CN101307106B

CN101307106B - Endothelial cell proliferation inhibitor and method of use

Info

Publication number: CN101307106B
Application number: CN 200810092130
Authority: CN
Inventors: Y·考; M·J·佛克曼; M·S·奥赖利
Original assignee: ALBERT LAB; Childrens Medical Center Corp
Current assignee: ALBERT LAB; Abbott Laboratories; Childrens Medical Center Corp
Priority date: 1995-12-13
Filing date: 1996-12-13
Publication date: 2013-06-12
Anticipated expiration: 2016-12-13
Also published as: CN100389126C; CN101307106A; CN1204343A

Abstract

The present invention comprises an endothelial inhibitor and method of use therefor. The endothelial cell proliferation inhibitor is a protein having a molecular weight of approximately 14 kD and having a N-terminal sequence of GPVGAGEPKCPLMVKVLDAV, that has the ability to inhibit endothelial cell proliferation in in vitro assays.

Description

Endothelial cell proliferation inhibitor and application method thereof

The application be that December 13, application number in 1996 are 96199011.2 the applying date, invention and created name divides an application for the application of " endothelial cell proliferation inhibitor and application method thereof ".

Technical field

The present invention relates to novel endothelial cell proliferation inhibitor.This inhibitor can suppress the Angiogenesis disease and regulate angiogenesis.In addition, the present invention relates to measure diagnositc analysis and the test kit of the inhibition dosage that exists in biological fluid sample, relate to the High iron-diamine method box of locating this inhibitor, relate to the DNA sequence dna of this inhibitor of encoding and the molecular probe of monitoring inhibitor biosynthesizing and degraded, the specific antibody that relates to this inhibitor, relate to peptide stimulant and the antagonist of developing this inhibitor acceptor, relate to specific antibody stimulant and the antagonist of counter inhibitor acceptor, also relate to the cytotoxic agent that is connected with this inhibitor.

Background technology

The term " vasculogenesis " of using herein refers to growth neovascularity in tissue or organ, and comprises endothelial cell proliferation.Under normal physiological conditions, the human or animal just just experiences vasculogenesis under quite special limitation circumstances.For example, vasculogenesis generally sees wound healing, the formation of fetus and embryo's growth and corpus luteum, uterine endometrium and placenta.Term " endothelium " expression lining is affixed on the smooth epithelial cell thin layer of serous cavity, lymphatic vessel and blood vessel.

It is believed that controlled and not controlled vasculogenesis is undertaken by similar manner.Formed capillary vessel by basic membrane-enclosed endotheliocyte and perithelial cells.Vasculogenesis starts from the enzyme of endotheliocyte and white corpuscle release to the erosion of basement membrane.Then the endotheliocyte that is lining in lumen of vessels passes basement membrane and stretches out.The stimulator of vasculogenesis lures that endothelial cell migration passes the basement membrane that is etched into.The cell of migration forms " sprouting " that breaks away from parent blood vessel, endotheliocyte experience mitotic division herein and propagation.This endothelium sprouting formation Capillary loops that mutually combines generates neovascularity.

That continue, not modulated vasculogenesis comes across in a large amount of morbid state, metastases and endotheliocyte abnormality growth, and is that the pathology damage that sees in these diseases is produced evidence.Wherein exist the various pathology morbid state of not modulated vasculogenesis to be classified as angiogenesis-dependent disease or Angiogenesis disease.

Be set forth at first 1971 (Folkman J., Tumor angiogenesis:Therapeutic implications., N.Engl.Jour.Med.285:1182-1186,1971) about " tumor growth is angiogenesis-dependent " this hypothesis.Set forth in its simplest statement:

In case " tumour occurs and ' capture ', the increase of the new capillary vessel on the tumour of being focused at must be arranged before each tumor cell number increases." tumour ' captures ' the front blood vessel phase (prevascular phase) that is understood to now tumor growth, wherein tumor cell group occupies several cubic millimeters of volumes and no more than millions of cells, and they can be survived on existing host's capillary blood vessel.Gross tumor volume needs inducing of new capillary vessel beyond expanding this phase to.For example, the micrometastasis knurl of the interim lung of blood vessel before mouse is early stage, unless examine art to the tissue slice inspection with powerful microscope, inspection be can not check.

Support the circumstantial evidence example of this concept to comprise:

(1) slowly and be linear, but speed is very fast and near exponential form after forming neovascularity before forming neovascularity for the speed of growth of implanting the tumour in the subcutaneous transparent chambers of mouse.(AlgireGH etc., Vascular reactions of normal and malignant tumors in vivo.I.Vascular reactions of mice to wounds and to normal and neoplastictransplants.J.Natl.Cancer Inst.6:73-85,1945).

