CN106519033A

CN106519033A - Anti-uPAR (urokinase plasminogen activator receptor)-antigen humanized antibody H2DL2 and application thereof

Info

Publication number: CN106519033A
Application number: CN201611023825.0A
Authority: CN
Inventors: 曹诚; 李成功; 靳彦文; 戴维·威孚
Original assignee: Institute of Bioengineering Chinese Academy of Military Medical Sciences
Current assignee: Institute of Bioengineering Chinese Academy of Military Medical Sciences
Priority date: 2016-11-14
Filing date: 2016-11-14
Publication date: 2017-03-22
Anticipated expiration: 2036-11-14
Also published as: CN106519033B

Abstract

The invention discloses an anti-uPAR (urokinase plasminogen activator receptor)-antigen humanized antibody H2DL2 and an application thereof. An IgG named antibody H2DL2 is provided firstly, CDR1, CDR2 and CDR3 in a heavy chain variable region of the IgG are shown sequentially as 50th-54th-position amino acid residues, 69th-85th-position amino acid residues and 118th-126th-position amino acid residues from the N terminal in the sequence 1 of the sequence table, and CDR1, CDR2 and CDR3 in a light chain variable region of the IgG are shown sequentially as 45th-54th-position amino acid residues, 70th-76th-position amino acid residues and 109th-116th-position amino acid residues from the N terminal in the sequence 3 of the sequence table. The provided antibody H2DL2 has good binding activity with human uPAR protein, can remarkably inhibit tumor cell migration and has broad application prospect in the field of tumor treatment.

Description

The humanized antibody H2DL2 of anti-uPAR antigens and its application

Technical field

The invention belongs to biological technical field, and in particular to a kind of humanized antibody H2DL2 of anti-uPAR antigens and its should With.

Background technology

The infiltration and transfer of tumour cell is which leaves initial site, moves to the group that the basilar memebrane for closing on invades surrounding Knit, or penetrate blood wall is shifted and infiltrates the process of position farther out.Urokinase type plasminogen activation system (uPAS) is in tumour Play an important role in the infiltration of cell and transfer process.The system is by plasma urokinase-type plasminogen activator (urokinase Plasminogen activator, uPA), uPAR Research (urokinase plasminogen Activator receptor, uPAR) and two PAI (PAI-1 and PAI-2) compositions.UPA is at the beginning of which It is inactive pepsinogen when beginning and secreting, it is 50KD glycoprotein that subsequent pepsinogen is hydrolyzed into activated molecular weight.uPAR Also known as CD87, which lacks cross-film and intracellular region, is that glycosyl-phosphatidyl inositol anchor determines protein, by tri- homeodomains of D1, D2 and D3 Composition.

Both at home and abroad many studies have shown that, uPAR many tumour cells especially SCCHN, colorectal cancer, High expression in the solid tumor cells such as prostate cancer and in normal cell low expression.Based on uPAR sticking, moving in tumour cell Important function in shifting, immune response, injury repair, infiltration and transfer, therefore which can be used as the important target of antineoplastic.

The content of the invention

It is an object of the invention to provide a kind of humanized antibody H2DL2 of anti-uPAR antigens and its application.

Present invention firstly provides a kind of IgG (being named as H2DL2 antibody), CDR1, CDR2 in its weight chain variable district and CDR3 is successively if the sequence 1 of sequence table is from N-terminal 50-54 amino acids residues, 69-85 amino acids residue and 118- Shown in 126 amino acids residues, CDR1, CDR2 and the CDR3 in its light chain variable district is successively if the sequence 3 of sequence table is from N-terminal Shown in 45-54 amino acids residues, 70-76 amino acids residue and 109-116 amino acids residues.

The weight chain variable district is specifically as shown in the sequence 1 from N-terminal 20-137 amino acids residues of sequence table.

The light chain variable district is specifically as shown in the sequence 3 from N-terminal 21-127 amino acids residues of sequence table.

The heavy chain of the IgG is concretely following (a) or (b)：A the sequence 1 of () sequence table is from N-terminal 20-467 positions ammonia The protein of base acid residue composition；Protein shown in the sequence 1 of (b) sequence table.

The light chain of the IgG is specially following (c) or (d)：C the sequence 3 of () sequence table is from N-terminal 21-233 bit aminos The protein of sour residue composition；Protein shown in the sequence 3 of (d) sequence table.

The gene for encoding the IgG falls within protection scope of the present invention.

