HRP enzyme substrates reaction solution A: sodium-acetate 13.6g, citric acid 1.6g, 30%H ₂O ₂, be settled to 500mL.HRP enzyme substrates reaction solution B:EDTA-Na 0.2g, citric acid 0.95g, glycerine 50mL, tetramethyl benzidine (TMB) 0.2g is settled to 500mL.), with flat board 37 ℃ hatch 15 minutes after with the sulfuric acid stopped reaction of 2M.Detect with Mutiskan MK3 (Thermo) microplate reader.Detected result is greater than the monoclonal antibody for VEGFR-3/Ig positive normal human serum feminine gender of 2.5 times of normal mouse serum OD values.With positive colony amplification cultivation in 24 hole tissue culturing plates (FALCON), and detect with above-mentioned identical method.The hybridoma of anti-VEGFR-3 monoclonal antibody is secreted in final acquisition 17 strains, the monoclonal antibody specific combination VEGFR-3 antigen that their produce and the debond human IgG screens that obtain as shown in table 1 with monoclonal antibody and essential information thereof the VEGFR-3 specific reaction.

The anti-VEGFR-3 monoclonal antibody that table 117 strain of hybridoma produces

The Mab clone	Subclass	Immunoblotting	IH (paraffin section)		IH (frozen section)
							The tire kidney	The tire lung	Fetal rhythm	The tire kidney
			BAB045	IgG 1	P	N					N	P	P
BDD092	IgG 1	N					N	N	N	N
			BDF016	IgG 1	N	N					N	N	N
BBE022	IgG 1	N					N	N	N	N

BCE024	IgG 1	N	N	N	N	N
							BBG047	IgG 1	N	N	N	N	N
BCD062	IgG 1	N	P	P	P	P
							BCB118	IgG 1	N	N	N	N	N
BDD024	IgG 1	N	N	N	N	N
							BDA076	IgG 1	P	P	P	P	P
BCH102	IgG 1	P	N	N	N	N
							BCF013	IgG 1	N	N	N	P	P
ABDD073	IgG2a	P	P	P	P	P
							BAD045	IgG 1	P	N	N	N	N
ABA112	IgG 1	P	N	N	N	N
							AAD016	IgG 1	P	N	N	N	N
AEH081	IgG 1	P	N	N	N	N

Three, the screening that has the active anti-VEGFR-3 monoclonal antibody of neutralization

1, the expression of recombinant human GST-VEGF-D and evaluation

According to the segmental encoding sequence of the VHD of VEGF-D (be for GenBank number: NM657920), design primer PCR this fragment that increases, primer sequence is as follows:

P1 (forward primer): 5 '-CCCAAGCTTATGTTTGCGGCAACTTTCTATGACATTG-3 ';

P2 (reverse primer): 5 '-CCGGAATTCTCTTCTGATAATTGAGTATGGATGG-3 '

Extract total RNA from HFL's tissue, its cDNA is synthesized in reverse transcription, and as template, under the guiding of primer P1 and P2, pcr amplification purpose fragment.The PCR reaction conditions: 94 ℃ of 30s, 58 ℃ of 30s, 72 ℃ of 1min carry out 30 circulations altogether.Reaction is carried out 1% agarose gel electrophoresis to the PCR product after finishing, and reclaims the also dna fragmentation of purifying 348bp, and it is cloned among the carrier pGEX-5X-1 (Amersham company).With recombinant products transformed into escherichia coli DH5 α competent cell, screening positive recombinant, upgrading grain, carrying out double digestion with restriction enzyme NotI and EcoRI identifies, the result cuts the endonuclease bamhi that has obtained 348bp through enzyme, conforms to expected results, shows that the purpose fragment correctly is connected in the carrier.With the method for order-checking positive plasmid is done further evaluation again, sequencing result shows that the insertion fragment sequence is correct, has obtained the plasmid of the reorganization table of VEGF-D gene, called after pGEX-5x-1/VEGF-D.

