CN1919874B - Antibody molecule ATF-Fc fusion albumen of antiurokinase type fibrinolysin activator receptor and use thereof - Google Patents
Antibody molecule ATF-Fc fusion albumen of antiurokinase type fibrinolysin activator receptor and use thereof Download PDFInfo
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Abstract
The invention discloses an antibody molecule ATF-Fc merge protein and application of antiurokinase type profibrinolytic activator acceptor in biological medicine area. which is characterized by the following: the ATF-Fc merge protein is ATF area of uPA and Fc of human immune globulin IgG1; heavy chain constants region 2 and heavy chain constants region 3 through Ser-Gly-Ser-Gly-Ser joint form to dimer merge protein; possessing 742 amino acids; single-chain possesses amino acids sequence of sequence table SEQ ID No.1, wherein the front 20 amino acids is uPA signal peptide.
Description
Technical field
The present invention relates to the antibody molecule ATF-Fc fusion rotein of a kind of antiurokinase type fibrinolysin activator receptor (uPAR) and the purposes in oncotherapy thereof, belong to field of biological pharmacy.
Background technology
Urokinase type fibrinolysin activator (urokinase-type plasminogen activator, uPA) protein of forming by 411 amino-acid residues, comprise the double-stranded uPA (tcu-PA) that does not have the strand of enzymatic activity uPA (scu-PA) and enzymatic activity is arranged, strand uPA also claims uPA (Pro-UK), under effects such as plasmin, trypsinase and kallikrein, the double-stranded uPA that the plasminogen activation function is arranged that forms after the peptide bond rupture between Lys158-Ile159 (is a urokinase, UK).
The glycoprotein of the about 52-54kD of molecular weight that natural uPA is made up of 411 amino acid, 12 pairs of disulfide linkage are arranged, a N-glycosylation site (Asn302) and an O-glycosylation site (Thr18), have three protein structure domains (Fig. 1): (EGF-like district, (1) skins somatomedin district, 5-49 amino acids residue is formed), main being responsible for combines with uPA acceptor (uPAR); (2) Kringle district (50-135aa) may be relevant with chemotactic activity; (3) serine protease position district is the primary structure territory that uPA brings into play its fibrinolytic activity, and wherein His204, Asp255 and Ser356 constitute the active centre of enzyme.
The EGF-like district of uPA and Kringle district be collectively referred to as ATF (Amino-Terminal Fragment, ATF).Many critical functions of uPA are all relevant with ATF, such as combining and biological chemotaxis with uPA acceptor (uPAR).The performance of a series of critical functions of uPA depends on and uPAR (urokinase-type plasminogen activator receptor, combination uPAR).UPAR is made up of three structural domains that (D1, D2 D3), fix on cell surface by glycosyl-phosphatidyl inositol anchor.D1 in conjunction with uPA after, uPAR changes active condition into, have the height chemotactic activity.UPA combines with uPAR by the EGF structural domain, attached to cell surface, plasminogen activation (plasminogen), make it to change into the plasmin (plasmin) that catalytic activity is arranged, then plasmin then activates and extracellular matrix degraded involved enzyme, as metal matrix proteolytic enzyme etc., the degraded extracellular matrix promotes tumor cell migration.Much studies show that recently, nearly all malignant tumour, especially late period, overexpression uPAR all on its tumor cell membrane, uPA and acceptor uPAR thereof play keying action in multiple malignant tumour Invasion and Metastasis such as colorectal carcinoma, ovarian cancer, mammary cancer, cancer of the stomach, prostate cancer and lung cancer.The uPA-uPAR system has participated in tumor growth, vasculogenesis and transfer, and uPAR has become the new target of anticancer therapy.
Also do not develop the report of ATF-Fc fusion rotein at present both at home and abroad.
Summary of the invention
The main technical problem to be solved in the present invention provides the human antibody class artificial protein ATF-Fc fusion rotein of a kind of safe and effective anti-uPAR.
Another technical problem that the present invention will solve provides a kind of stabilization characteristics of genetics, ATF-Fc Expression of Fusion Protein carrier that expression amount is high.
Last problem that the present invention will solve provides the ATF-Fc fusion rotein and contains the purposes of expression carrier in the medicine of preparation treatment cancer of this fusion rotein of encoding.
For achieving the above object, the present invention is by the following technical solutions:
A kind of ATF-Fc fusion rotein, it is (the EGF-like district+Kringle district, ATF district of uPA, the 1-134 amino acids of high molecular uPA) dimer fusion protein (Fig. 2) that is connected to form by Ser-Gly-Ser-Gly-Ser joint (linker) with the Fc section (hinge area (Hinge region), CH2 (CH2) and CH3 (CH3)) of human normal immunoglobulin IgG1, strand ATF-Fc molecule has the aminoacid sequence shown in the sequence table SEQ ID No.1, totally 391 amino-acid residues; Wherein preceding 20 signal peptides that amino acid is uPA, so sophisticated strand ATR-Fc has 371aa, dimer is 742aa.