The gene for encoding the heavy chain of the IgG is following (1) or (2) or (3)：

(1) DNA molecular shown in the sequence 2 of sequence table from 5 ' end 77-1420 positions nucleotides；

(2) DNA molecular shown in the sequence 2 of sequence table from 5 ' end 20-1423 positions nucleotides；

(3) DNA molecular shown in the sequence 2 of sequence table；

The gene for encoding the light chain of the IgG is following (4) or (5) or (6)：

(4) DNA molecular shown in the sequence 4 of sequence table from 5 ' end 80-718 positions nucleotides；

(5) DNA molecular shown in the sequence 4 of sequence table from 5 ' end 20-721 positions nucleotides；

(6) DNA molecular shown in the sequence 4 of sequence table.

The present invention also protects application of the H2DL2 antibody in the medicine for inhibition cancer cell migration is prepared.The cancer is thin Cancer cell of the born of the same parents for high expression uPAR Research.The cancer cell concretely colon cancer cell, more Concretely human colon cancer cell, such as SW480 cells.

The present invention also protects application of the H2DL2 antibody in the medicine for treating cancer is prepared.The cancer in particular can For colon cancer.The cancer in particular can be the cancer that the cancer cell of high expression uPAR Research causes. The cancer that the cancer in particular can cause for colon cancer cell.The colon cancer cell concretely human colon cancer cell, for example SW480 cells.

The present invention also protects application of the H2DL2 antibody in product is prepared；The purposes of the product for following (f1) or Or (f3) or (f4) (f2)：

(f1) detect cancer cell；

(f2) combine cancer cell；

(f3) detect carrying human urokinase-type plasminogen activator acceptor (people's uPAR albumen)；

(f4) with reference to carrying human urokinase-type plasminogen activator acceptor (people's uPAR albumen).

The cancer cell is the cancer cell of high expression uPAR Research.The cancer cell specifically may be used For colon cancer cell, can more specifically be human colon cancer cell, such as SW480 cells.

The present invention also protects a kind of product, and its active component is H2DL2 antibody；The purposes of the product for following (f1) or Or (f3) or (f4) (f2)：

(f1) detect cancer cell；

(f2) combine cancer cell；

The H2DL2 antibody that the present invention is improved has good binding activity with people's uPAR albumen, can significantly inhibit tumour Cell migration, has broad application prospects in the field for the treatment of tumour.

Description of the drawings

Fig. 1 is elution curve.

10%SDS-PAGE collection of illustrative plates of the Fig. 2 for H2DL2 solution.

Results of the Fig. 3 for cell scratch experiment.

Specific embodiment

Below example facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments Method, if no special instructions, is conventional method.Test material used in following embodiments, if no special instructions, is certainly What routine biochemistry reagent shop was commercially available.Quantitative test in following examples, is respectively provided with three repetitions and tests, as a result make even Average.

PcDNA-3.3 carriers (linear plasmid)：Invitrogen companies, catalog number (Cat.No.) K8300-01.POptiVEC carrier (lines Property grain)：Invitrogen companies, catalog number (Cat.No.) 12744-017.293F cells：Thermo Products, catalog number (Cat.No.) R79007. SW480 cells (human colon cancer cell)：ATCC, catalog number (Cat.No.)CCL-228。I culture medium (Opti-MEMI Reduced Serum Medium)：Invitrogen companies, catalog number (Cat.No.) 31985-062.Liposome (293fectin^TMTransfection Reagent)：Invitrogen companies, catalog number (Cat.No.) 12347-019.Freestyle^TM 293 expression culture medium (FreeStyle^TM293Expression Medium)：Invitrogen companies, catalog number (Cat.No.) 12338-018. Recombined human uPAR albumen (abbreviation uPAR-ECD)：Yi Qiao Divine Land Bioisystech Co., Ltd, article No. 10925-H08H-100.Such as nothing Specified otherwise, the PBS in embodiment are the PBS of pH7.4,0.01M.

The discovery of embodiment 1, antibody

Inventor carries out microcomputer modelling and transformation based on groping in a large number, analyzing, verifying, devises a kind of new resisting The antibody (being named as H2DL2 antibody) of uPAR antigens.H2DL2 antibody is IgG, and, as shown in the sequence 1 of sequence table, which is light for its heavy chain Chain is as shown in the sequence 3 of sequence table.