(2) tumour that grows in the organ of isolation that blood vessel do not breed, perfusion is limited to 1-2mm ³, but when they are transplanted will expand to rapidly during to mouse this volume more than 1000 times and become the tumour of neovascularization.(Folkman J, Deng, Tumor behavior in isolated perfusedorgans:In vitro growth and metastasis of biopsy material in rabbitthyroid and canine intestinal segments.Annals of Surgery 164:491-502,1966)

(3) grow in slow without the intracorneal tumor growth rate of blood vessel and be linear, but transfer the growth of exponential form to after neovascularization.(Gimbrone, M.A., Jr. etc., Tumor growthand neovascularization:An experimental model using the rabbitcornea.J.Natl.Cancer Institute 52:41-427,1974)

(4) tumour that is suspended in the aqueous fluids of lagophthalmos anterior eye can be survived, and without blood vessel, size is limited to＜1mm ³But in case implanted iris vessels bed, they will become neovascularization and ramp, just reach 16,000 times of their original volumes within 2 weeks.(Gimbrone M.A.Jr. etc., Tumor dormancy in vivo by prevention of neovascularization.J.Exp.Med.136:261-276)

(5) when tumour being implanted chick embryo villi chorioallantoic membrane, they＞72 hours without the blood vessel phase during poor growth, mean diameter is no more than 0.93+0.29mm.Begin to form that in 24 hours after neovascularity, tumour enlarges rapidly, reach 8.0+2.5mm to the tumor average diameter of these vascularizations in the 7th day.(Knighton D.，Avascular and vascular phases of tumorgrowth in the chick embryo.British J.Cancer，35：347-356，1977)

(6) the blood vessel cast of rabbit Intrahepatic metastasis knurl shows metastatic tumor size unhomogeneity, but shows as the point cut-off of relative homogeneous at the size place that forms blood vessel.The tumour of diameter in 1mm be usually without blood vessel, but just form neovascularity greater than this diameter.(Lien W., Deng, The blood supply ofexperimental liver metastases.II.A microcirculatory study of normaland tumor vessels of the liver with the use of perfused silicone rubber.Surgery 68:334-340,1970)

(7) in the transgenic mice of the β of pancreas islet Intracellular growth cancer, the hyperplasia island size before blood vessel is limited to＜1mm.During age in week, the island of 4-10% becomes neovascularization, grows the tumour of large vascularization from these islands at 6-7, and gross tumor volume is larger more than 1000 times than the volume on the front island of blood vessel.(Folkman J, etc., Induction of angiogenesis during the transition fromhyperplasia to neoplasia.Nature 339:58-61,1989)

(8) specific antibody of anti-VEGF (vascular endothelial growth factor) reduces microvessel density, and can " significantly or strong " suppresses 3 kinds and depend on VEGF as the growth of people's tumour of their the unique amboceptor of vasculogenesis (in nude mice).This antibody is in the growth of external not inhibition tumor cell.(Kim K J, etc., Inhibition of vascular endothelial growth factor-induced angiogenesis suppresses tumor growth in vivo.Nature362:841-844,1993).

(9) anti-bFGF monoclonal anti physical efficiency 70% suppresses to depend on secretion bFGF as the growth of the mouse tumor of its unique vascularization amboceptor.This antibody is in the growth of external not inhibition tumor cell.(Hori A, Deng, Suppression of solid tumor growth byimmunoneutralizing monoclonal antibody against human basicfibroblast growth factor.Cancer Research, 51:6180-6184,1991)

(10) peritoneal injection bFGF can strengthen by the growth that stimulates capillary endotheliocyte in tumour the growth of primary tumor and metastatic tumor thereof.These tumour cells itself lack the acceptor of bFGF, and bFGF is not the mitogen of tumor cell in vitro.(Gross JL, etc., Modulationof solid tumor growth in vivo by bFGF.Proc.Amer.Assoc.Canc.Res.31:79,1990)

(11) specificity angiogenesis inhibitor (AGM-1470) suppresses growth and metastasis of tumours in vivo, but active much smaller in extracorporeal suppression tumor cell propagation.Little 4 orders of magnitude when its inhibition tumor cell of concentration ratio when it suppresses vascular endothelial cell proliferation half maximum value is bred.(Ingber D, Deng, Angioinhibins:Synthetic analogues of fumagillin whichinhibit angiogenesis and suppress tumor growth.Nature, 48:555-557,1990). also have indirect clinical indication to show that tumor growth is relevant to vasculogenesis.

(12) these spheroplasts are transferred to Vitrea human retinoblastoma and develop into without the blood vessel spherule, although can be survived and combination ³H-thymidine (when removing from the eye that enucleates during analyzed in vitro), but be limited to＜1mm ³

Minimum without blood vessel white seed (1-3mm when (13) ovarian cancer is transferred to peritonaeum ³).Unless one or more becomes neovascularization, otherwise that these grafts seldom can grow to is larger.

