CN106432498A - Humanized antibody H2BL2 for resisting uPAR (urokinase plasminogen activator receptor) antigen and application of humanized antibody H2BL2 - Google Patents

Humanized antibody H2BL2 for resisting uPAR (urokinase plasminogen activator receptor) antigen and application of humanized antibody H2BL2 Download PDF

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CN106432498A
CN106432498A CN201611023722.4A CN201611023722A CN106432498A CN 106432498 A CN106432498 A CN 106432498A CN 201611023722 A CN201611023722 A CN 201611023722A CN 106432498 A CN106432498 A CN 106432498A
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戴维·威孚
鲁亚芳
张部昌
徐昌志
朱林
王萌
李萌
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Anhui University
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Abstract

The invention discloses a humanized antibody H2BL2 for resisting an uPAR (urokinase plasminogen activator receptor) antigen and application of the humanized antibody H2BL2. The invention provides IgG (named H2BL2 antibody) firstly, CDR1, CDR2 and CDR3 in a heavy chain variable region of the IgG is sequentially shown as amino acid residues at sites from 45 to 54, from 69 to 85 and from 118 to 126 from an N terminus in a sequence 1 in a sequence table, and CDR1, CDR2 and CDR3 in a light chain variable region of the IgG is sequentially shown as amino acid residues at sites from 45 to 54, from 70 to 76 and from 109 to 116 from an N terminus in a sequence 3 in the sequence table. The improved H2BL2 antibody is good in combination activity with a human uPAR protein, can remarkably inhibit tumor cell migration and has wide application prospect in the field of treating tumors.

Description

The humanized antibody H2BL2 of anti-uPAR antigen and its application
Technical field
The invention belongs to biological technical field and in particular to a kind of humanized antibody H2BL2 of anti-uPAR antigen and its should With.
Background technology
The infiltration of tumor cell and transfer are that it leaves initial site, move to the group that the basement membrane closing on invades surrounding Knit, or penetrate blood wall shifts and infiltrates the process of position farther out.Urokinase type plasminogen activation system (uPAS) is in tumor Play an important role in the infiltration of cell and transfer process.This system is by plasma urokinase-type plasminogen activator (urokinase Plasminogen activator, uPA), uPAR Research (urokinase plasminogen Activator receptor, uPAR) and two plasminogen activator inhibitor (PAI-1 and PAI-2) compositions.UPA is at the beginning of it 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, it 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 tumor cells especially squamous cell carcinoma of the head and neck, colorectal cancer, High expression in the solid tumor cells such as carcinoma of prostate and in normal cell low expression.Based on uPAR sticking, moving in tumor cell Important function in shifting, immunne response, injury repairing, infiltration and transfer, therefore it can be used as the important target of antitumor drug.
Content of the invention
It is an object of the invention to provide a kind of humanized antibody H2BL2 of anti-uPAR antigen and its application.
Present invention firstly provides a kind of IgG (being named as H2BL2 antibody), CDR1, the 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 residue, 69-85 amino acids residue and 118- Shown in 126 amino acids residues, CDR1, CDR2 and CDR3 in its light chain variable district are successively if the sequence 3 of sequence table is from N-terminal Shown in 45-54 amino acids residue, 70-76 amino acids residue and 109-116 amino acids residue.
Described weight chain variable district specifically as sequence table sequence 1 from N-terminal 20-137 amino acids residue shown in.
Described light chain variable district specifically as sequence table sequence 3 from N-terminal 21-127 amino acids residue shown in.
The heavy chain of described IgG is concretely following (a) or (b)：A the sequence 1 of () sequence table is from N-terminal 20-467 position ammonia The protein of base acid residue composition；The protein shown in sequence 1 of (b) sequence table.
The light chain of described IgG is specially following (c) or (d)：C the sequence 3 of () sequence table is from N-terminal 21-233 bit amino The protein of sour residue composition；The protein shown in sequence 3 of (d) sequence table.
The gene encoding described IgG falls within protection scope of the present invention.