In the sequence 1 of sequence table, leader peptide, 20-137 amino acids are constituted from N-terminal 1-19 amino acids residue Residue composition weight chain variable district VL (wherein, 50-54 amino acids residue composition CDR1,69-85 amino acids residue composition CDR2,118-126 amino acids residue composition CDR3), 138-235 amino acids residue composition CH CH1, the 236-250 amino acids residue composition heavy chain hinge region Hinge, 251-361 amino acids residue composition CH CH2,362-467 amino acids residue composition CH CH3.

Polypeptide shown in the sequence 1 of the DNA molecular polynucleotide shown in the sequence 2 of sequence table.The sequence 2 of sequence table In, from 5 ' end 20-76 positions nucleotide coding leader peptides, 77-430 positions nucleotide coding VH, 431-724 positions nucleotides Coding CH1,725-769 positions nucleotide coding Hinge, 770-1102 positions nucleotide coding CH2,1103-1420 positions core Thuja acid encodes CH3, and 1421-1423 positions nucleotides is terminator codon.

In the sequence 3 of sequence table, leader peptide, 21-127 amino acids are constituted from N-terminal 1-20 amino acids residue Residue composition light chain variable district VL (wherein, 45-54 amino acids residue composition CDR1,70-76 amino acids residue composition CDR2,109-116 amino acids residue composition CDR3), 128-233 amino acids residue composition constant region of light chain CL.

Polypeptide shown in the sequence 3 of the DNA molecular polynucleotide shown in the sequence 4 of sequence table.4 institute of sequence of sequence table The DNA molecular for showing is from 5 ' end 20-79 positions nucleotide coding leader peptides, 80-400 positions nucleotide coding VL, 401-718 Position nucleotide coding CL, 719-721 positions nucleotides are terminator codon.

The preparation of embodiment 2, antibody

First, the structure of recombinant plasmid

1st, by the DNA molecular insertion pOptiVEC carriers shown in the sequence 2 of sequence table, obtain recombinant plasmid pOptiVEC/ H2D。

2nd, by the DNA molecular insertion pcDNA-3.3 carriers shown in the sequence 4 of sequence table, obtain recombinant plasmid pcDNA- 3.3-L2。

2nd, the preparation of H2DL2 antibody

1st, 15 μ g recombinant plasmids pOptiVEC/H2D and 15 μ g recombinant plasmid pcDNA-3.3-L2 are taken, is usedI Culture medium is settled to 1ml, gently mixes.

2nd, 60 μ l liposomes are taken, is usedI culture mediums are settled to 1ml, are incubated at room temperature 5min after gently mixing.

3rd, the solution of the solution and step 2 of step 1 is mixed, is incubated at room temperature 20-30min.

4th, 125ml Tissue Culture Flasks are taken, Freestyle is used in addition^TM293F cell (the 293F that 293 expression culture mediums suspend Cell content is 3 × 10⁷Individual cell).

5th, the solution that 2ml completes step 3 is added in the Tissue Culture Flask for completing step 4, in 37 DEG C, 8%CO₂、 Cultivate 6-9 days under conditions of 150rpm vibrations.

6th, after completing step 5,12000rpm centrifugation 15min collect supernatant, adjust pH to 6.0-7.0, then filtered with 0.45 μm Membrane filtration, collects filtrate.

7th, the filtrate that step 6 is obtained is taken, is carried out using Bio-Rad Duo-Flow protein purification systems (760-0135) pure Change.

Bio-Scale Mini Affi-prep proteinA affinity columns (Bole company, article No. 732-4602)：Post Volume is 5 milliliters of prepacked columns.

Combination buffer (pH7.5)：The phosphate buffer of the 20mM containing 3M NaCl.

Elution buffer (pH2.8)：0.1M citrate buffers.

Flow velocity：2ml/min.

Process：(1) pillar is balanced with 50ml combination buffers；(2) filtrate that loading 3ml step 6 is obtained；(3) use 50ml Combination buffer washs pillar；(4) destination protein is eluted with 25ml elution buffers, collect and retain UV280 more than 0.05AU's Cross solution after post.The elution curve of whole process is shown in Fig. 1.

8th, solution after the post excessively that step 7 is obtained is taken, and pH to 7.0 is adjusted with Tris-HCl solution (pH9.0) at once, is then used Molecular cut off for 10kD super filter tube (Millipore companies, Amicon Ultra-4) carry out concentration change liquid process (concentration is changed The buffer solution that liquid is adopted is for PBS), the solution for the obtaining as solution containing H2DL2 antibody is named as H2DL2 solution. The 10%SDS-PAGE collection of illustrative plates of H2DL2 solution is shown in Fig. 2 (swimming lane 1 and swimming lane 2 in Fig. 2 is all H2DL2 solution).

