CN1478541A - Design of high effect blood vessel production inhibitor and its application - Google Patents
Design of high effect blood vessel production inhibitor and its application Download PDFInfo
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- CN1478541A CN1478541A CNA021381887A CN02138188A CN1478541A CN 1478541 A CN1478541 A CN 1478541A CN A021381887 A CNA021381887 A CN A021381887A CN 02138188 A CN02138188 A CN 02138188A CN 1478541 A CN1478541 A CN 1478541A
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Abstract
An efficient angiogenesis depressant with the affinity and binding power to integrin, its structure form, its preparing process, and its application in suppressing endothelial cell growth and angiogenesis are disclosed.
Description
The invention belongs to the biotech medicine product technical field.
Angiogenesis is meant the blood vessel of deriving, growing and make new advances from existing blood vessel.Angiogenesis, as malignant tumor, rheumatoid arthritis, retina and choroidal artery new life and plays an important role in many dermatosis at many human important diseases.The angiogenesis inhibitor treatment, promptly angiogenesis suppresses therapy, is (Folkman, J.N.Engl.J.Med., 285,1182-1186,1971 that taken the lead in proposing by Judah doctor Folkman before 30 years; Folkman, J.Ann.Surg., 175,409-416,1972).He proposes: can reach the purpose of eliminating tumor by cutting off the blood supply of tumor.In the process of angiogenesis, there are many regulatory factors playing a role, as somatomedin, extracellular matrix molecule, the bonded protein of cell membrane, growth factor receptors etc.Anti-angiogenic medicaments one angiogenesis inhibitor that is at present carrying out clinical experiment also can be divided into because of it acts on target: endotheliocyte inhibitor, matrix metallo-proteinase inhibitor, angiogenesis growth factor antagonist etc.Wherein famous is the angiogenesis inhibitor that Judah doctor Folkman laboratory is found: (Cell 79 for O ' Reilly, M.S. etc. for angiostatin, 315-328,1994), endostatin (O ' Reilly, M.S. etc., Cell88,277-285,1997), human plasminogen kringle 5 (Cao, Y.H. etc., J.Biol.Chem.272,22924-22928,1997), and the vasostatin of other laboratory discovery, restin, arresten, canstatin, tumstain, prolactin N end 16kD fragment, troponin I, pigment epithelium derived factor, platelet factor 4 etc.Although these angiogenesis inhibitors present very tempting application prospect, but its defective is also very obvious: when mouse model is tested, the using dosage of angiostatin is up to milligram/kg body weight up to a hundred, the dosage of endostatin also is tens milligrams/kg body weight, the using dosage of kringle 5 is also at tens milligrams--milligram/kg body weight up to a hundred, according to estimates when these angiogenesis inhibitors when human body uses, using dosage will reach a few at least gram/people's dosage.Big like this drug use dosage will certainly increase the probability of such poisonous side effect of medicine generation from now on, causes such drug quality control difficulty increasing, production scale and production cost increase and drug price to occupy high.
The transmembrane protein heterodimer that integrin (integrin) is made up of two subunits of α, β, they pass through to control with the interaction of extracellular matrix molecule migration, differentiation and the propagation of cell.Most of molecules in more than 20 kind of integrin can both discern contain RGD (arginine-glycine-aspartic acid, Arg-Gly-Asp) or NGR (agedoite-glycine-arginine, the extracellular matrix aglucon of sequence such as Asn-Gly-Arg).Some integrins can play regulating action to angiogenesis, also are found the effect with angiogenesis inhibiting at the antibody of some integrin.