The ATF-Fc fusion rotein of the present invention's development is the human antibody class artificial protein of a kind of anti-uPAR, ATF is responsible for combining with uPAR, human IgG1's Fc section is then brought into play the physiological function of antibody molecule, as cellulotoxic effect (ADCC), complement-dependent cellulotoxic effect (CDC) or the immune opsonization of antibody dependent cellular mediation, thereby target kills the tumour cell of uPAR overexpression.
Coding has the gene of the ATF-Fc fusion rotein strand of aminoacid sequence shown in the sequence table SEQ ID No.1, has the nucleotide sequence shown in the sequence table SEQ ID No.2.
Coding has the gene of fusion rotein strand of the ATF-Fc of aminoacid sequence shown in the sequence table SEQ IDNo.1, includes but not limited to SEQ ID No.2.The utilization techniques well known, the coding nucleotide sequence that can design its preference according to the expression system that is adopted for example can adopt the preference codon of yeast expression system or escherichia expression system etc. to improve expression efficiency.All can expressed sequence table SEQ ID No.1 shown in the coding nucleotide sequence of aminoacid sequence all belong to and be equal to replacement of the present invention.In addition, the utilization techniques well known can be used other linker to connect ATF and two sections peptide sections of Fc, even can connect ATF and Fc without linker.All ATF-Fc homodimer fusion roteins that comprise ATF and human IgG Fc composition all are considered as the albumen that is equal to of the present invention.
The expression vector that contains nucleotide sequence shown in the SEQ ID No.2, these expression vectors can be commercial source or obtain through texture improvement and making up.
The cell that contains above-mentioned expression vector includes but not limited to protokaryon or eukaryotic cell, for example intestinal bacteria, yeast and Chinese hamster ovary celI etc.
The ATF-Fc fusion rotein that the present invention makes up is to adopt gene recombination technology to express in Chinese hamster ovary celI (Chinese hamster ovary cell), and detailed process is:
(1) ATF gene clone, with the pIRES-dhfr/HMW-ProUK carrier is that template (preserve at China Committee for Culture Collection of Microorganisms common micro-organisms center by this expression vector, preservation day is on February 24th, 2006, preserving number is CGMCC No.1623, the microbial strain of ginseng certificate is pIRES-dhfr/HMW-ProUK, the classification called after colon bacillus Escherichia coli of suggestion, Chinese invention patent 200610065813.4), with primer ATFs and ATFx is the upstream and downstream primer, pcr amplification ATF gene reclaims the ATF gene.
ATFs：5’-TGCGCTAGCCCACCATGAGAGCCCTGCTGGCGCG-3’
ATFx：5’-CGCGGATCCACTTACCTGTACTGCCAGATCCGCTTCCATCTGCGCAGTCATGCAC-3’
(2) carrier for expression of eukaryon of structure ATF-Fc fusion rotein, with Nhe I and BamH I double digestion ATF gene, use Nhe I and BamH I double digestion carrier pcDNA3.1/ATR-Fc (to preserve at China Committee for Culture Collection of Microorganisms common micro-organisms center simultaneously, register on the books and be numbered: CGMCC No.1399, China's invention 200510084233.5), reclaim expression vector pcDNA3.1/Fc, two segment DNAs that reclaim are connected with the T4 ligase enzyme, transformed into escherichia coli, select positive colony and carry out enzyme and cut and identify and two-way sequencing, obtain expressing the carrier for expression of eukaryon pcDNA3.1/ATF-Fc of ATF-Fc fusion rotein.
(3) transfection CHO cell and screen the positive colony of high expression level, using the liposome method transfection to the CHO-K1 cell carrier for expression of eukaryon pcDNA3.1/ATF-Fc that expresses the ATR-Fc fusion rotein, is 10-15 μ g/ (10 with G418 screening and acquisition ATF-Fc expression level
6Cells.d) genetically engineered Chinese hamster ovary celI is ATF-Fc-H6.
(4) cell cultures and purification of Recombinant ATF-Fc albumen, with rolling bottle serum-free culture ATF-Fc-H6 cell, collect supernatant and adopt the albumin A purification of recombinant proteins, and identify that with the ELISA method ATF-Fc had both had and anti-uPA polyclonal antibody bonded characteristic, show that the ATF among the ATF-Fc has kept the identical structural performance of ATF among the uPA, can combine with the polyclonal antibody of anti-human IgG1 Fc again, show that ATF-Fc has the structural performance of human IgG Fc.
(5) the antineoplastic biologic activity of ATF-Fc is estimated in cell and experimentation on animals, estimate ATF-Fc with MCF-7, stomach cancer cell system and esophageal cancer cell system and suppressed growth of tumour cell and transfer character, the result shows, ATF-Fc has very strong lethal effect to breast cancer cell line, stomach cancer cell system there is certain lethal effect and can suppresses cell migration, system does not have lethal effect to esophageal cancer cell, but the effect of certain its migration of inhibition is arranged.In nude mice lotus knurl experiment, ATF-Fc shows the curative effect that suppresses tumor growth and transfer preferably, and curative effect and dosage are tangible positive correlation.