Recombinant expression plasmid pGEX-5x-1/VEGF-D transformed into escherichia coli BL21 (DE3) competent cell with above-mentioned structure, the screening positive transformant carries out prokaryotic expression, expression product is carried out 10%SDS-PAGE to be detected, obtained the recombinant protein of 38KD, conform to the proteic molecular weight size of VEGF-D, (Amersham company) carries out purifying to target protein with the GST affinity column.The reorganization GST-VEGF-D that analyzes after purified carries out following analysis:

1) reorganization GST-VEGF-D and VEGFR-3/Fc's in conjunction with activation analysis

With concentration is the VEGFR-3/Fc (R﹠amp of 1,2 μ g/mL; D research system corp.) mixes with the GST-VEGF-D of 20 μ g/mL, with the pure product of VEGFR-3/Fc is contrast, it is active with combining of VEGFR-3/Fc to analyze GST-VEGF-D by the OD value that detects the 450nm place, the result shows that recombinant human GST-VEGF-D can interact with VEGFR-3/Ig as shown in Figure 1.

2) GST-VEGF-D promotes hel cell propagation

(change the pGEX-5x-1 carrier over to BL21 (DE3) competent cell with GST, 0.2mM IPTG abduction delivering 3h, have children outside the state plan broken bacterium, use the Amersham GST of company prepackage purification column purifying behind the 12000g centrifuging and taking supernatant) for contrasting, use 0.8 respectively, 1.6,3.2,6.4,12.8 the GST-VEGF-D of μ g/mL handles human erythroleukemia (HEL) cell, detect GST-VEGF-D has the hel cell growth of VEGFR-3 to expression influence by the OD value of measuring the 492nm place, (result is the mean value of three measurement results to the result as shown in Figure 2, * represent P＜0.01), rising along with GST-VEGF-D concentration, the hel cell amount also increases thereupon, and along with the rising of GST concentration, the hel cell amount does not have considerable change, shows that GST-VEGF-D can stimulate the growth of hel cell in the finite concentration scope.

2, the screening that has the active anti-VEGFR-3 monoclonal antibody of neutralization

Select step 2 screening that obtain with the monoclonal antibody VEGFR-3 specific reaction (with the hybridoma that produces these antibody), therefrom screen with the ELISA method and to have the active anti-VEGFR-3 monoclonal antibody of neutralization.Concrete grammar is: the purified 8 μ g/mL GST-VEGF-D bag that obtains with step 1 is by 96 orifice plates, and sealing mixes VEGFR-3/IgG and GST-VEGF-D to hatch to add in 96 orifice plates behind the 1h and hatched one hour, adds anti-VEGF-D antibody (R﹠amp after washing plate; D research system corp.), adds the colour developing of two anti-sheep anti-mouse igg-HRP antibody (Jackson company) at last, measure OD ₄₅₀Value.Otherwise detect with the VEGFR-3/IgG wrapper sheet.The result as shown in Figure 3, use this system successfully screen have the neutralization active anti-VEGFR-3 monoclonal antibody ABDD073.Produce the hybridoma cell strain of this monoclonal antibody, name is called BDD073, is preserved in Chinese common micro-organisms culture presevation management committee common micro-organisms center on November 25th, 2005, and deposit number is CGMCC No.1537.

Embodiment 2, has the evaluation of monoclonal antibody ABDD073 of the active anti-VEGFR-3 of neutralization

One, immunoblotting (Western blot) is identified

The monoclonal antibody ABDD073 with the active anti-VEGFR-3 of neutralization to embodiment 1 screening carries out the western immunoblotting assay.Concrete grammar is: with 10 ⁷The cracking in RAPI (1%NP-40,0.15M NaCl, 1 * PBS PH 7.2,2nM EDTA, 50mM Sodium Fluoride, 0.2mM vanadic acid sodium, proteinase inhibitor 1: 50) of individual hel cell.Cell lysate is carried out the 10%SDS-PAGE electrophoresis and electricity forwards on the nitrocellulose filter.This film is hatched with the monoclonal antibody ABDD073 of anti-VEGFR-3, sheep anti mouse antiserum(antisera) (company limited of China fir Golden Bridge in Beijing) and ECL reagent (Amersham) with the coupling horseradish peroxidase make the colour developing of bonded antibody, the colour developing result is (NM IgG represents normally to belong to IgG) as shown in Figure 4, at 175kDa and 125kDa place two obvious bands appear, corresponding to the VEGFR-3 polypeptide.