3rd, the preparation of ATN615 antibody

ATN615 antibody is a kind of monoclonal antibody of targeting uPAR, and the antibody is higher with people's uPAR affinity and does not affect The combination of uPAR albumen and uPA.ATN615 antibody, its heavy chain as shown in the sequence 5 of sequence table, the sequence 7 of light chain such as sequence table It is shown.Polypeptide shown in the sequence 5 of the double chain DNA molecule polynucleotide shown in the sequence 6 of sequence table.The sequence 8 of sequence table Polypeptide shown in the sequence 7 of shown double chain DNA molecule polynucleotide.

With reference to step one and step 2, ATN615 solution is prepared.

4th, empty carrier test

Replace recombinant plasmid pOptiVEC/H2D with pOptiVEC carriers, replace recombinant plasmid with pcDNA-3.3 carriers PcDNA-3.3-L2, carries out the 1 to 7 of step 2 successively, and any eluting peak is not shown in elution process.

Embodiment 3, affinity is detected

First, ELISA detections

1st, it is coated with

ELISA Plate is taken, 100 μ l coating buffers, 4 DEG C of overnight incubations are added per hole.The preparation method of coating buffer：With pH9.0, UPAR-ECD is diluted by 0.05M carbonate buffer solutions, obtains the solution that protein concentration is 2ng/ μ l.

2nd, close

After completing step 1, the ELISA Plate is taken, with PBST solution board-washing three times (every time 350 μ l, every time 3 minutes), then 200 μ l, 10% calf serums are added per hole, 37 DEG C is put and is incubated 2 hours.

3rd, it is loaded

After completing step 2, take the ELISA Plate, per hole add 100 μ l sample to be tested solution, 37 DEG C be incubated 2 hours, then With PBST solution board-washing three times (every time 350 μ l, every time 3 minutes).Sample to be tested solution is H2DL2 solution prepared by embodiment 2 Dilution or embodiment 2 prepare ATN615 solution dilution, be diluted as solvent with PBS.Treat test sample The protein concentration of this solution is 0.125 μ g/ml or 0.0625 μ g/ml.

4th, enzyme-added labeling antibody

After completing step 3, the ELISA Plate is taken, 100 μ l enzyme labelled antibodies (Goat anti human IgG-HRP, middle China fir gold are added per hole Bridge, article No. ZB-2304), 37 DEG C are incubated 2 hours, then with PBST solution board-washing three times (every time 350 μ l, every time 3 minutes).

5th, develop the color

After completing step 4, the ELISA Plate is taken, 100 μ l tmb substrate solution are added per hole, and (Tiangeng is biological, PA107- 01), 37 DEG C are incubated 15 minutes.

6th, terminating reaction

After completing step 4, the ELISA Plate is taken, 50 μ l 2M sulfuric acid solutions are added per hole.

7th, result judgement

ELIASA (MD190) scanning wavelength is adjusted to into OD450, each hole OD values are detected, if more than the negative control OD of regulation 2.1 times of value, it is as positive.

Carry out five repetitions to test, results averaged.

Used as solution to be measured, corresponding OD450 values are the dilution (protein concentration is 0.125 μ g/ml) of H2DL2 solution 2.11.Used as solution to be measured, corresponding OD450 values are the dilution (protein concentration is 0.0625 μ g/ml) of H2DL2 solution 1.44.Used as solution to be measured, corresponding OD450 values are the dilution (protein concentration is 0.125 μ g/ml) of ATN615 solution 1.94.Used as solution to be measured, corresponding OD450 values are the dilution (protein concentration is 0.0625 μ g/ml) of ATN615 solution 1.26。

As a result show, the affinity of H2DL2 antibody on human uPAR albumen is significantly higher than ATN615 antibody.

2nd, Fortebio detections

Fortebio methods of operating are briefly as follows:(1) 30s is balanced to system baseline with the PBS of pH7.2；(2) will The protein concentration of test antibodies (test antibodies are ATN615 solution prepared by H2DL2 solution prepared by embodiment 2 or embodiment 2) Adjust to 0.2mg/ml, be fixed on the chip of anti-human igg Fc by Loading loading steps；(3) with pH7.2's PBS carries out releveling to system baseline；(4) by antigen uPAR-ECD be adjusted to concentration for 1 μM, 0.75 μM, 0.5 μM with 0.25 μM of four concentration, and the above-mentioned combination for carrying out 60s；(5) the antigen 1 20s that PBS dissociation is combined, machine detection reaction signal, Binding constant Ka and dissociation constant Kd value that analysis antigen-antibody is combined, and then calculate equilibrium dissociation constant KD value (KD=Kd/ Ka).The bigger explanation dissociation of KD is more, it is weaker to represent affinity, and the less explanation dissociation of KD is less, to represent affinity stronger.