The carrier for expression of eukaryon pcDNA3.1/ATF-Fc of the ATF-Fc fusion rotein that the present invention makes up preserves at China Committee for Culture Collection of Microorganisms common micro-organisms center, preservation date is on August 3rd, 2006, register on the books and be numbered: CGMCC No.1774, the microbial strain name of preserving is called colon bacillus (Escherichia coli), (address: No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City, Institute of Microorganism, Academia Sinica, 100080).Adopt LipofectamineTM2000 cationic-liposome transfection reagent box (Invitrogen Co) with this carrier (CGMCCNo.1774) transfection CHO-K1 cell (ATCCNo.CRL9096), the genetically engineered Chinese hamster ovary celI that has obtained the higher expression ATF-Fc of expression level through the G418 screening and cloning is ATF-Fc-H6, the about 10-15 μ of the expression level of this clone g/ (10
6Cellsd), and continuous passage cultivated 1 year, and expression level does not change.
The carrier for expression of eukaryon carrier for expression of eukaryon pcDNA3.1/ATF-Fc (CGMCC No.1774) that ATF-Fc fusion rotein and being used to is expressed ATF-Fc is used to prepare the purposes of the medicine for the treatment of tumour.
Described tumour is mammary cancer, cancer of the stomach and other uPAR high expression level tumours.
The ATF-Fc fusion rotein that the CH Fc of urokinase type plasminogen activator of the present invention (uPA) and its acceptor (uPAR) bonded key structure territory ATF and human normal immunoglobulin IgG1 is connected to form can be used for preparing the application of the tumor treatment medicine of uPAR overexpression.
Because the ATF-Fc fusion rotein had both kept among the uPA and uPAR bonded key structure territory ATF, lacked the enzyme active center of plasminogen activation (plasminogen) among the uPA again, ATF-Fc can combine uPAR with uPA competitiveness like this, stop uPA to combine with uPAR, the activation of blocking-up plasminogen, thus the degraded of extracellular matrix and the migration of cell suppressed.On the other hand, because ATF-Fc has the Fc section, after being attached to the tumor cell surface of uPAR high expression level, can be by CDC effect, ADCC effect, promote phagolysis and bring out effects such as apoptosis, the kill tumor cell, promptly the human antibody of ATF-Fc and anti-uPAR has identical function, and just ATF-Fc has higher avidity, and the uPAR site of sealing just with uPA bonded zone, thereby have better therapeutic than anti-uPAR antibody.Cell and experimentation on animals confirm: ATF-Fc can obviously suppress breast cancer cell and vitro growth of gastric cancer cell and transfer, and this fusion rotein is worth further studying and being developed to the antibody class medicine of the tumour for the treatment of mammary cancer, stomach cancer cell and other uPAR overexpressions.
Advantage of the present invention is: this fusion rotein can combine with the antigen high-affinity, be equivalent to the Fab fragment of antibody, and the Fc section provides the biologic activity of antibody, and it has following characteristics: (1) no antigen, and two sections albumen of ATF and Fc are all from human body; (2) avidity height, all higher (Kd～10 of avidity of general part and acceptor
-10M), uPA and uPAR bonded affinity costant Kd～10
-10M; (3) block site is special, because uPAR has 308aa, have a plurality of epitopes, the anti-uPAR monoclonal antibody that therefore might be sieved to high-affinity may not stop uPA to combine with uPAR, and ATF-Fc specificity sealing uPAR is in conjunction with the structural domain of uPA, thereby stops combining of uPA and uPAR; (4) also have the ADDC effect and the CDC effect of antibody; (5) long half time, close with human antibody (about 10～20 days).
The present invention will be further described below in conjunction with the drawings and specific embodiments, the enforcement that does not limit the present invention in any way.Every any this area of carrying out according to content disclosed by the invention be equal to replacement, all belong to protection scope of the present invention.
Description of drawings
Fig. 1 is urokinase type plasminogen activator (u-PA) primary structure synoptic diagram.
Fig. 2 is the ATF-Fc structural representation.
Fig. 3 cuts the evaluation electrophorogram for carrier for expression of eukaryon pcDNA3.1/ATF-Fc Nhe I/Xho I enzyme.
Fig. 4-A is the aspect graph of ATF-Fc-H6 for the rCHO engineering cell that obtains.
Fig. 4-B is that 32 monoclonal cells that screen are measured the ELISA photo that it expresses the ATF-Fc level.
Fig. 5 detects the ATF-Fc albumen of purifying for SDS-PAGE.
Fig. 6-A kills and wounds the breast cancer cell line MCF-7 of normal growth in the experiment of breast cancer cell line MCF-7 for the ATF-Fc fusion rotein.
Fig. 6-B kills and wounds in the experiment of breast cancer cell line MCF-7 for the ATF-Fc fusion rotein: the form of breast cancer cell line MCF-7 when ATF-Fc concentration is 1.25 μ g/ml in the substratum, visible all cells is the atrophy apoptosis.
Fig. 6-C kills and wounds in the experiment of breast cancer cell line MCF-7 for the ATF-Fc fusion rotein: the form of breast cancer cell line MCF-7 when ATF-Fc concentration is 2.5 μ g/ml in the substratum, visible all cells is the atrophy apoptosis.