Two, tissue positioned

Utilize frozen tissue section, paraffin organization section and immunohistochemistry technique that the positive vascular of VEGFR-3 is identified.Concrete grammar is: with fetal rhythm, rehydration is carried out in the section of tire kidney, tire lung tissue sample in phosphate buffered saline buffer, and with sheep blood serum incubation 30 minutes at room temperature.With described section and concentration be then the ABDD073 monoclonal antibody that obtains of the embodiment 1 of 1.0 μ g/mL in malaria 4 ℃ hatched 12-24 hour, other monoclonal antibody BDA076 with the different antigenic determinants of anti-VEGFR-3, BCD062, BAB045, BCF013 is contrast.Use the reagent incubation 50 minutes of PV-9000 test kit (company limited of China fir Golden Bridge in Beijing) subsequently.With 3,3-diaminobenzene diamine (DAB, company limited of China fir Golden Bridge in Beijing) makes peroxidase colour developing 1 minute.At last, with phenodin with section statining 5 minutes.By omit first antibody or with normal mouse IgG as negative control.The result shows that ABDD073 can discern fetal rhythm, the blood vessel endothelium of tire kidney, tire lung tissue as shown in Figure 5.Other monoclonal antibody BDA076, BCD062, BAB045, BCF013 also can discern fetal rhythm, the blood vessel endothelium of tire kidney, tire lung tissue.

Three, flow cytometry is identified

Adopt FACS flow cytometry analysis ABDD073 and hel cell surface expression VEGFR-3 combine activity.Concrete grammar is: respectively with 1 * 10 ⁶Individual hel cell and ABDD073, control antibodies were hatched 1 hour altogether, gave a baby a bath on the third day after its birth time with PBS, added FITC-sheep anti-mouse igg antibody (company limited of China fir Golden Bridge in Beijing), hatched 1 hour, give a baby a bath on the third day after its birth all over after cell is resuspended among the 500 μ l PBS.Carry out immunofluorescence analysis and measure mean fluorecence density on FACS, with the negative contrast of PBS, the result shows that ABDD073 can control antibodies have significant reaction with the VEGFR-3 specific combination of HEL expression as shown in Figure 6.

But above-mentioned experimental result proves the active monoclonal antibody ABDD073 specific recognition VEGFR-3 of neutralization that has of embodiment 1 acquisition.

Example 3, ABDD073 blocking VEGF R-3 and the interactional experiment in vitro of VEGF-D

Have the used ELISA interaction system of monoclonal antibody of the active anti-VEGFR-3 of neutralization with step 3 screening among the embodiment 1, it is active to VEGFR-3 and the interactional inhibition of VEGF-D to investigate ABDD073.With antibody A BDD073, BAB045 (contrast) respectively with VEGFR-3/Ig 37 ℃ hatch 1 hour after, mixed solution adds to have wrapped and was continued to hatch 1 hour in the 96 hole elisa plates of GST-VEGF-D (8 μ g/mL), PBST washes plate three times; To wherein adding anti-VEGF-D monoclonal antibody, hatch after 30 minutes with washings for 37 ℃ and give a baby a bath on the third day after its birth time.The sheep anti mouse Ig-HRP antibody of dilution in 1: 5000 is joined in the microtitre cylindrical void, hatch after 20 minutes for 37 ℃ and wash described cylindrical void as stated above.Add HRP enzyme substrates reaction solution A and B (each 80 μ l/ hole), with flat board 37 ℃ hatch 15 minutes after with the sulfuric acid stopped reaction of 2M, detect with Mutiskan MK3 (Thermo) microplate reader, detected result as shown in Figure 7, show that ABDD073 can suppress the interaction of VEGF-D and VEGFR-3, and along with the rising of ABDD073 dosage, it is strong more to suppress VEGF-D and the interactional ability of VEGFR-3.