The gene encoding the heavy chain of described IgG is following (1) or (2) or (3)：
(1) DNA molecular shown in from 5 ' end 77-1420 position nucleotide for the sequence 2 of sequence table；
(2) DNA molecular shown in from 5 ' end 20-1423 position nucleotide for the sequence 2 of sequence table；
(3) DNA molecular shown in sequence 2 of sequence table；
The gene encoding the light chain of described IgG is following (4) or (5) or (6)：
(4) DNA molecular shown in from 5 ' end 80-718 position nucleotide for the sequence 4 of sequence table；
(5) DNA molecular shown in from 5 ' end 20-721 position nucleotide for the sequence 4 of sequence table；
(6) DNA molecular shown in sequence 4 of sequence table.
The present invention also protects H2BL2 antibody in preparation for the application in the medicine of anticancer migration.Described cancer is thin Born of the same parents are the cancerous cell of high expression uPAR Research.Described cancerous cell concretely colon cancer cell, more Concretely human colon cancer cell, such as SW480 cell.
The present invention also protects H2BL2 antibody to be used for the application in the medicine for the treatment of cancer in preparation.Described cancer in particular can For colon cancer.The cancer that described cancer in particular can cause for the cancerous cell of high expression uPAR Research. The cancer that described cancer in particular can cause for colon cancer cell.Described colon cancer cell concretely human colon cancer cell, for example SW480 cell.
The present invention also protects application in preparing product for the H2BL2 antibody；The purposes of described product be following (f1) or Or (f3) or (f4) (f2)：
(f1) detect cancerous cell；
(f2) combine cancerous cell；
(f3) detection carrying human urokinase-type plasminogen activator receptor (people's uPAR albumen)；
(f4) combine carrying human urokinase-type plasminogen activator receptor (people's uPAR albumen).
Described cancerous cell is the cancerous cell of high expression uPAR Research.Described cancerous cell specifically may be used For colon cancer cell, can be more specifically human colon cancer cell, such as SW480 cell.
The present invention also protects a kind of product, and its active component is H2BL2 antibody；The purposes of described product be following (f1) or Or (f3) or (f4) (f2)：
(f1) detect cancerous cell；
(f2) combine cancerous cell；
(f3) detection carrying human urokinase-type plasminogen activator receptor (people's uPAR albumen)；
(f4) combine carrying human urokinase-type plasminogen activator receptor (people's uPAR albumen).
Described cancerous cell is the cancerous cell of high expression uPAR Research.Described cancerous cell specifically may be used For colon cancer cell, can be more specifically human colon cancer cell, such as SW480 cell.
The H2BL2 antibody that the present invention improves and people's uPAR albumen have good binding activity, can significantly inhibit tumor Cell migration, has broad application prospects in the field for the treatment of tumor.
Brief description
Fig. 1 is elution curve.
Fig. 2 is the 10%SDS-PAGE collection of illustrative plates of H2BL2 solution.
Fig. 3 is the result of 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 Routine biochemistry reagent shop is commercially available.Quantitative test in following examples, is respectively provided with three times and repeats to test, result is made even Average.
PcDNA-3.3 carrier (linear plasmid)：Invitrogen company, catalog number (Cat.No.) K8300-01.POptiVEC carrier (line Property grain)：Invitrogen company, catalog number (Cat.No.) 12744-017.293F cell：Thermo Products, catalog number (Cat.No.) R79007. SW480 cell (human colon cancer cell)：Purchased from ATCC, catalog number (Cat.No.)CCL-228.Opti-I culture medium (Opti- MEMI Reduced Serum Medium)：Invitrogen company, catalog number (Cat.No.) 31985-062.Liposome (293fectinTMTransfection Reagent)：Invitrogen company, catalog number (Cat.No.) 12347-019.FreestyleTM 293 expression culture medium (FreeStyleTM293Expression Medium)：Invitrogen company, 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.As no Specified otherwise, the PBS in embodiment is the PBS of pH7.4,0.01M.
Embodiment 1, the discovery of antibody
Inventor is based on and gropes in a large number, analyzes, verifying, carries out microcomputer modelling and transformation, devises a kind of new resisting The antibody (being named as H2BL2 antibody) of uPAR antigen.H2BL2 antibody is IgG, and, as shown in the sequence 1 of sequence table, it is light for its heavy chain Chain is as shown in the sequence 3 of sequence table.