Fig. 6-D kills and wounds for the ATF-Fc fusion rotein in the experiment of breast cancer cell line MCF-7 in the substratum: the form of breast cancer cell line MCF-7 when ATF-Fc concentration is 5 μ g/ml, visible all cells is the atrophy apoptosis.
Fig. 7 is the inhibition test result of ATF-Fc fusion rotein to several tumor cell migration.
Fig. 8-A suppresses stomach cancer cell BGC-823 migration test for the ATF-Fc fusion rotein: show the control group test-results that does not add the ATF-Fc fusion rotein, newborn tumor cell migration is arranged to the growth of cut place.
Fig. 8-B suppresses stomach cancer cell BGC-823 migration test for the ATF-Fc fusion rotein: show the test group that adds the ATF-Fc fusion rotein, do not have newborn tumour cell, show and can suppress tumor cell migration.
Fig. 8-C suppresses stomach cancer cell BGC-823 migration test for the ATF-Fc fusion rotein: show the test group that adds Pro-UK, a large amount of newborn tumor cell migration are arranged to the growth of cut place, the migration of prompting Pro-UK inducing tumor cell.
Fig. 8-D suppresses stomach cancer cell BGC-823 migration test for the ATF-Fc fusion rotein: show the test group that adds ATF-Fc fusion rotein and Pro-UK, do not have newborn tumor cell migration to the growth of cut place, prompting ATF-Fc fusion rotein has suppressed the migration of Pro-UK inducing tumor cell.
Fig. 9 is the ATF-Fc fusion rotein suppresses tumor growth to nude inoculation stomach cancer cell BGC-823 a exercising result.
Embodiment
Test materials and source:
The competence bacillus coli DH 5 alpha is a Time Inc. available from the sky, CHO-K1 cell strain (ATCC No.CRL 9096), dhfr gene, this research department of pro-UK gene preserve (Chinese patent CN96119836.2), plasmid pUC19 (TakaraBiotechnology Co., production number No.D3219), pcDNA3.1 carrier for expression of eukaryon (Invitrogen company) is derived from the gene (U.S. R﹠amp of the coding human normal immunoglobulin IgG1 Fc section (hinge area, CH2 district and CH3 district) of genome (containing intron); D Systems company, carrier is by name: pIG1, production code member is: pIg vectors, MBV-002-10).
Restriction enzyme Nhe I, BamH I, Hind III, Xho I and Not I etc. are available from NEB company, and T4 dna ligase and high-fidelity Taq enzyme Pyrobest are available from precious biotechnology company limited (TaKaRa).Plasmid extracts, the PCR product reclaims and enzyme is cut product recovery test kit all available from Promega company.DNA Marker is a Time Inc. available from the sky.
DMEM substratum, DMEM/F12 substratum and reinforced calf serum are available from Hyclone company; The serum free medium additive is this chamber preparation (Chinese patent ZL 200310124257.X); G418 is available from Sigma company; Lipofectamine
TM2000 available from Invitrogen company; Enzyme connection detector is Bio-Rad550; NProtein A Sepharose4 Fast Flow separating medium is available from Amersham Bioscience.
The primer of all oligonucleotide is synthetic finishes (China Invitrogen company) by Chinese Ying Jun biotech company.
The clone of embodiment 1.ATF gene and pcDNA3.1/ATF-Fc construction of eukaryotic expression vector
With the pIRES-dhfr/HMW-ProUK carrier is that template (preserve at China Committee for Culture Collection of Microorganisms common micro-organisms center by this expression vector, preservation day is on February 24th, 2006, preserving number is CGMCC No.1623, the microbial strain of ginseng certificate is pIRES-dhfr/HMW-ProUK, the classification called after colon bacillus Escherichia coli of suggestion, the Chinese invention patent application number is 200610065813.4), with primer ATFs and ATFx is the upstream and downstream primer, pcr amplification ATF gene reclaims the ATF gene.
ATFs：5’-TGCGCTAGCCCACCATGAGAGCCCTGCTGGCGCG-3’
ATFx：5’-CGCGGATCCACTTACCTGTACTGCCAGATCCGCTTCCATCTGCGCAGTCATGCAC-3’
The PCR reaction system is: 8 μ LdNTP (2.5mmol/L), 0.5 μ L Pyrobest polysaccharase, 2 μ LpIRES-dhfr/HMW-ProUK carriers, 10 μ L, 10 * Buffer, 2 μ L ATFs upstream primers (10 μ mol/L), 2 μ L ATFx downstream primers (10 μ mol/L), 75.5 μ L water, the total reaction system is 100 μ L, PCR reaction conditions: 94 ℃, 2min (pre-sex change) → [94 ℃, 30s → 60 ℃, 60s → 72 ℃, 1min] * → 72 ℃ of 25 circulations, 5min → 4 ℃, 5min.Reclaim PCR product (Promega, Agarose LMP) with 0.8%-1% low melting-point agarose glue.