Carry out five repetitions to test, results averaged.

The ka values of ATN615 antibody and uPAR-ECD are 7.733e4 (1/Ms), and kd values are 5.757e-4 (1/s), and KD values are 7.445e-9(M).The ka values of H2DL2 antibody and uPAR-ECD are 5.469e4 (1/Ms), and kd values are 3.023e-4 (1/s), KD values For 5.528e-9 (M).

3rd, Flow cytometry

1st, take the logarithm the SW480 cells in growth period, concentration is prepared into for 5 × 10⁵The cell suspension of cell/ml.

2nd, take 1 EP to manage, the cell suspension for adding 1ml steps 1 to obtain, 4 DEG C, 1000rpm centrifugation 5min abandon supernatant, use PBSs of the 1ml containing 1%FBS washed once.

3rd, after completing step 2, take the EP and manage, 200 μ l of addition solution to be measured, 4 DEG C of incubation 60min, then 4 DEG C, 1000rpm is centrifuged 5min, abandons supernatant, washed once with PBSs of 500 μ containing 1%FBS.

Solution to be measured is the dilute of ATN615 solution prepared by the dilution or embodiment 2 of H2DL2 solution prepared by embodiment 2 Liquid is released, and protein concentration is 5 μ g/ml, protein concentration is adjusted with PBS.

4th, after completing step 3, take EP pipe, add 100 μ l FITC marks goat anti-human igg (GAH-FITC, 1：32 Dilution), 4 DEG C of lucifuges are incubated 45min.

5th, after completing step 4, take EP pipe, 4 DEG C, 1000rpm, centrifugation 5min, abandon supernatant, contain 1%FBS with 500 μ l PBS washed once.

6th, after completing step 5, the EP pipes are taken, is flowed after adding PBS re-suspended cells of the 300 μ l containing 1%FBS Formula cell detection.

Carry out five repetitions to test, results averaged.

The positive rate combined with SW480 cells by H2DL2 antibody is 99.91%, and average fluorescent strength is 9.13.ATN615 resists The positive rate combined with SW480 cells by body is 98.69%, and average fluorescent strength is 8.87.

As a result show, H2DL2 antibody can be effectively combined SW480 cells, and binding ability is higher than ATN615 antibody.

Embodiment 4, cell scratch experiment

The most basic biological property of tumour cell is, with invasion and attack and the ability for shifting, to study invasion and metastasis of tumor Rule and its mechanism, the preventing and treating to malignant tumour are significant.Cell scarification is to determine Tumor Cell Migration characteristic A kind of method.

1st, compared in six orifice plate behind rulers with Marker pens, uniformly draw horizontal line, per 5, hole line.

2nd, six orifice plates of step 1 are taken, 5 × 10 is added per hole⁵Individual SW480 cells, are placed in 37 DEG C, 5%CO₂Train in incubator Support 12 hours (cell is paved with bottom hole).

3rd, after completing step 2, six orifice plate is taken, compares ruler with pipette tips, as far as possible perpendicular to Marker line cuts, pipette tips Will be perpendicular to ware bottom.

4th, after completing step 3, six orifice plate is taken, washs three times to remove the cell under drawing with PBS.

5th, after completing step 4, six orifice plate is taken, DMEM culture mediums of the 2ml containing 10%FBS is added per hole, 5 μ L are added (solution to be measured is H2DL2 solution or ATN615 solution prepared by embodiment 2 so that the AC in system is for solution to be measured 50 μ g/ml), it is placed in 37 DEG C, 5%CO₂Cultivate in incubator.Using equal-volume PBS as solution to be measured negative control. Sample after culture 0h or 48h and taken pictures, measure cut spacing.

As a result see Fig. 3.

Carry out five repetitions to test, results averaged.

When solution to be measured is PBS, 0h moment average scratches distance is 5.5cm, and after 48h, average scratches distance is 1cm.When solution to be measured is H2DL2 solution, 0h moment average scratches distance is 5.5cm, and after 48h, average scratches distance is 5.2cm. When solution to be measured is ATN615 solution, 0h moment average scratches distance is 5.5cm, and after 48h, average scratches distance is 4cm.