In the sequence 1 of sequence table, form leader peptide, 20-137 amino acids 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 forms 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.
The polypeptide shown in sequence 1 of the DNA molecular polynucleotide shown in sequence 2 of sequence table.The sequence 2 of sequence table In, from 5 ' end 20-76 position nucleotide coding leader peptides, 77-430 position nucleotide coding VH, 431-724 position nucleotide Coding CH1,725-769 position nucleotide coding Hinge, 770-1102 position nucleotide coding CH2,1103-1420 position core Thuja acid encodes CH3, and 1421-1423 position nucleotide is termination codon.
In the sequence 3 of sequence table, form leader peptide, 21-127 amino acids 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 forms constant region of light chain CL.
The polypeptide shown in sequence 3 of the DNA molecular polynucleotide shown in sequence 4 of sequence table.Sequence 4 institute of sequence table The DNA molecular showing is from 5 ' end 20-79 position nucleotide coding leader peptides, 80-400 position nucleotide coding VL, 401-718 Position nucleotide coding CL, 719-721 position nucleotide is termination codon.
Embodiment 2, the preparation of antibody
First, the structure of recombiant plasmid
1st, by the DNA molecular insertion pOptiVEC carrier shown in the sequence 2 of sequence table, obtain recombiant plasmid pOptiVEC/ H2B.
2nd, by the DNA molecular insertion pcDNA-3.3 carrier shown in the sequence 4 of sequence table, obtain recombiant plasmid pcDNA- 3.3-L2.
2nd, the preparation of H2BL2 antibody
1st, take 15 μ g recombiant plasmid pOptiVEC/H2B and 15 μ g recombiant plasmid pcDNA-3.3-L2, use Opti-I trains Foster base is settled to 1ml, gently mixes.
2nd, take 60 μ l liposomees, use Opti-I culture medium is settled to 1ml, incubated at room 5min after gently mixing.
3rd, the solution of the solution of step 1 and step 2 is mixed, incubated at room 20-30min.
4th, take 125ml Tissue Culture Flask, add and use FreestyleTM293F cell (the 293F that 293 expression culture medium suspend Cell content is 3 × 107Individual cell).
5th, the solution that 2ml is completed step 3 adds in the Tissue Culture Flask completing step 4, in 37 DEG C, 8%CO2、 Cultivate 6-9 days under conditions of 150rpm vibration.
6th, after completing step 5,12000rpm is centrifuged 15min, collects supernatant, adjusts pH to 6.0-7.0, is then filtered with 0.45 μm Membrane filtration, collects filtrate.
7th, take the filtrate that step 6 obtains, carried out using Bio-Rad Duo-Flow protein purification system (760-0135) pure Change.
Bio-Scale Mini Affi-prep proteinA affinity column (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 buffer.
Flow velocity：2ml/min.
Process：(1) 50ml combination buffer is used to balance pillar；(2) filtrate that loading 3ml step 6 obtains；(3) use 50ml Combination buffer washs pillar；(4) use 25ml elution buffer eluting destination protein, 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, take what step 7 obtained to cross solution after post, use Tris-HCl solution (pH9.0) to adjust pH to 7.0 at once, then use Molecular cut off be 10kD super filter tube (Millipore company, Amicon Ultra-4) carry out concentration change liquid process (concentration is changed The buffer that liquid adopts is PBS), the solution obtaining is the solution containing H2BL2 antibody, is named as H2BL2 solution. Fig. 2 (swimming lane 1 in Fig. 2 and swimming lane 2 are all H2BL2 solution) is shown in by the 10%SDS-PAGE collection of illustrative plates of H2BL2 solution.
3rd, the preparation of ATN615 antibody
ATN615 antibody is a kind of monoclonal antibody of targeting uPAR, and this antibody is higher with people's uPAR affinity and does not affect UPAR albumen and the combination of 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 Shown.The polypeptide shown in sequence 5 of the double chain DNA molecule polynucleotide shown in sequence 6 of sequence table.The sequence 8 of sequence table The polypeptide shown in sequence 7 of shown double chain DNA molecule polynucleotide.