The monoclonal antibody ABDD073 of embodiment 4, anti-VEGFR-3 suppresses the confirmatory experiment of hel cell propagation

With hel cell with 10 ⁴The concentration in individual/hole is inoculated in 96 orifice plates, to the ABDD073 antibody purification and the control antibodies normal mice IgG of the GST-VEGF-D that wherein adds 64 μ g/mL respectively and different concns (1,10,40,80,100,150,200,250,300nM), at 37 ℃, 5%CO ₂Incubator was cultivated after 72 hours, and every hole adds MTT solution 20 μ l, hatched for 37 ℃ to add the dmso solution precipitation after 4 hours, surveyed OD _492nmValue is analysed hel cell inhibition of proliferation rate with the SAS8.0 credit that takes statistics, and the result show that the monoclonal antibody ABDD073 of anti-VEGFR-3 can significantly suppress hel cell propagation, and control antibodies does not have obvious effect as shown in Figure 8.

The monoclonal antibody ABDD073 of embodiment 5, anti-VEGFR-3 suppresses the confirmatory experiment by the generation of reorganization GST-VEGF-D inductive chick chorioallantoic membrane blood vessel

Chick chorioallantoic membrane is the outer skim of chicken embryo, and major function provides the surface of carrying out gaseous interchange, and the enforcement of chick chorioallantoic membrane function simultaneously also depends on the support of fine and close vasoganglion.Because lymphatic vessel is accompanied by the formation of a large amount of chorioallantoic membrane blood vessels in the chick embryo development process, therefore chick chorioallantoic membrane is good model (the Mandriota SJ that the research blood vessel takes place and forms, Jussila L, Jeltsch M, et al.Vascular endothelial growth factor-c-mediated lymphangiogenesis promotes tumor metastasis[J] .EMBOJ, 2001,20 (4), 672-682.).Now the monoclonal antibody ABDD073 with anti-VEGFR-3 carries out the inhibition experiment by reorganization GST-VEGF-D inductive chick chorioallantoic membrane vasculogenesis, concrete grammar is: will hatch 3 days for 39 ℃ available from the zygote egg of Institute of Animal Husbandry, China Academy of Agriculture Scinces, aseptic condition adds the GST-VEGF-D albumen of the solubility of various dose (5,10,20 μ g/mL) down, with GST is contrast, observe its influence to the chick chorioallantoic membrane blood vessel, (A figure is the microscopic examination result to the result as shown in Figure 9; B figure is that (X-coordinate is a sample sets to blood vessel area density analytical results, ordinate zou is the blood vessel area density)), in contrast, only can obviously see the main blood vessel (a of chicken embryo, b arrow place), can see that in the experimental group that has added GST-VEGF-D a large amount of capillary vesseies generates (c, d, e), and along with the rising of GST-VEGF-D dosage, the quantity of the capillary vessel of generation also increases, and The above results shows that reorganization GST-VEGF-D has the effect that promotes chicken embryo vasculogenesis.The anti-VEGFR-3 monoclonal antibody ABDD073 that adds various dose (0,1.25,10 μ g/mL) again in 20 μ g/mL GST-VEGF-D dosage groups, with GST is contrast, detect it to restraining effect by reorganization GST-VEGF-D inductive chick chorioallantoic membrane vasculogenesis, after continuing to hatch 3 days with paraffin sealing, observe chicken embryo capillary blood vessel and generate situation, (A figure is the microscopic examination result to the result as shown in figure 10; B figure is that (X-coordinate is a sample sets to blood vessel area density analytical results, ordinate zou is the blood vessel area density)), after GST-VEGF-D joins the chick embryo allantois film edge, there is a large amount of capillary vesseies to generate, and after adding ABDD073, induce the blood vessel of generation to be suppressed by GST-VEGF-D by ABDD073 antibody.Above-mentioned experimental result proves that the monoclonal antibody ABDD073 of anti-VEGFR-3 has the effect that suppresses vasculogenesis.