Sequence table

<110>Biologic Engineering Inst., Academy of Millitary Medical Sciences of P.L.A

<120>The humanized antibody H2DL2 of anti-uPAR antigens and its application

<130> GNCYX161890

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<210> 1

<211> 467

<212> PRT

<213>Artificial sequence

<400> 1

Met Asp Trp Thr Trp Arg Val Phe Cys Leu Leu Ala Val Ala Pro Gly

1 5 10 15

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

20 25 30

Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe

35 40 45

Ser Asn Phe Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu

50 55 60

Glu Trp Ile Gly Trp Ile Phe His Gly Ser Asp Asn Thr Glu Tyr Asn

65 70 75 80

Glu Lys Phe Lys Ser Lys Ala Thr Ile Thr Ala Asp Glu Ser Thr Ser

85 90 95

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

100 105 110

Phe Tyr Cys Ala Arg Trp Gly Pro His Trp Tyr Phe Asp Ala Trp Gly

115 120 125

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

130 135 140

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

145 150 155 160

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

165 170 175

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

180 185 190

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

195 200 205

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

210 215 220

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

225 230 235 240

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

245 250 255

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

260 265 270

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

275 280 285

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

290 295 300

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

305 310 315 320

Arg Val Val Ser Val Leu Ser Val Leu His Gln Asp Trp Leu Asn Gly

325 330 335

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

340 345 350

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

355 360 365

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

370 375 380

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

385 390 395 400

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

405 410 415

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

420 425 430

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

435 440 445

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

450 455 460

Pro Gly Lys

465

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<213>Artificial sequence

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aagcttaatt gccgccacca tggactggac ctggagggtc ttctgcttgc tggctgtagc 60

tccaggtgct caatccgagg ttcagctggt tcagtccggt gctgaggtta agaagccagg 120

ttcctctgtt aaggtttctt gcaaggcttc tggctacacc ttctctaact tctacatcca 180

ctgggttcgt caggctccag gccagggtct ggagtggatc ggttggatct tccacggttc 240

tgacaacact gagtacaacg agaagttcaa gtctaaggct actatcactg ctgacgagtc 300

cacttctacc gcttacatgg agctgtcctc tctgcgttcc gaggacactg ctgttttcta 360

ctgcgctcgt tggggtccac actggtactt cgacgcttgg ggtcgtggta ctctggttac 420

tgtttcctca gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag 480

cacctctggg ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt 540

gacggtgtcg tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct 600

acagtcctca ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg 660

cacccagacc tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa 720

agttgagccc aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact 780

cctgggggga ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc 840

ccggacccct gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa 900

gttcaactgg tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga 960

gcagtacaac agcacgtacc gtgtggtcag cgtcctctcc gtcctgcacc aggactggct 1020

gaatggcaag gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa 1080

aaccatctcc aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcctccatc 1140

tcgggatgag ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc 1200

cagcgacatc gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac 1260

gcctcccgtg ctggactccg acggctcctt cttcctctat agcaagctca ccgtggacaa 1320

gagcaggtgg cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa 1380

ccactacacg cagaagagcc tctccctgtc tccgggtaaa tga 1423

<210> 3

<211> 233

<212> PRT

<213>Artificial sequence

<400> 3

Met Glu Thr Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro

1 5 10 15

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

20 25 30

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

35 40 45

Ser Val Ser Tyr Ile His Trp Tyr Gln Gln Lys Pro Gly Arg Ala Pro

50 55 60

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

65 70 75 80

Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Tyr Thr Leu Thr Ile Ser

85 90 95

Ser Leu Gln Ser Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Asn

100 105 110

Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr

115 120 125

Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu

130 135 140

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

145 150 155 160

Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly

165 170 175

Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr

180 185 190

Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His

195 200 205

Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val

210 215 220

Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230

<210> 4

<211> 721

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<213>Artificial sequence

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aagcttaatt gccgccacca tggagacacc cgcccagctg ctgttcctgc tgctgctgtg 60