The PCR product reclaims ATF gene Nhe I and the BamH I double digestion that test kit reclaims, use Nhe I and BamHI double digestion carrier pcDNA3.1/ATR-Fc (to preserve at China Committee for Culture Collection of Microorganisms common micro-organisms center simultaneously, register on the books and be numbered: CGMCC No.1399, the Chinese invention patent application number is 200510084233.5), reclaim expression vector pcDNA3.1/Fc, two segment DNAs that reclaim are connected with the T4 ligase enzyme, transformed into escherichia coli DH5 α, select positive colony through shake-flask culture, carry out enzyme with primer behind the extraction plasmid and cut evaluation and two-way sequencing, the swimming result shows ATF-Fc gene fragment size about 2000bp (Fig. 3), sequencing result proves, ATF-Fc recombination sequence is in full accord with the design gene, this gene contains NheI and BamHI restriction enzyme site, the Kozak sequence, the ATF gene obtains expressing the carrier for expression of eukaryon pcDNA3.1/ATF-Fc of ATF-Fc fusion rotein.
The Lipofectamine that transfection adopts Invitrogen company to produce
TM2000 cationic-liposome transfection reagent boxes are operated according to the test kit specification sheets.The CHO-K1 cell cultures of transfection is in 5%CO2,37 ℃ of incubators.Behind the transfection 12h, change the DMEM/F12 substratum (Hyclone) that contains 10% reinforced calf serum (Hyclone).Behind the 48h, adopt 750 μ g/mL G418 (Sigma) screening culture medium pressurization screening, every 2-3d changes liquid once, pressurize behind about 12d, with resistant cell clone with 0.25% trysinization, in 96 hole micropore Tissue Culture Plates (NUNC company), carry out the mono-clonal cultivation with limiting dilution assay, promptly with the DMEM/F12 substratum that contains 10% calf serum and 300 μ g/mL G418 with cell dilution to 10/mL, inoculation 200 μ L substratum (on average each Kong Zhonghan have an appointment 2 cells) cell clone occurs after 2 weeks in each micropore.Change serum free medium and continue to cultivate 3d, collect culture supernatant and select positive colony with direct competitive ELISA, being about to serum-free culture supernatant direct coated NUNCTM elisa plate spends the night for 4 ℃, 3%BSA sealing back adds the HRP anti-human IgG of mark goat (H+L), hatch the back and add the TMB reagent colour development, and 405nm survey OD value (Fig. 4-B).With the negative contrast of serum free medium, the probability that the higher cell clone of expression level occurs is about 10%-20% (Fig. 4 (B)).
Select the highest gene recombination CHO engineering cell of expression level be ATF-Fc-H6 (Fig. 4-A) in the 1000mL rolling bottle with the DMEM/F12 culture medium culturing that contains 2% calf serum, culture temperature is 37 ℃, when treating that cell covers with bottle wall (about 3 days), be changed to serum free medium (Chinese patent ZL200310124257.X), receive liquid every other day, (cultural method please refer to: Gao Lihua can to receive liquid continuously 3-4 time, Hu Xianwen, Chen Wei etc. anthrax toxin acceptor and human IgG1's the expression of Fc section fusion rotein in Chinese hamster ovary celI. the biotechnology journal, 2005,21 (5): 826).
The purifying of embodiment 3. expressing proteins and SDS-PAGE analyze
The ATF-Fc-H6 serum-free culture supernatant of collecting albumin A affinity column (Pharmacia) separation and purification.Utilize the specific adsorption effect of albumin A, adopt nProtein A Sepharose 4 Fast Flow separating mediums (Amersham Biosciences) to carry out affinity chromatography, purifying expressing protein IgG.Working method is referring to the description of product.Bonded albumen has a tangible elution peak (Fig. 4 (A)) with pH2.5,0.1mol/L glycine-HCl buffer solution elution, and the wash-out composition transfers to 7 with the Tris-HCl damping fluid of 2mol/L with elutriant pH.Behind the purifying, measure A with ultraviolet spectrophotometer
260And A
280Absorbancy is calculated protein concentration, and the protein quantification formula is: protein content (mg/mL)=1.45 * A
260-0.74 * A
280Measure proteic purity, molecular weight with SDS-PAGE.
If the ATF-Fc fusion rotein can correctly be expressed in Chinese hamster ovary celI, it should be the homodimer antibody molecule that expressed proteins is estimated, promptly the ATF-Fc strand connects into dimer in the Hinge district by interchain disulfide bond.Strand ATF-Fc molecule has 391 amino-acid residues.Because the Fc section has glycosylation site, so the molecular weight of strand ATF-Fc is expected at 45-50kD.The ATR-Fc fusion rotein that affinitive layer purification obtains carries out SDS-PAGE respectively and analyzes coomassie brilliant blue staining under reduction and non-reduced condition.Under non-reduced condition, a protein band is arranged at the 97kD place, and under reductive condition, a tangible protein band is arranged at the 45kD place, and do not have other protein band (Fig. 5).Because all disulfide linkage comprise that interchain disulfide bond is all interrupted under the reductive condition, the molecular weight that records is a strand ATF-Fc molecule, but not different peptide chains still connect by disulfide linkage under the reductive condition, at the molecular weight under the non-reduced condition just in time is the twice of molecular weight under the reductive condition, show that the ATF-Fc fusion rotein that we obtain is the homodimer antibody molecule, conform to expected result.