The clone and the sequencing of embodiment 6, anti-VEGFR-3 monoclonal antibody ABDD073 gene order

Separation and purification mRNA from the hybridoma BDD073CGMCC No.1537 that produces ABDD073, the first chain cDNA is synthesized in reverse transcription, the primer of synthetic its variable region of light chain (VL) of design and variable region of heavy chain (VH), primer sequence is as follows:

The variable region of light chain primer:

PL1：5’-CTGCCATGGAGTCAGACACACTGCTG-3’

PL2：5’-TGGATGGTGGGAAGATGGA-3’；

The variable region of heavy chain primer:

PH1：5’-CAGGTGCAGCTTCAGGAATCTGG-3’

PH2：5’-CCAGGGGCCAGTGGATAGACAAGCTTGGGTGTCGTTTT-3’

PH3：5’-CCAGGGGCCAGTGGATAGACGGGTGG-3’

The eDNA that obtains with the total RNA of reverse transcription hybridoma BDD073 CGMCC No.1537 is a template, under the guiding of primer PL1 and PL2, with PCR method amplification ABDD073 chain variable region gene, 50 μ l PCR reaction systems are: 10 * PCR damping fluid, 5 μ l, dNTP mixture (2.5mM) 4 μ l, precious biological Ex Taq enzyme 0.2 μ l, template 2 μ l, PL1, each 1 μ l of PL2, ddH ₂O 36.8 μ l, reaction conditions are 94 ℃ of sex change 30s, 55 ℃ of annealing 45s, and 72 ℃ are extended 1min, totally 30 circulations; The cDNA that obtains with the total RNA of reverse transcription hybridoma BDD073 CGMCC No.1537 is a template, under the guiding of primer PH1, PH2 and PH3, with nested PCR method its heavy chain variable region gene that increases, 50 μ l PCR reaction systems are: 10 * PCR damping fluid, 5 μ l, dNTP mixture (2.5mM) 4 μ l, precious biological Ex Taq enzyme 0.2 μ l, template 2 μ l, PH1, PH2, each 1 μ l of PH3, ddH ₂O 36.8 μ l, reaction conditions is: 94 ℃ of sex change 30s, 60 ℃ of annealing 45s, 72 ℃ are extended 1min, totally 30 circulations.After reaction finishes, pcr amplification product is carried out 1% agarose gel electrophoresis to be detected, (swimming lane M is DNA Marker to detected result as shown in figure 11, swimming lane 1 is the chain variable region gene of amplification, and swimming lane 2 is the heavy chain variable region gene of amplification), the purpose fragment of recovery and purifying 396bp and 351bp, it is cloned into respectively among the carrier pGEM-T (Promega company), with recombinant products transformed into escherichia coli DH5 α competent cell, the screening positive recombinant, the upgrading grain checks order.Sequencing result shows that the ABDD073 chain variable region gene has SEQ ID № in the sequence table: 3 nucleotide sequence, SEQ ID № in the sequence table: 3 by 351 based compositions, its encoding sequence is that coding has SEQ ID № in the sequence table: the protein of 1 amino acid residue sequence from 5 ' end the 1st to 351 bit base; Heavy chain variable region gene has SEQ ID № in the sequence table: 4 nucleotide sequence, SEQ ID № in the sequence table: 4 by 396 based compositions, its encoding sequence is that coding has SEQ ID № in the sequence table: the protein of 2 amino acid residue sequence from 5 ' end the 1st to 396 bit base.

The preparation of embodiment 7, anti-VEGFR-3 small molecules single-chain antibody

Anti-VEGFR-3 small molecules single-chain antibody is the smallest molecule that keeps antibodies specific and biologic activity.Because its molecular weight is little, immunogenicity is low, helps becoming as preparing carriers the immunotherapeutical medicine of diseases such as treatment autoimmune disorder, infectious diseases and tumour.The preparation method of anti-VEGFR-3 small molecules single-chain antibody is as follows:

Design amplification ABDD073 is light, the primer of heavy chain variable region gene, wherein light chain downstream primer and heavy chain upstream primer have 20 bases to mate mutually, flexibly connect son (Gly4ser) 3 to guarantee that VL and VH fragment merge mutually and insert, form VL-linker-VH ScFv fragment; In addition, also contain restriction enzyme EcoRI recognition site at the upstream primer of light chain, the heavy chain downstream primer contains restriction enzyme NotI recognition site, so that the ScFv fragment cloning that merges is to expression vector, primer sequence is:

The variable region of light chain primer:

PL3 (upstream primer): 5 '-GC GAATTCATGGAGTCAGACACACTGCTG-3 ' (band underscore base is the EcoRI recognition site)