Angiogenesis is a complex physical process, just has multiple molecular marker in neonatal blood vessels, but not single labelling makes them be different from the blood vessel that has generated.Therefore the angiogenesis inhibitor of a good angiogenesis inhibitor should have the selectivity to the multiple labelled molecule of new vessels, so just can reach the effect that new vessels is had guidance quality, selectively acting; A plurality of labelled molecule of new vessels or target spot are had an effect simultaneously, will improve the inhibitory action of medicine on the whole angiogenesis; And to the many target spots of new vessels and guidance quality, optionally exercising result just can cause just bringing into play the effect of angiogenesis inhibitor efficiently as long as use than low-dose drugs.Anti-angiogenic medicaments up to now, such as above-mentioned angiostatin, endostatin, kringle 5 etc. all only at the molecule of the single target spot effect of new vessels, the inhibition that they are still disliked inadequately, new vessels is generated the specificity and the selectivity of blood vessel also just from a target spot carry out individual combat, effect is limited, so the result causes needs high using dosage like this.
The objective of the invention is to develop a kind of new high effect blood vessel production inhibitor, promptly make up a kind of angiogenesis inhibitor that integrin (integrn) is had combination, make existing angiogenesis inhibitor have affinity or binding ability, give the affinity of existing angiogenesis inhibitor with integrin to integrin.This kind high effect blood vessel production inhibitor can significantly improve activity and the efficient that existing angiogenesis inhibitor suppresses endothelial cell growth and angiogenesis, improve specificity and the selectivity of angiogenesis inhibitor, reduce the using dosage of existing angiogenesis inhibitor endotheliocyte and new vessels.
Purpose of the present invention can reach by following measure:
Structure contains the angiogenesis inhibitor of RGD (arginine-glycine-aspartic acid) or NGR integrin binding sequence peptide sections such as (agedoite-glycine-arginine) (as angiostatin, endostatin, kringle 5 etc.) gene-be the high effect blood vessel production inhibitor gene, can with this high effect blood vessel production inhibitor gene clone in gene therapy vector, be directly used in gene therapy; Can utilize recombinant DNA technology to carry out the high effect blood vessel production inhibitor that gene engineering expression contains integrin binding sequence peptide sections such as RGD or NGR, separation and purification recombination high efficiency angiogenesis inhibitor protein; Or with the synthetic peptide section that contains integrin binding sequences such as RGD or NGR of polypeptide synthesis method, the peptide section that will contain sequences such as RGD or NGR with the method for chemical coupling is connected on angiogenesis inhibitor natural origin or the recombinant DNA source then, formation can have the angiogenesis inhibitor of combination, high effect blood vessel production inhibitor promptly of the present invention with integrin.Have combination with integrin, contain N end or C end that the peptide section of sequences such as RGD or NGR can merge at angiogenesis inhibitor, or the N that merges simultaneously at angiogenesis inhibitor end and C hold:
A peptide, B peptide are respectively that length is polypeptide fragment 3 to 90 amino acid residues, that contain integrin binding sequences such as RGD or NGR, and A peptide, B peptide have respectively and the bonded ability of integrin.High effect blood vessel production inhibitor of the present invention can significantly improve the biological activity of existing angiogenesis inhibitor inhibition of endothelial cell proliferation and angiogenesis, can be as the medicine of treatment disease, more effectively treat human or animal's the disease relevant with angiogenesis, as malignant tumor, rheumatoid arthritis, retina and choroidal artery new life, and relevant dermatosis etc., the perhaps medicine of effect diagnosis usefulness positions and diagnoses human or animal's the disease relevant with angiogenesis.Synthetic and the chemical coupling method tradition of the genetic engineering production of high effect blood vessel production inhibitor of the present invention or polypeptide, routine, simple, easily to go, the output height has important directive significance and practical value to the development and production high effect blood vessel production inhibitor.