Whether have the effect of killing tumor cell in order to detect expressed ATF-Fc fusion rotein, done the tumor cytotoxicity experiment.Method: in 24 porocyte culture plates (Costar company product), cultivate breast cancer cell MCF-7 (ATCC with the DMEM substratum (Hyclone) that contains 10% foetal calf serum (Hyclone company product), Cat.No.HTB-22), 37 ℃ of cultivations, when treating cell length to fusion rate 80%, add ATF-Fc albumen (final concentration is respectively 1.25,2.5 and 5 μ g/ml), continuation after 24 hours, is observed the fragmentation effect of ATF-Fc pair cell 37 ℃ of cultivations.The result: find that almost 100% breast cancer cell is killed, and the normal (Fig. 6-A to Fig. 6-D), prove that the ATF-Fc fusion rotein that we make up has the very strong effect that kills and wounds breast cancer cell MCF-7 of the breast cancer cell of control group growth.Therefore the present invention can be used for the exploitation of the antibody class medicine of human breast carcinoma treatment.
Embodiment 5. biologic activity two: the ATF-Fc fusion rotein is to the retarding effect of several tumor cell migration
Because it is very important that uPAR plays a part in the migration of tumour cell, sealing uPAR may suppress the migration of tumour cell.ATF-Fc has the effect of sealing uPAR, has observed the influence of ATF-Fc to tumor cell migration.
Method: in 24 porocyte culture plates (Costar company product), cultivate breast cancer cell line MCF-7 respectively with the DMEM substratum (Hyclone) that contains 10% foetal calf serum (Hyclone company product), stomach cancer cell is that BGC-823 (can obtain by following laboratory, Istituto Nazionale per la Ricerca sul Cancro c/o CBA (ICLC, Genova), clone is numbered ICLC HTL98007, is EC109 cell strain (available from Shanghai cell institute of the Chinese Academy of Sciences) with esophageal cancer cell www.biotech.ist.unige.it/cldb/c14930.html).37 ℃ of cultivations, when treating cell length to fusion rate 80%, cut, add ATF-Fc albumen (final concentration is 5 μ g/ml), set up pro-UK (source: Chinese patent CN96119836.2) and lower molecular weight UK (originate: (final concentration is 1 μ g/ml) control group Chinese patent 200610065813.4), continuation after 24 hours, is observed the influence of ATF-Fc on cell migration 37 ℃ of cultivations.Result's (seeing Fig. 7 and Fig. 8): (1) is for breast cancer cell MCF-7, as long as contain the culture hole of ATF-Fc fusion rotein, cell wherein is all dead, can't observe the migration that ATF-Fc suppresses breast cancer cell MCF-7, and the control group of adding pro-UK and UK, all breast cancer cells are all survived, and density is very high, and there is the cell that newly grows at the cut place, and illustrates that pro-UK and UK all can stimulate growth and the migration of breast cancer cell MCF-7; (2) for stomach cancer cell, after cultivation in 24 hours, the culture hole that contains the ATF-Fc fusion rotein has the part cell to be killed, cell does not wherein all have the cell of new growth at the cut place, illustrate that ATF-Fc has very strong inhibition stomach cancer cell migration and certain lethal effect is arranged, it is more remarkable to cultivate after 48 hours the lethal effect performance, on the contrary, the culture hole that does not add ATF-Fc, especially the culture hole that has added pro-UK or UK, the growth of wherein cell is fine and close, and the cut director gone out many new cells, illustrates that pro-UK and UK can promote the migration of stomach cancer cell; (3) for esophageal cancer cell, no matter whether contain ATF-Fc, the growth of cell is not all suppressed, and ATF-Fc can slightly suppress the migration of nasopharyngeal carcinoma cell.This experiment shows that ATF-Fc has the effect of very strong inhibition stomach cancer cell migration, can be used for developing the biotech drug of treatment mammary cancer and cancer of the stomach.
Selection is done the experiment of lotus knurl to the stomach cancer cell BGC-823 of ATF-Fc medium sensitivity, the reason of selecting is: if ATF-Fc has very strong inhibition growth and the effect of shifting to stomach cancer cell in the mouse body, can infer that ATF-Fc should have better therapeutic for mammary cancer.Method: the BGC-823 cell in the vegetative period of taking the logarithm is mixed with 5 * 10 with fresh medium DMEM (Hyclone)
6The cell suspension of individual/mL, every mouse armpit subcutaneous vaccination 0.2ml, inoculation back mouse is divided into the physiological saline group at random, CTX (endoxan) organizes (100mg/kg), three dosage groups of ATF- Fc 100,40 and 8mg/kg, totally 5 groups, every group of 7 mouse.Treat that it is administration that tumour is grown to about 0.3cm * 0.3cm volume size, administering mode: the next day subcutaneous injection, totally 7 times.The administration volume is the 0.2ml/20g body weight, takes off neck execution animal in 2 days after the last administration, dissects and gets the knurl piece, claims knurl heavy, calculates the tumor growth tumour inhibiting rate according to following formula:
ATF-Fc when dosage 100,40,8mg/kg, the next day subcutaneous injection 7 times, the tumour inhibiting rate of nude mice BGC-823 cancer of the stomach is respectively 73.59,60.92,53.87%, the results are shown in Table 1 and Fig. 9.This result shows that ATF-Fc has the effect that suppresses the cancer of the stomach growth in vivo.