With reference to step one and step 2, prepare ATN615 solution.
4th, empty carrier test
Replace recombiant plasmid pOptiVEC/H2B with pOptiVEC carrier, replace recombiant plasmid with pcDNA-3.3 carrier PcDNA-3.3-L2, carries out the 1 to 8 of step 2 successively, does not show any eluting peak in elution process.
Embodiment 3, affinity detection
First, ELISA detection
1st, it is coated
Take ELISA Plate, every hole adds 100 μ l to be coated liquid, 4 DEG C of overnight incubation.It is coated the preparation method of liquid：With pH9.0, UPAR-ECD is diluted by 0.05M carbonate buffer solution, obtains the solution that protein concentration is 2ng/ μ l.
2nd, close
After completing step 1, take described ELISA Plate, wash plate three times (every time 350 μ l, 3 minutes every time) with PBST solution, then Every hole adds 200 μ l 10% calf serum, puts 37 DEG C and is incubated 2 hours.
3rd, it is loaded
After completing step 2, take described ELISA Plate, every hole adds 100 μ l sample to be tested solution, 37 DEG C are incubated 2 hours, then Wash plate three times (every time 350 μ l, 3 minutes every time) with PBST solution.Sample to be tested solution is the H2BL2 solution of embodiment 2 preparation Diluent or embodiment 2 preparation ATN615 solution diluent, 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, take described ELISA Plate, every hole adds 100 μ l enzyme labelled antibodies (Goat anti human IgG-HRP, middle China fir gold Bridge, article No. ZB-2304), 37 DEG C are incubated 2 hours, then wash plate three times (every time 350 μ l, 3 minutes every time) with PBST solution.
5th, develop the color
After completing step 4, take described ELISA Plate, every hole adds 100 μ lTMB substrate solutions (Tiangeng is biological, PA107-01), 37 DEG C are incubated 15 minutes.
6th, terminating reaction
After completing step 4, take described ELISA Plate, every hole adds 50 μ l2M sulfuric acid solutions.
7th, result judgement
Microplate reader (MD190) scanning wavelength is adjusted to OD450, detects each hole OD value, if the negative control OD more than regulation 2.1 times of value, as positive.
Carry out five times repeating to test, results averaged.
As solution to be measured, corresponding OD450 value is the diluent (protein concentration is 0.125 μ g/ml) of H2BL2 solution 1.60.As solution to be measured, corresponding OD450 value is the diluent (protein concentration is 0.0625 μ g/ml) of H2BL2 solution 1.38.As solution to be measured, corresponding OD450 value is the diluent (protein concentration is 0.125 μ g/ml) of ATN615 solution 1.55.As solution to be measured, corresponding OD450 value is the diluent (protein concentration is 0.0625 μ g/ml) of ATN615 solution 1.26.
Result shows, the affinity of H2BL2 antibody on human uPAR albumen is significantly higher than ATN615 antibody.
2nd, Fortebio detection
Fortebio operational approach is briefly as follows:(1) PBS using pH7.2 balances 30s to system baseline；(2) will The protein concentration of test antibodies (test antibodies are the H2BL2 solution of embodiment 2 preparation or the ATN615 solution of embodiment 2 preparation) Adjust to 0.2mg/ml, be fixed on the chip of anti-human igg Fc by Loading loading step；(3) use pH7.2's PBS carries out reequilibrate to system baseline；(4) by antigen uPAR-ECD be adjusted to concentration be 1 μM, 0.75 μM, 0.5 μM with 0.25 μM of four concentration, with the above-mentioned combination carrying out 60s；(5) the antigen 1 20s that PBS dissociation combines, machine detects reaction signal, Binding constant Ka and dissociation constant Kd value that analysis antigen-antibody combines, and then calculate equilibrium dissociation constant KD value (KD=Kd/ Ka).KD bigger explanation dissociation is more, it is weaker to represent affinity, and KD less explanation dissociation is less, to represent affinity stronger.
Carry out five times repeating to test, results averaged.