gctgcccgac accaccggca tggacatcca gatgactcag tccccatcta ctctgtctgc 120

ttccgttggc gaccgtgtta ccatcacttg ccgtgcttct agctccgttt cttacatcca 180

ctggtaccag caaaagccgg gtcgtgctcc gaagccactg atgtacgagg cttcctctcg 240

cgctactgga gttccatctc gcttctctgg ttccggttct ggcactgagt acactctgac 300

tatctcctct ctgcagagcg acgacttcgc tacttactac tgccagcagt ggaactaccc 360

attcactttc ggacagggta ctaagctgga gatcaagcgg accgtggcgg cgccatctgt 420

cttcatcttc ccgccatctg atgagcagtt gaaatctggt accgctagcg ttgtgtgcct 480

gctgaataac ttctatccca gagaggccaa agtacagtgg aaggtggata acgccctcca 540

atcgggtaac tcccaggaga gtgtcacaga gcaggacagc aaggacagca cctacagcct 600

cagcagcacc ctgacgctga gcaaagcaga ctacgagaaa cacaaagtct acgcctgcga 660

agtcacccat cagggcctga gctcgcccgt cacaaagagc ttcaacaggg gagagtgtta 720

g 721

<210> 5

<211> 468

<212> PRT

<213>Artificial sequence

<400> 5

Met Asp Trp Thr Trp Arg Val Phe Cys Leu Leu Ala Val Ala Pro Gly

1 5 10 15

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

20 25 30

Lys Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser

35 40 45

Phe Thr Asn Phe Tyr Ile His Trp Val Lys Gln Arg Pro Gly Gln Gly

50 55 60

Leu Glu Trp Ile Gly Trp Ile Phe His Gly Ser Asp Asn Thr Glu Tyr

65 70 75 80

Asn Glu Lys Phe Lys Asp Lys Ala Thr Leu Thr Ala Asp Thr Ser Ser

85 90 95

Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala

100 105 110

Val Tyr Phe Cys Ala Arg Trp Gly Pro His Trp Tyr Phe Asp Val Trp

115 120 125

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

130 135 140

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

145 150 155 160

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

165 170 175

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

180 185 190

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

195 200 205

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

210 215 220

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

225 230 235 240

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

245 250 255

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

260 265 270

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

275 280 285

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

290 295 300

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

305 310 315 320

Tyr Arg Val Val Ser Val Leu Ser Val Leu His Gln Asp Trp Leu Asn

325 330 335

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

340 345 350

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

355 360 365

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

370 375 380

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

385 390 395 400

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

405 410 415

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

420 425 430

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

435 440 445

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

450 455 460

Ser Pro Gly Lys

465

<210> 6

<211> 1426

<212> DNA

<213>Artificial sequence

<400> 6

aagcttaatt gccgccacca tggactggac ctggagggtc ttctgcttgc tggctgtagc 60

tccaggtgct caatccatgg gcgttaagct gcagcaatcc ggcccagagg ttgttaagcc 120

aggtgcttct gttaagatct cctgcaaggc ttctggatac tccttcacta acttctacat 180

ccactgggtt aagcagcgtc caggtcaggg tctggagtgg atcggttgga tcttccacgg 240

ttctgacaac actgagtaca acgagaagtt caaggacaag gctactctga ctgctgacac 300

ttcctctagc actgcttaca tgcagctgtc ctctctgact agcgaggatt ccgctgttta 360

cttctgcgct cgctggggcc cacactggta cttcgacgtt tggggtcagg gtaccactgt 420

taccgtttcc tctgctagca ccaagggccc atcggtcttc cccctggcac cctcctccaa 480

gagcacctct gggggcacag cggccctggg ctgcctggtc aaggactact tccccgaacc 540

ggtgacggtg tcgtggaact caggcgccct gaccagcggc gtgcacacct tcccggctgt 600

cctacagtcc tcaggactct actccctcag cagcgtggtg accgtgccct ccagcagctt 660

gggcacccag acctacatct gcaacgtgaa tcacaagccc agcaacacca aggtggacaa 720

gaaagttgag cccaaatctt gtgacaaaac tcacacatgc ccaccgtgcc cagcacctga 780

actcctgggg ggaccgtcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat 840

ctcccggacc cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt 900

caagttcaac tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga 960

ggagcagtac aacagcacgt accgtgtggt cagcgtcctc tccgtcctgc accaggactg 1020

gctgaatggc aaggagtaca agtgcaaggt ctccaacaaa gccctcccag cccccatcga 1080

gaaaaccatc tccaaagcca aagggcagcc ccgagaacca caggtgtaca ccctgcctcc 1140

atctcgggat gagctgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta 1200

tcccagcgac atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac 1260

cacgcctccc gtgctggact ccgacggctc cttcttcctc tatagcaagc tcaccgtgga 1320

caagagcagg tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca 1380

caaccactac acgcagaaga gcctctccct gtctccgggt aaatga 1426

<210> 7

<211> 233

<212> PRT

<213>Artificial sequence

<400> 7

Met Glu Thr Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Lys Pro Trp Ile Phe Glu Ile Ser Lys Leu Ala Ser Gly Val Pro Ala