Embodiment 1:
1. composite coding contains the PCR primer of the polypeptide fragment of integrin binding sequences such as RGD or NGR, press angiogenesis inhibitor gene 5 ' or 3 ' gene order design PCR primer, obtain to contain the angiogenesis inhibitor gene of integrin binding sequence peptide sections such as RGD or NGR by PCR reaction amplification.As synthetic primer 1:5 ' CGGGATCGATGACGACGACAAGGCATGCGACTGCCGTG 3 ', primer 2: 5 ' GACTGCCGTGGTGACTGCTTCTGCGGTGTCCTGCTTCCAGATGTAGAGAC 3 ', primer 3:5 ' GCGAATTCTAGGCTGCACACTGAGGGAC 3 '.Wherein, primer 1 encoded BamHI site and enterokinase recognition site sequence, in the primer 2: the peptide section that GACTGCCGTGGTGACTGCTTCTGCGGT has encoded and contained the RGD sequence, GTCCTGCTTCCAGATGTAGAGAC coding human plasminogen 449-456 amino acids, the i.e. N of handle domain 5 (kringle 5) end; Primer 3 coding human plasminogen 538-543 amino acids.
With the human plasminogen gene cDNA is template, with primer 2 and 3 is primer, carry out pcr amplification reaction, be primer with primer 1 and 3 again, with primary PCR product is template, carry out pcr amplification reaction, obtain to contain handle domain 5 genes of RGD sequence, i.e. high effect blood vessel production inhibitor of the present invention molecule--high effect blood vessel production inhibitor molecule 1: RGD-Kringle 5 (the human plasminogen handle domain 5 that contains arginine-glycine-aspartic acid sequence peptide section).The aminoacid sequence of this high effect blood vessel production inhibitor molecule 1: RGD-Kringle 5 is: ACDCRGDCFCGVLLPDVETPSEEDCMFGNGKGYRGKRATTVTGTPCQDWAAQEPHR HSIFTPETNPRAGLEKNYCRNPDGDVGGPWCYTTNPRKLYDYCDVPQCAA
2. for the ease of the relatively designed high effect blood vessel production inhibitor molecule 1 of the present invention--actual effect and the superior part of RGD-Kringle5, we have cloned, have expressed the handle domain 5 that does not contain the RGD sequence simultaneously in this example, with in contrast and relatively.
The primer of difference composite coding human plasminogen handle domain 5N end and C end, primer 4:5 ' CGGGATCGATGACGACGACAAGGTCCTGCTTCCAGATGTAGAGAC3 ', primer 4 coding human plasminogen 449-456 amino acids, be the N end of handle domain 5, its 5 ' end has enterokinase recognition site sequential coding sequence.With the human plasminogen gene cDNA is template, is primer with primer 4 and 3, carries out pcr amplification reaction, obtains handle domain 5 genes.
3. will be from 1,2. the gene of acquisition is cleared up through restricted enzyme respectively, be cloned in the range gene engineering expression vector, as the PCR product that will from 1., obtain through BamHI, EcoRI enzyme action, be cloned among the escherichia coli GST fusion expression vector pALEX, at first carry out dna sequence analysis, the correctness of checking PCR product D NA sequence, is carried out recombinant expressed bacterium SDS-PAGE and is analyzed expression after 2 hours through 0.5mM IPTG abduction delivering respectively then.
4. the expression bacterium in 3 is carried out ultrasonication respectively, respectively ultrasonic supernatant is carried out the Glutathione-Agarose affinity chromatograph, collect eluting peak, a step affinitive layer purification can obtain purer gst fusion protein.For the GST-kringle 5 and GST-RGD-kringle 5 fusion rotein that obtain, with enterokinase fusion rotein is cut that (enzyme: substrate=1: 1000～4000), two kinds of fusion rotein almost all can be cut after 10 hours through 37 ℃ of enzyme action fully.
GST-Kringle 5 and GST-RGD-Kringle 5 fusion rotein enzyme action products carry out the Glutathione-Agarose affinity chromatograph respectively once more, collect and pass the peak.One of Kringle 5 that obtains and high effect blood vessel production inhibitor example RGD-Kringle 5 is through SDS-PAGE electrophoresis and C18 reversed-phase HPLC checking purity.