PL4 (downstream primer): 5 '-GAACCACCGCCGCCCGAACCGCCACCACCCCGTTTTATTTCCAGCTTG-3 ';

The variable region of heavy chain primer:

PH4 (upstream primer): 5 '-TCGGGCGGCGGTGGTTCCGGGGGTGGCGGCTCCCAGGTGCAGCTTCAGG-3 ';

PH5 (downstream primer): 5 '-ATAAGAAT GCGGCCGCAGAGACAGTGACCAGAGTCC-3 ' (band underscore base is the NotI recognition site)

Light, heavy chain variable region gene with the antibody A BDD073 that amplifies among the embodiment 6 is template respectively, respectively at above-mentioned primer to (PL3﹠amp; PL4, PH4﹠amp; PH5) under the guiding, with light, the heavy chain variable region gene of PCR method amplification antibody A BDD073.Get light, each 1 μ l of heavy chain variable region gene of the ABDD073 that is increased again, merge the PCR reaction, reaction system and reaction conditions are: VH, each 1 μ l of VL gene fragment of getting amplification, 94 ℃ of sex change 30s of elder generation, 70 ℃ of annealing 45s, 72 ℃ are extended 3min, totally 3 circulations; 94 ℃ of sex change 30s then, 55 ℃ of annealing 45s, 72 ℃ are extended 1min, totally 27 circulations.After reaction finishes, the PCR product is carried out 1% agarose gel electrophoresis detect, (swimming lane M is DNA Marker to detected result, and molecular weight ranges is identical with Figure 11 as shown in figure 12; Swimming lane 1 is the heavy chain variable region gene of amplification; Swimming lane 2 is the chain variable region gene of amplification; Swimming lane 3 is light, the heavy chain fusion rotein VL-linker-VH gene fragment of ABDD073), reclaim the also fusion gene fragment of purifying 792bp, it is cloned among the carrier PET32a (Novagen), obtain the efficient expression plasmid of ABDD073 soluble single-chain antibody, called after PET32a/VL-linker-VH, with recombinant products transformed into escherichia coli BL21 competent cell, screen the positive recombinant of secretory antibody with the ammonia benzyl mycin of 100mg/L.The picking positive recombinant, at 30 ℃, 0.2mM inducing culture under the IPTG, with the e. coli bl21 is contrast, and centrifugal collection contains supernatant, the precipitation of soluble antibody function fragment, carries out 10%SDS-PAGE and detects, (BL21 is contrast to detected result as shown in figure 13, Trx (TRX) is the empty carrier contrast, and Total Ivsisi is a whole cell), obtain the molecular weight size through expression and be the scfv fusion protein of 48KD.With affinity chromatography separation and purification single-chain antibody VL-linker-VH, use the ELISA system identical to identify specificity and the biological activity of the anti-VEGFR-3 single-chain antibody of the solubility VL-linker-VH that expresses again with embodiment 1, the result is as shown in table 2, shows that the single-chain antibody that gives expression to has the activity that combines with VEGFR-3.PET32a/VL-linker-VH is checked order, sequencing result shows that the anti-VEGFR-3 antibody gene of institute's synthetic strand has SEQ ID № in the sequence table: 6 nucleotide sequence, SEQ ID № in the sequence table: 6 by 792 based compositions, its encoding sequence is from 5 ' end the 1st to 792 bit base, flexibly connect son (Gly4ser) 3 from 5 ' end 397-441 bit base for coding, coding has a SEQ ID № in the sequence table: the protein of 5 amino acid residue sequence, the SEQ ID № in the sequence table: 5 are made up of 264 amino-acid residues.