65 70 75 80

Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser

85 90 95

Ser Met Glu Ala Glu Asp Ala Ala Ile Tyr Tyr Cys Gln Gln Trp Asn

100 105 110

Tyr Pro Phe Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr

115 120 125

Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu

130 135 140

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

145 150 155 160

Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly

165 170 175

Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr

180 185 190

Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His

195 200 205

Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val

210 215 220

Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230

<210> 8

<211> 721

<212> DNA

<213>Artificial sequence

<400> 8

aagcttaatt gccgccacca tggagacacc cgcccagctg ctgttcctgc tgctgctgtg 60

gctgcccgac accaccggca tggacatcgt gctgacccag tccccagaca tcactgctgc 120

ttctctgggc cagaaggtta ctatcacttg ctctgcttct tccagcgttt cctacatgca 180

ctggtaccag cagaagtccg gcactagccc taagccttgg atcttcgaga tctccaagct 240

ggcttccggc gtgccggctc gcttctccgg ctctggttcc ggcactagct actccctgac 300

catctcttcc atggaggctg aggacgctgc tatctactac tgccagcagt ggaactaccc 360

attcactttc ggcggtggta ctaagctgga gatcaagcgg accgtggcgg cgccatctgt 420

cttcatcttc ccgccatctg atgagcagtt gaaatctggt accgctagcg ttgtgtgcct 480

gctgaataac ttctatccca gagaggccaa agtacagtgg aaggtggata acgccctcca 540

atcgggtaac tcccaggaga gtgtcacaga gcaggacagc aaggacagca cctacagcct 600

cagcagcacc ctgacgctga gcaaagcaga ctacgagaaa cacaaagtct acgcctgcga 660

agtcacccat cagggcctga gctcgcccgt cacaaagagc ttcaacaggg gagagtgtta 720

g 721

Claims

1. a kind of IgG, CDR1, CDR2 and the CDR3 in its weight chain variable district are successively if the sequence 1 of sequence table is from N-terminal 50- Shown in 54 amino acids residues, 69-85 amino acids residue and 118-126 amino acids residues, in its light chain variable district CDR1, CDR2 and CDR3 successively as sequence table sequence 3 from N-terminal 45-54 amino acids residues, 70-76 bit aminos Shown in sour residue and 109-116 amino acids residues.

2. IgG as claimed in claim 1, it is characterised in that：

The weight chain variable district is as shown in the sequence 1 from N-terminal 20-137 amino acids residues of sequence table；

The light chain variable district is as shown in the sequence 3 from N-terminal 21-127 amino acids residues of sequence table.

3. IgG as claimed in claim 2, it is characterised in that：

The heavy chain is following (a) or (b)：A the sequence 1 of () sequence table is constituted from N-terminal 20-467 amino acids residue Protein；Protein shown in the sequence 1 of (b) sequence table；

The light chain is following (c) or (d)：C the sequence 3 of () sequence table is constituted from N-terminal 21-233 amino acids residue Protein；Protein shown in the sequence 3 of (d) sequence table.

4. the gene of IgG described in claim 3 is encoded.

5. gene as claimed in claim 4, it is characterised in that：

The gene for encoding the heavy chain is following (1) or (2) or (3)：

(3) DNA molecular shown in the sequence 2 of sequence table；

The gene for encoding the light chain is following (4) or (5) or (6)：

(6) DNA molecular shown in the sequence 4 of sequence table.

6. applications of the IgG described in claim 1 or 2 or 3 in the medicine for inhibition cancer cell migration is prepared.

7. applications of the IgG described in claim 1 or 2 or 3 in the medicine for treating cancer is prepared.

8. applications of the IgG in product is prepared described in claim 1 or 2 or 3；The purposes of the product is following (f1) or (f2) Or (f3) or (f4)：

(f1) detect cancer cell；

(f2) combine cancer cell；

(f3) detect carrying human urokinase-type plasminogen activator acceptor；

(f4) with reference to carrying human urokinase-type plasminogen activator acceptor.

9. a kind of product, its active component are IgG described in claim 1 or 2 or 3；The purposes of the product for following (f1) or Or (f3) or (f4) (f2)：

(f1) detect cancer cell；

(f2) combine cancer cell；

(f3) detect carrying human urokinase-type plasminogen activator acceptor；

10. the application as described in claim 6 or 7 or 8 or product as claimed in claim 9, it is characterised in that：The cancer is thin Born of the same parents are colon cancer cell；The cancer is colon cancer.