Table 1:ATF-Fc continuous subcutaneous injection is to nude mice BGC-823 efficacy in treating gastric carcinoma
The invention provides a kind of treat late malignant tumour, safe and effective anti-uPAR human antibody class ATF-Fc fusion rotein.
Sequence table
＜110〉Academy of Military Medicine, PLA
＜120〉antibody molecule ATF-Fc fusion rotein of a kind of antiurokinase type fibrinolysin activator receptor and uses thereof
<130>
<160>2
<170>PatentIn?version?3.3
<210>1
<211>391
<212>PRT
＜213〉Genus Homo, ethnic group
<220>
＜221〉signal peptide of uPA
<222>(1)..(20)
<400>1
Met?Arg?Ala?Leu?Leu?Ala?Arg?Leu?Leu?Leu?Cys?Val?Leu?Val?Val?Ser
1 5 10 15
Asp?Ser?Lys?Gly?Ser?Asn?Glu?Leu?His?Gln?Val?Pro?Ser?Asn?Cys?Asp
20 25 30
Cys?Leu?Asn?Gly?Gly?Thr?Cys?Val?Ser?Asn?Lys?Tyr?Phe?Ser?Asn?Ile
35 40 45
His?Trp?Cys?Lys?Cys?Pro?Lys?Lys?Phe?Gly?Gly?Gln?His?Cys?Glu?Ile
50 55 60
Asp?Lys?Ser?Lys?Thr?Cys?Tyr?Glu?Gly?Asn?Gly?His?Phe?Tyr?Arg?Gly
65 70 75 80
Lys?Ala?Ser?Thr?Asp?Thr?Met?Gly?Arg?Pro?Cys?Leu?Pro?Trp?Asn?Ser
85 90 95
Ala?Thr?Val?Leu?Gln?Gln?Thr?Tyr?His?Ala?His?Arg?Ser?Asp?Ala?Leu
100 105 110
Gln?Leu?Gly?Leu?Gly?Lys?His?Asn?Tyr?Cys?Arg?Asn?Pro?Asp?Asn?Arg
115 120 125
Arg?Arg?Pro?Trp?Cys?Tyr?Val?Gln?Val?Gly?Leu?Lys?Pro?Leu?Val?Gln
130 135 140
Glu?Cys?Met?Val?His?Asp?Cys?Ala?Asp?Gly?Ser?Gly?Ser?Gly?Ser?Glu
145 150 155 160
Pro?Lys?Ser?Cys?Asp?Lys?Thr?His?Thr?Cys?Pro?Pro?Cys?Pro?Ala?Pro
165 170 175
Glu?Leu?Leu?Gly?Gly?Pro?Ser?Val?Phe?Leu?Phe?Pro?Pro?Lys?Pro?Lys
180 185 190
Asp?Thr?Leu?Met?Ile?Ser?Arg?Thr?Pro?Glu?Val?Thr?Cys?Val?Val?Val
195 200 205
Asp?Val?Ser?His?Glu?Asp?Pro?Glu?Val?Lys?Phe?Ash?Trp?Tyr?Val?Asp
210 215 220
Gly?Val?Glu?Val?His?Asn?Ala?Lys?Thr?Lys?Pro?Arg?Glu?Glu?Gln?Tyr
225 230 235 240
Asn?Ser?Thr?Tyr?Arg?Val?Val?Ser?Val?Leu?Thr?Val?Leu?His?Gln?Asp
245 250 255
Trp?Leu?Asn?Gly?Lys?Glu?Tyr?Lys?Cys?Lys?Val?Ser?Asn?Lys?Ala?Leu
260 265 270
Pro?Ala?Pro?Ile?Glu?Lys?Thr?Ile?Ser?Lys?Ala?Lys?Gly?Gln?Pro?Arg
275 280 285
Glu?Pro?Gln?Val?Tyr?Thr?Leu?Pro?Pro?Ser?Arg?Asp?Glu?Leu?Thr?Lys
290 295 300
Asn?Gln?Val?Ser?Leu?Thr?Cys?Leu?Val?Lys?Gly?Phe?Tyr?Pro?Ser?Asp
305 310 315 320
Ile?Ala?Val?Glu?Trp?Glu?Ser?Asn?Gly?Gln?Pro?Glu?Asn?Asn?Tyr?Lys
325 330 335
Thr?Thr?Pro?Pro?Val?Leu?Asp?Ser?Asp?Gly?Ser?Phe?Phe?Leu?Tyr?Ser
340 345 350
Lys?Leu?Thr?Val?Asp?Lys?Ser?Arg?Trp?Gln?Gln?Gly?Asn?Val?Phe?Ser
355 360 365
Cys?Ser?Val?Met?His?Glu?Ala?Leu?His?Asn?His?Tyr?Thr?Gln?Lys?Ser
370 375 380
Leu?Ser?Leu?Ser?Pro?Gly?Lys
385 390
<210>2
<211>1176
<212>DNA
＜213〉Genus Homo, ethnic group
<400>2
atgagagccc?tgctggcgcg?cctgcttctc?tgcgtcctgg?tcgtgagcga?ctccaaaggc 60
agcaatgaac?ttcatcaagt?tccatcgaac?tgtgactgtc?taaatggagg?aacatgtgtg 120
tccaacaagt?acttctccaa?cattcactgg?tgcaagtgcc?caaagaaatt?cggagggcag 180
cactgtgaaa?tagataagtc?aaaaacctgc?tatgagggga?atggtcactt?ttaccgagga 240
aaggccagca?ctgacaccat?gggccggccc?tgcctgccct?ggaactctgc?cactgtcctt 300
cagcaaacgt?accatgccca?cagatctgat?gctcttcagc?tgggcctggg?gaaacataat 360
tactgcagga?acccagacaa?ccggaggcga?ccctggtgct?atgtgcaggt?gggcctaaag 420
ccgcttgtcc?aagagtgcat?ggtgcatgac?tgcgcagatg?gaagcggatc?tggcagtgag 480
cccaaatctt?gtgacaaaac?tcacacatgc?ccaccgtgcc?cagcacctga?actcctgggg 540
ggaccgtcag?tcttcctctt?ccccccaaaa?cccaaggaca?ccctcatgat?ctcccggacc 600