The specificity of the anti-VEGFR-3 single-chain antibody of table 2 solubility VL-linker-VH and bioactive ELISA qualification result

	OD 450
								ScFv 6.5μg/mL	ScFv 13μg/mL	ScFv 65μg/mL	Normal mouse serum (1: 1000)	Anti-His (1∶1000)	PBS	The BDD073 supernatant
	VEGFR-3/Fc (0.5μg/mL)	0.515± 0.022	0.7296± 0.0495	1.007± 0.0799	0.05± 0.001	0.082± 0.050	0.081± 0.038								0.889± 0.109
Normal human IgG (0.5 μ g/mL)								0.0805± 0.0035	0.0066± 0.022	0.066± 0.008	0.064± 0.011	0.054± 0.013	0.061± 0.006	0.074± 0.018

Sequence table

<160>6

<210>1

<211>117

<212>PRT

＜213〉Mus mouse (Mus musculus)

<400>1

Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Arg Pro Arg Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Ala Arg Ile Arg Asn Lys Ser Asn Asn Tyr Ala Thr Tyr Tyr Ala Asn

50 55 60

Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Gly Asp Ser Gln Ser Met

65 70 75 80

Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr

85 90 95

Tyr Cys Val Arg Asp Gly Tyr Ser Leu Val His Trp Gly His Gly Thr

100 105 110

Leu Val Thr Val Ser

115

<210>2

<211>132

<212>PRT

＜213〉Mus mouse (Mus musculus)

<400>2

Met Glu Ser Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

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

20 25 30

Val Ser Leu Gly Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Lys Ser

35 40 45

Val Ser Thr Phe Gly Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro

50 55 60

Gly Gln Pro Pro Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser

65 70 75 80

Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

85 90 95

Leu Asn Ile His Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys

100 105 110

Gln His Ser Arg Glu Leu Met Tyr Thr Phe Gly Gly Gly Thr Lys Leu

115 120 125

Glu Ile Lys Arg

130

<210>3

<211>351

<212>DNA

＜213〉Mus mouse (Mus musculus)

<400>3

caggtgcagc ttcaggaatc tggtggagga ttggtgcggc ctagagggtc attgaaactc 60

tcatgtgcag cctctggatt caccttcaat acctacgcca tgaactgggt ccgccaggct 120

ccaggaaagg gtttggaatg gattgctcgc ataagaaata aaagtaataa ttatgcaaca 180

tattatgcca attcagtgaa agacaggttc accatctcca gaggtgattc acaaagcatg 240

ctctatctac aaatgaacaa cttgaaaact gaggacacag ccatgtatta ctgtgtgaga 300

gatggttact cccttgttca ctggggccat gggactctgg tcactgtctc t 351

<210>4

<211>396

<212>DNA

＜213〉Mus mouse (Mus musculus)