5. in order to compare the bioactive difference of one of Kringle 5 and high effect blood vessel production inhibitor example RGD-Kringle 5, we have carried out endothelial cell proliferation and have suppressed determination of activity.In the DMEM culture medium of the people's recombinant bfgf (deriving from Bio-engineering Institute of Jinan University) that adds 10% calf serum, antibiotic and 3ng/ml, cultivate ECV304 cell (human endothelial cell strain).Experiment is carried out in 24 porocyte culture plates, and the concentration range of sample is 20nM～1280nM, and each concentration is done 3 repetitions, is blank with the phosphate buffer, and 37 ℃, 5%CO
2Cultivated 48 hours, and used trypsin digestion cell, the blood counting chamber counting, living cells number in comparative sample and the blank well calculates the suppression ratio of 5 pairs of ECV304 cell growths of one of variable concentrations Kringle 5 and high effect blood vessel production inhibitor example RGD-Kringle with this.
The result shows: Kringle 5 is when 320nM in reorganization, and the suppression ratio that the ECV304 cell is grown is 31%, and during 1280nM, suppression ratio is 55%; And as the RGD-Kringle 5 of one of designed high effect blood vessel production inhibitor example of the present invention when the 320nM, be 58% to the suppression ratio of ECV304 cell growth, during 1280nM, suppression ratio is 100%.
From example 1, we can see in vitro endothelial cell growth suppresses to test, the inhibition activity of 5 pairs of human endothelial cell strains of RGD-Kringle ECV304 cell growth of one of high effect blood vessel production inhibitor example that the present invention is designed is 4 times of Kringle 5 at least, and its ED50 only is 1/4th of Kringle 5 ED50.The designed high effect blood vessel production inhibitor of the present invention has significantly improved the biological activity of existing angiogenesis inhibitor Kringle 5 really.
Embodiment 2:
Angiostatin is first successful Angiostatin that doctor Folkman of Harvard University laboratory is found.Angiostatin contains four kringle molecules, kringle 1-4, wherein kringle's 1 is maximum to the active contribution of angiostatin vascular endothelial cell inhibition, and independent kringle 1 vascular endothelial cell inhibition activity accounts for the angiostatin vascular endothelial cell and suppresses active more than 50%.In order further to verify the actual effect and the superior part of the high effect blood vessel production inhibitor that the present invention is designed, we have cloned, have expressed the handle structure ring handle structure territory 1 (kringle 1) of containing the RGD sequence simultaneously in this example 2, and the biological activity to their angiogenesis inhibitor in the experimental model animal body compares.
1. composite coding contains the segmental PCR primers of polypeptide of sequence such as RGD or NGR, press angiogenesis inhibitor gene 5 ' or 3 ' gene order design PCR primer, obtain to contain the angiogenesis inhibitor gene of integrin binding sequence peptide sections such as RGD or NGR by PCR reaction amplification.Synthetic primer 1:5 ' CGGGATCCATGCCATGGCATGCGACTGCCGTG 3 '; Primer 2: 5 ' GACTGCCGTGGTGACTGCTTCTGCGGTGTGTATCTCTCAGAGTGCAAGACTGGG 3 '; Primer 3:5 ' GCGAATTCTAAGCACACTCAAGAATGTCGCAGTAGTC 3 '; Primer 4:5 ' CGGGATCCATGCCATGGTGTATCTCTCAGAGTGCAAG 3 '.Wherein, encoded BamH I and Nco I restriction enzyme enzyme recognition site and start codon ATG of primer 1; In the primer 2: the peptide section that GACTGCCGTGGTGACTGCTTCTGCGGT has encoded and contained the RGD sequence, GTGTATCTCTCAGAGTGCAAGACTGGG coding human plasminogen 80-88 amino acids, i.e. 8 aminoacid of the N of handle domain 1 (kringle 1) end; Primer 3 coding human plasminogen 157-164 amino acids, i.e. 9 aminoacid of the C of handle domain 1 (kringle 1) end, and termination codon, and an EcoRI restriction enzyme enzyme recognition site; 4 of primers contain BamH I and Nco I restriction enzyme enzyme recognition site, the sequence of start codon ATG and one section coding human plasminogen 80-88 amino acids.