ATN615 antibody is 7.733e4 (1/Ms) with the ka value of uPAR-ECD, and kd value for 5.757e-4 (1/s), KD value is 7.445e-9(M).H2BL2 antibody is 7.179e4 (1/Ms) with the ka value of uPAR-ECD, and kd value is 4.538e-4 (1/s), KD value For 6.321e-9 (M).
Result shows, the affinity of H2BL2 antibody on human uPAR albumen is significantly higher than ATN615 antibody.
3rd, Flow cytometry
1st, take the logarithm the SW480 cell of trophophase, being prepared into concentration is 5 × 105The cell suspension of cell/ml.
2nd, take 1 EP pipe, add the cell suspension that 1ml step 1 obtains, 4 DEG C, 1000rpm centrifugation 5min, abandon supernatant, use The PBS that 1ml contains 1%FBS washed once.
3rd, after completing step 2, described EP is taken to manage, 4 DEG C of incubation 60min of addition 200 μ l solution to be measured, then 4 DEG C, 1000rpm is centrifuged 5min, abandons supernatant, be washed once with the PBS that 500 μ contain 1%FBS.
Solution to be measured be embodiment 2 preparation the diluent of H2BL2 solution or embodiment 2 preparation ATN615 solution dilute Release liquid, protein concentration is 5 μ g/ml, adjusts protein concentration with PBS.
4th, after completing step 3, take described EP pipe, add 100 μ lFITC labellings goat anti-human igg (GAH-FITC, 1：32 is dilute Release), 4 DEG C of lucifuges are incubated 45min.
5th, after completing step 4, take described 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, take described EP pipe, add 300 μ l to be flowed after containing the PBS re-suspended cell of 1%FBS Formula cell detection.
Carry out five times repeating to test, results averaged.
The positive rate that H2BL2 antibody is combined with SW480 cell is 99.64%, and average fluorescent strength is 4.44.ATN615 resists The positive rate that body is combined with SW480 cell is 98.64%, and average fluorescent strength is 4.96.
Result shows, H2BL2 antibody can be effectively combined SW480 cell, and binding ability is higher than ATN615 antibody.
Embodiment 4, cell scratch experiment
The most basic biological property of tumor cell is the ability with invasion and attack and transfer, research invasion and metastasis of tumor Rule and its mechanism, the preventing and treating to malignant tumor is significant.Cell scarification is to measure Tumor Cell Migration characteristic A kind of method, for identifying the ability of Drug inhibition tumor cell migration.
1st, compared in six orifice plate behind rulers with Marker pen, uniformly draw horizontal line, 5 lines in every hole.
2nd, six orifice plates of step 1 are taken, every hole adds 5 × 105Individual SW480 cell, is placed in 37 DEG C, 5%CO2Train in incubator Support 12 hours (cell is paved with bottom hole).
3rd, after completing step 2, take described six orifice plates, compare ruler with pipette tips, as far as possible perpendicular to Marker line cut, pipette tips Will be perpendicular to ware bottom.
4th, after completing step 3, take described six orifice plates, wash three times with PBS to remove the cell under drawing.
5th, after completing step 4, described six orifice plates are taken, every hole adds the DMEM culture medium that 2ml contains 10%FBS, adds 5 μ L (solution to be measured is the H2BL2 solution of embodiment 2 preparation or ATN615 solution so that the antibody concentration 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.
Result is shown in Fig. 3.
Carry out five times repeating 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 H2BL2 solution, 0h moment average scratches distance is 5.5cm, and after 48h, average scratches distance is 4.5cm. 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>University of Anhui
<120>The humanized antibody H2BL2 of anti-uPAR antigen and its application
<130> GNCYX161889
<160> 8
<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
Thr Asp Tyr 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
<210> 2
<211> 1423
<212> DNA
<213>Artificial sequence
<400> 2
aagcttaatt gccgccacca tggactggac ctggagggtc ttctgcttgc tggctgtagc 60
tccaggtgct caatccgagg ttcagctggt tcagtccggt gctgaggtta agaagccagg 120
ttcctctgtt aaggtttctt gcaaggcttc tggctacact ttcaccgact actacatcca 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
<212> DNA
<213>Artificial sequence
<400> 4
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 (10)

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 residue, in its light chain variable district CDR1, CDR2 and CDR3 successively as sequence table sequence 3 from N-terminal 45-54 amino acids residue, 70-76 bit amino Shown in sour residue and 109-116 amino acids residue.