<400>4

atggagtcag acacactgct gttatgggta ctgctgctct gggttccagg ttccactggt 60

gacattgtgc tgacacagtc tcctgcttcc ttagctgtat ctctggggca gagggccacc 120

atctcatgca gggccagcaa aagtgtcagt acatttggct atagttatat gcactggtac 180

caacagaaac caggacagcc acccaaactc ctcatctatc ttgcatccaa cctagaatct 240

ggggtccctg ccaggttcag tggcagtggg tctgggacag acttcaccct caacatccat 300

cctgtggagg aggaggatgc tgcaacctat tactgtcagc acagtaggga acttatgtac 360

acgttcggag gggggaccaa gctggaaata aaacgg 396

<210>5

<211>264

<212>PRT

＜213〉artificial sequence

<220>

<223>

<400>5

Met Glu Ser Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

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

20 25 30

Val Ser Leu Gly Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Lys Ser

35 40 45

Val Ser Thr Phe Gly Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro

50 55 60

Gly Gln Pro Pro Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser

65 70 75 80

Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

85 90 95

Leu Asn Ile His Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys

100 105 110

Gln His Ser Arg Glu Leu Met Tyr Thr Phe Gly Gly Gly Thr Lys Leu

115 120 125

Glu Ile Lys Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

Glv Glv Ser Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Arg

145 150 155 160

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

165 170 175

Asn Thr Tyr Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

180 185 190

Glu Trp Ile Ala Arg Ile Arg Asn Lys Ser Asn Asn Tyr Ala Thr Tyr

195 200 205

Tyr Ala Asn Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Gly Asp Ser

210 215 220

Gln Ser Met Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr

225 230 235 240

Ala Met Tyr Tyr Cys Val Arg Asp Gly Tyr Ser Leu Val His Trp Gly

245 250 255

His Gly Thr Leu Val Thr Val Ser

260

<210>6

<211>792

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>6

atggagtcag acacactgct gttatgggta ctgctgctct gggttccagg ttccactggt 60

gacattgtgc tgacacagtc tcctgcttcc ttagctgtat ctctggggca gagggccacc 120

atctcatgca gggccagcaa aagtgtcagt acatttggct atagttatat gcactggtac 180

caacagaaac caggacagcc acccaaactc ctcatctatc ttgcatccaa cctagaatct 240

ggggtccctg ccaggttcag tggcagtggg tctgggacag acttcaccct caacatccat 300

cctgtggagg aggaggatgc tgcaacctat tactgtcagc acagtaggga acttatgtac 360

acgttcggag gggggaccaa gctggaaata aaacggggtg gtggcggttc gggcggcggt 420

ggttccgggg gtggcggctc ccaggtgcag cttcaggaat ctggtggagg attggtgcgg 480

cctagagggt cattgaaact ctcatgtgca gcctctggat tcaccttcaa tacctacgcc 540

atgaactggg tccgccaggc tccaggaaag ggtttggaat ggattgctcg cataagaaat 600

aaaagtaata attatgcaac atattatgcc aattcagtga aagacaggtt caccatctcc 660

agaggtgatt cacaaagcat gctctatcta caaatgaaca acttgaaaac tgaggacaca 720

gccatgtatt actgtgtgag agatggttac tcccttgttc actggggcca tgggactctg 780

gtcactgtct ct 792

Claims (10)

1, hybridoma cell strain BDD073 CGMCC No.1537.

2, the monoclonal antibody of the human VEGFR-3 resistant-3 of the described hybridoma cell strain BDD073 of claim 1 CGMCC No.1537 generation.

3, the monoclonal antibody of human VEGFR-3 resistant according to claim 2-3 is characterized in that: the variable region of heavy chain of described monoclonal antibody has the SEQ ID № in the sequence table: 1 amino acid residue sequence or with SEQ ID № in the sequence table: 1 amino acid residue sequence is through replacement, disappearance or the interpolation of one to ten amino-acid residue and have in the human VEGFR-3 resistant-3 and active polypeptide; Variable region of light chain has the SEQ ID № in the sequence table: 2 amino acid residue sequence or with SEQ ID № in the sequence table: 2 amino acid residue sequence is through replacement, disappearance or the interpolation of one to ten amino-acid residue and have in the human VEGFR-3 resistant-3 and active polypeptide.

4, the monoclonal antibody of human VEGFR-3 resistant according to claim 3-3 is characterized in that: the variable region of heavy chain of described monoclonal antibody has the SEQ ID № in the sequence table: 1 amino acid residue sequence; Variable region of light chain has the SEQ ID № in the sequence table: 2 amino acid residue sequence.

5, be derived from the single-chain antibody of claim 2 described human VEGFR-3 resistant-3 monoclonal antibody, Fab antibody and human mouse chimeric antibody.

6, the gene of coding claim 2 described human VEGFR-3 resistant-3 monoclonal antibody, its variable region of heavy chain encoding gene has SEQ ID № in the sequence table: SEQ ID № in 3 dna sequence dna or the code sequence tabulation: 1 dna sequence dna or under the rigorous condition of height can with SEQ ID № in the sequence table: the nucleotide sequence of the 3 dna sequence dnas hybridization that limit; The variable region of light chain encoding gene has SEQ ID № in the sequence table: SEQ ID № in the tabulation of 4 dna sequence dna or code sequence: 2 dna sequence dna or under the rigorous condition of height can with SEQ ID № in the sequence table: the nucleotide sequence of the 4 dna sequence dnas hybridization that limit.

7, gene according to claim 6 is characterized in that: described variable region of heavy chain encoding gene has SEQ ID № in the sequence table: 3 dna sequence dna; The variable region of light chain encoding gene has SEQ ID № in the sequence table: 4 dna sequence dna.

8, contain the described expression carrier of claim 6, transgenic cell line and host bacterium.

9, the described single-chain antibody that is derived from human VEGFR-3 resistant-3 monoclonal antibody of coding claim 5, the gene of Fab antibody and human mouse chimeric antibody.

10, the application of the monoclonal antibody of claim 2 described human VEGFR-3 resistant-3 in the curative drug of preparation inhibition tumor lympha transfer and angiogenesis inhibitor.

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