cctgaggtca?catgcgtggt?ggtggacgtg?agccacgaag?accctgaggt?caagttcaac 660
tggtacgtgg?acggcgtgga?ggtgcataat?gccaagacaa?agccgcggga?ggagcagtac 720
aacagcacgt?accgtgtggt?cagcgtcctc?accgtcctgc?accaggactg?gctgaatggc 780
aaggagtaca?agtgcaaggt?ctccaacaaa?gccctcccag?cccccatcga?gaaaaccatc 840
tccaaagcca?aaggtcagcc?ccgagaacca?caggtgtaca?ccctgccccc?atcccgggat 900
gagctgacca?agaaccaggt?cagcctgacc?tgcctggtca?aaggcttcta?tcccagcgac 960
atcgccgtgg?agtgggagag?caatgggcag?ccggagaaca?actacaagac?cacgcctccc 1020
gtgctggact?ccgacggctc?cttcttcctc?tacagcaagc?tcaccgtgga?caagagcagg 1080
tggcagcagg?ggaacgtctt?ctcatgctcc?gtgatgcatg?aggctctgca?caaccactac 1140
acgcagaaga?gcctctccct?gtctccgggt?aaatga 1176
Claims (8)
1. ATF-Fc fusion rotein, it is characterized in that: it be Fc section, CH2 and the CH3 of the ATF district of uPA and human normal immunoglobulin IgG1 by the dimer fusion protein that the Ser-Gly-Ser-Gly-Ser joint is connected to form, have 742 amino acid; The aminoacid sequence of strand ATF-Fc molecule shown in sequence table SEQ ID No.1, wherein preceding 20 signal peptides that amino acid is uPA.
2. coding gene of the fusion rotein strand of the ATF-Fc of aminoacid sequence shown in sequence table SEQ ID No.1.
3. the gene of coding according to claim 2 fusion rotein strand of the ATF-Fc of aminoacid sequence shown in sequence table SEQ ID No.1 is characterized in that: the nucleotide sequence shown in sequence table SEQ ID No.2.
4. the expression vector that contains nucleotide sequence shown in the SEQ ID No.2.
5. expression vector according to claim 4 is characterized in that: described expression vector is kept in the colon bacillus, and preserving number is CGMCC No.1774.
6. the cell that contains claim 4 or 5 described expression vectors.
7. the described ATF-Fc fusion rotein of claim 1 is used to prepare the purposes of the medicine for the treatment of tumour, it is characterized in that: described tumour is mammary cancer and cancer of the stomach.
8. claim 4 or 5 described expression vectors are used for the purposes of the medicine of transfection CHO cell preparation treatment tumour, and described tumour is mammary cancer and cancer of the stomach.
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| CN101337992B (en) * | 2007-07-04 | 2012-12-19 | 吉林圣元科技有限责任公司 | Fusion protein of urokinase type plasminogen activator a chain and melittin and preparation 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 |
| CN101798349B (en) * | 2009-12-09 | 2012-07-04 | 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 coding gene thereof |
| CN102721817B (en) * | 2012-06-27 | 2014-08-13 | 中国医学科学院输血研究所 | Method for detecting biological activity of Fc segment of human immunoglobulin by high throughput method |
| CN109925509B (en) * | 2019-03-19 | 2021-08-10 | 大连医科大学 | Medicine for treating choriocarcinoma |
| CN114917329B (en) * | 2021-02-11 | 2023-07-21 | 兰州大学第二医院 | Combination of anti-CD 87 antibody and anti-PD 1 antibody for treating gastric cancer |
| EP4490203A1 (en) * | 2022-03-09 | 2025-01-15 | Immunofyx | Anti-cancer n-terminal fc conjugated immunotherapeutics |
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