With the human plasminogen gene cDNA is template, with primer 2 and 3 is primer, carry out pcr amplification reaction, be primer with primer 1 and 3 again, with primary PCR product is template, carry out pcr amplification reaction, obtain to contain handle domain 1 gene of RGD sequence, i.e. high effect blood vessel production inhibitor of the present invention molecule-high effect blood vessel production inhibitor molecule 2:RGD-Kringle 1 (the human plasminogen handle domain 1 that contains arginine-glycine-aspartic acid sequence peptide section).The aminoacid sequence of this high effect blood vessel production inhibitor molecule 2:RGD-Kringle 1 is: ACDCRGDCFCGVYLSECKTGNGKNYRGTMSKTKNGITCQKWSSTSPHRPRFSPATH PSEGLEENYCRNPDNDPQGPWCYTTDPEKRYDYCDILECA;
Being template with the human plasminogen gene cDNA simultaneously, is primer with primer 4 and 3, carries out pcr amplification reaction, obtains not contain handle domain 1 gene of RGD sequence.
2. will be from 1,2. the gene of acquisition is cleared up through restricted enzyme respectively, be cloned in the range gene engineering expression vector, as the PCR product that will from 1., obtain respectively through Nco I, EcoR I enzyme action, be cloned among the escherichia coli non-fusion expression carrier pNJUTRX-1, at first carry out dna sequence analysis, the correctness of checking PCR product D NA sequence, is carried out recombinant expressed bacterium SDS-PAGE and is analyzed expression after 2 hours through 0.5mM IPTG abduction delivering respectively then.
Carry out ultrasonication respectively to expressing bacterium, respectively ultrasonic supernatant is carried out Lysine Sepharose 4B affinity chromatograph, with buffer (0.5M NaCl, 0.5M arginine, 20mM Tris-HCl, pH 7.4) carry out eluting, collect eluting peak, after dialysing, a step affinitive layer purification receives purer Kringle 1 and RGD-Kringle 1 recombinant protein.Kringle 1 that obtains and two RGD-Kringle 1 of high effect blood vessel production inhibitor example are through SDS-PAGE electrophoresis and C18 reversed-phase HPLC checking purity, and carry out endothelial cell growth with cattle endothelial cell strain BCE cell and suppress activity experiment, determine recombinant products biological activity.
5. on the mice with tumor animal model of inoculation transitional cell carcinoma of bladder, behind the inoculation cancerous cell suspension the 5th day, carry out the experiment of Kringle 1 and RGD-Kringle 1 anti-tumor biological, the consumption of reorganization Kringle 1 and RGD-Kringle 1 is all the 20mg/kg body weight/day, lumbar injection, once a day, mice with lumbar injection 0.1ml normal saline group is contrast, behind the successive administration 15 days, measure the size and the weight of mouse tumor, calculate tumour inhibiting rate (tumor suppression efficient), tumour inhibiting rate=(1-administration group mice average tumor weight/normal saline control group mice average tumor weight) * 100%.The result shows: under the dosage of 20mg/kg body weight/day, the tumour inhibiting rate of Kringle 1 is 8%, and the tumour inhibiting rate of two RGD-Kringle 1 of high effect blood vessel production inhibitor example of the present invention is 68%, compares with Kringle 1, and the tumour inhibiting rate of RGD-Kringle 1 has improved 8 times.
From example 1 and 2, we can obviously see: the present invention has tangible advantage and innovation compared with prior art.Can both improve activity and the efficient that existing angiogenesis inhibitor suppresses endothelial cell growth and angiogenesis significantly in the certain experiment in vivo and in vitro of the affinity that the present invention is designed and the high effect blood vessel production inhibitor of binding ability, have prospect good, conduct treatment relevant diseases of angiogenesis (as tumor) medicine with integrin.Example 1 and 2 has proved the high effect blood vessel production inhibitor science, reasonable, feasible and effective of the present invention's design effectively.