2. IgG as claimed in claim 1 it is characterised in that：
Described weight chain variable district as sequence table sequence 1 from N-terminal 20-137 amino acids residue shown in；
Described light chain variable district as sequence table sequence 3 from N-terminal 21-127 amino acids residue shown in.
3. IgG as claimed in claim 2 it is characterised in that：
Described heavy chain is following (a) or (b)：A the sequence 1 of () sequence table forms from N-terminal 20-467 amino acids residue Protein；The protein shown in sequence 1 of (b) sequence table；
Described light chain is following (c) or (d)：C the sequence 3 of () sequence table forms from N-terminal 21-233 amino acids residue Protein；The protein shown in sequence 3 of (d) sequence table.
4. the gene of IgG described in coding claim 3.
5. gene as claimed in claim 4 it is characterised in that：
The gene encoding described heavy chain is following (1) or (2) or (3)：
(1) DNA molecular shown in from 5 ' end 77-1420 position nucleotide for the sequence 2 of sequence table；
(2) DNA molecular shown in from 5 ' end 20-1423 position nucleotide for the sequence 2 of sequence table；
(3) DNA molecular shown in sequence 2 of sequence table；
The gene encoding described light chain is following (4) or (5) or (6)：
(4) DNA molecular shown in from 5 ' end 80-718 position nucleotide for the sequence 4 of sequence table；
(5) DNA molecular shown in from 5 ' end 20-721 position nucleotide for the sequence 4 of sequence table；
(6) DNA molecular shown in sequence 4 of sequence table.
6. claim 1 or IgG described in 2 or 3 application in the medicine that preparation migrates for anticancer.
7. claim 1 or IgG described in 2 or 3 are used for the application in the medicine for the treatment of cancer in preparation.
8. claim 1 or application in preparing product for the IgG described in 2 or 3；The purposes of described product is following (f1) or (f2) Or (f3) or (f4)：
(f1) detect cancerous cell；
(f2) combine cancerous cell；
(f3) detect carrying human urokinase-type plasminogen activator receptor；
(f4) combine carrying human urokinase-type plasminogen activator receptor.
9. a kind of product, its active component is claim 1 or IgG described in 2 or 3；The purposes of described product be following (f1) or Or (f3) or (f4) (f2)：
(f1) detect cancerous cell；
(f2) combine cancerous cell；
(f3) detect carrying human urokinase-type plasminogen activator receptor；
(f4) combine carrying human urokinase-type plasminogen activator receptor.
10. the application as described in claim 6 or 7 or 8 or product as claimed in claim 9 it is characterised in that：Described cancer is thin Born of the same parents are colon cancer cell；Described cancer is colon cancer.
CN201611023722.4A 2016-11-14 2016-11-14 Humanized antibody H2BL2 for resisting uPAR (urokinase plasminogen activator receptor) antigen and application of humanized antibody H2BL2 Pending CN106432498A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023125842A1 (en) * 2021-12-29 2023-07-06 博生吉医药科技(苏州)有限公司 Development of novel upar single-domain antibody

Citations (2)

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Publication number Priority date Publication date Assignee Title
WO2011100620A2 (en) * 2010-02-12 2011-08-18 The Regents Of The University Of California Upar binding agents and methods of use thereof
CN101798348B (en) * 2009-12-09 2012-07-11 中国人民解放军军事医学科学院生物工程研究所 Anti-uPAR humanized antibody and application thereof

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CN101798348B (en) * 2009-12-09 2012-07-11 中国人民解放军军事医学科学院生物工程研究所 Anti-uPAR humanized antibody and application thereof
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023125842A1 (en) * 2021-12-29 2023-07-06 博生吉医药科技(苏州)有限公司 Development of novel upar single-domain antibody

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Application publication date: 20170222