Claims (10)
1. high effect blood vessel production inhibitor, it is characterized in that: this high effect blood vessel production inhibitor is to have the affinity of integrin and the angiogenesis inhibitor of binding ability.
2. according to the described high effect blood vessel production inhibitor of claim 1., it is characterized in that: be connected with the peptide section that integrin is had affinity and binding ability respectively in the one or both ends of angiogenesis inhibitor molecule.
3. according to claim 1 and 2. described high effect blood vessel production inhibitors, it is characterized in that: the peptide section that connects respectively in the one or both ends of angiogenesis inhibitor molecule, integrin is had affinity and a binding ability can be the section peptide that contains arginine-glycine-aspartic acid sequence, or contain agedoite-glycine-arginine sequence the section peptide, also can be the peptide section that contains above-mentioned two kinds of sequences simultaneously.
4. high effect blood vessel production inhibitor according to claim 3 is characterized in that: be connected with the section peptide that contains arginine-glycine-aspartic acid sequence on human plasminogen handle domain 1 protein.
5. high effect blood vessel production inhibitor according to claim 3 is characterized in that: be connected with the section peptide that contains arginine-glycine-aspartic acid sequence on human plasminogen handle domain 5 protein.
6. the application of a high effect blood vessel production inhibitor, it is characterized in that: this kind high effect blood vessel production inhibitor can significantly improve activity and the efficient that existing angiogenesis inhibitor suppresses endothelial cell growth and angiogenesis, reduces the using dosage of existing angiogenesis inhibitor; This kind high effect blood vessel production inhibitor can be as the medicine of treatment disease or diagnosis usefulness, treatment human or animal's the disease relevant with angiogenesis.
7. according to the application of claim 4 and 5 described high effect blood vessel production inhibitors, it is characterized in that: this kind angiogenesis inhibitor can effectively suppress endothelial cell growth, can be as the medicine of treatment disease or diagnosis usefulness, the disease relevant of more effectively treating the human or animal with angiogenesis.
8. the production method of high effect blood vessel production inhibitor, it is characterized in that: make up the angiogenesis inhibitor gene that contains arginine-glycine-aspartic acid or agedoite-glycine-integrin binding sequence peptide sections such as arginine, this gene is used for gene therapy; Or carry out gene engineering expression, separation and purification recombinant protein; Or with the synthetic peptide section that contains arginine-glycine-aspartic acid or agedoite-glycine-integrin binding sequences such as arginine of polypeptide synthesis method, the peptide section that will contain arginine-glycine-aspartic acid or agedoite-glycine-integrin binding sequences such as arginine with the method for chemical coupling is connected on angiogenesis inhibitor natural origin or the recombinant DNA source then, and formation can have the high effect blood vessel production inhibitor of combination with integrin.
9. the production method of high effect blood vessel production inhibitor according to claim 8 is characterized in that, obtains human plasminogen handle domain 1 gene that coding contains arginine-glycine-aspartic acid sequence peptide section, is used for gene therapy; Perhaps carry out gene engineering expression, separation, processing, purification, prepare the human plasminogen handle domain 1 that contains arginine-glycine-aspartic acid sequence peptide section.
10. the production method of high effect blood vessel production inhibitor according to claim 8 is characterized in that, obtains human plasminogen handle domain 5 genes that coding contains arginine-glycine-aspartic acid sequence peptide section, is used for gene therapy; Perhaps carry out gene engineering expression, separation, processing, purification, prepare the human plasminogen handle domain 5 that contains arginine-glycine-aspartic acid sequence peptide section.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009000137A1 (en) * | 2007-06-22 | 2008-12-31 | Hanmei Xu | A inhibiting agent for inhibition of angiogenesis, a method for preparing the agnet, a method for modifying the agent and its use for manufacturing a medicament for treating tumor |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009000137A1 (en) * | 2007-06-22 | 2008-12-31 | Hanmei Xu | A inhibiting agent for inhibition of angiogenesis, a method for preparing the agnet, a method for modifying the agent and its use for manufacturing a medicament for treating tumor |
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