(14) (Weidner N in mammary cancer, Deng, Tumor angiogenesis correlates withmetastasis in invasive breast carcinoma.N.Engl.J.Med.324:1-8, 1991, and Weidner N, Deng, Tumor angiogenesis:A new significant andindependent prognostic indicator in earlystage breast carcinoma, JNatl.Cancer Inst.84:1875-1887, 1992) with prostate cancer in (WeidnerN, Carroll PR, Flax J, Blumenfeld W, Folkman J.Tumor angiogenesiscorrelates with metastasis in invasive prostate carcinoma.AmericanJournal of Pathology, 143 (2): 401-409, 1993) intensity of neovascularization is with the dangerous height correlation that shifts in the future.

(15) rarer from the melanomatous transfer of human skin before forming neovascularity.Form at the beginning neovascularity and just cause damage to thicken, the danger of transfer increases.(Srivastava A, Deng, Theprognostic significance of tumor vascularity in intermediate thickness (0.76-4.0mm thick) skin melanoma.Amer.J.Pathol.133:419-423,1988)

(16) in bladder cancer, the urine content of angiogenesis peptides bFGF is than situation and the degree sensitiveer indicator of cytology to disease.(Nguyen M, etc., Elevated levels of anangiogenic peptide, basic fibroblast growth factor, in urine of bladdercancer patients.J.Natl.Cancer Inst.85:241-242,1993)

Therefore, vasculogenesis obviously plays a major role in the transfer of cancer.If can suppress or eliminate or control and regulate this angiogenic activity, even the tumour existence can not grown yet so.In morbid state, prevent that vasculogenesis from can avoid new Microvasculature to invade and the damage that causes.Can cause eliminating or alleviating this class disease for the therapy of controlling angiogenesis.

What therefore, need is can inhibition of endothelial cell proliferation such as undesirable angiogenic growth, the composition of particularly growing in tumour and method.Also need to check, measure and locate the method for said composition.Said composition should be able to suppress to shift the activity of Endogenous Growth Factors in pre-neoplastic and prevent the formation of capillary vessel in tumour, thereby suppresses the growth of tumour.The part of said composition, composition and the specific antibody of said composition also should be able to be regulated the formation of capillary vessel in other angiogenesis such as wound healing and regeneration.Suppress the said composition of vasculogenesis and method should be preferably nontoxic with the side effect that produces seldom.Also need to check, measure and locate the method in the biosynthesizing site of the binding site of said composition and said composition.The part of said composition and composition should be able to be puted together with other molecule that is used for radioactivity and nonradioactive labeling

Summary of the invention

The present invention includes the method that Profibrinolysin Kringel 5th district that use to separate suppress the endothelium proliferation activity.Kringle 5 peptide fragment with this separation that suppresses active comprise approximately 80 amino acid whose sequences:

CMFGNGKGYRGKRATTVTGTPCQDWAAQEPHRHSIFTP

ETNPRAGLEKNYCRNPDGDVGGPWCYTTNPRKLYDYC

DVPQ

C=Cys Y=Tyr D=Asp wherein

M＝Met R＝Arg W＝Trp

F＝Phe T＝Thr H＝His

G＝Gly V＝Val S＝Ser

N＝Asn P＝Pro I＝Ile

K＝Lys Q＝Gln A＝Ala

E＝Glu L＝Leu

Endothelial cell proliferation peptide of the present invention is equivalent to from the peptide fragment of human plasminogen generation, and the approximately amino acid 462 that it starts from human plasminogen extends approximately 80 amino acid again.

The present invention also comprises diagnostic method and methods for the treatment of, is used for this inhibiting peptide of check body fluid and whether exists, and use this peptide for this compounds of the upper significant quantity of needs treatment with the patient of adjusting endothelial cell proliferation or specifically in conjunction with the antibody of this peptide.In addition, this inhibiting peptide can at the external compound that uses to test the inhibition that alleviates this peptide together with propagation Endothelial cell culture thing, namely screen inhibiting other compound that somatomedin maybe can overcome or change endothelial cell proliferation.

Therefore, an object of the present invention is to provide the composition that contains endothelial cell proliferation inhibitor, this inhibitor comprises approximately 80 amino acid whose peptide fragment of human plasminogen, and this segment is equivalent to start from kringle 5th district of the amino acid 462 of human plasminogen basically.

Another object of the present invention is to provide treatment by the disease of endothelial cell proliferation, particularly angiogenesis-mediated and the method for process.

Another purpose of the present invention is to provide the existence of inhibitor in detection body fluid or tissue and diagnostic method or method of prognosis and the test kit of content thereof.

another purpose of the present invention is to provide treatment by the method and composition of disease and the process of angiogenesis-mediated, wherein disease and process include but not limited to: vascular tumor, solid tumor, leukemia, shift, telangiectasis, psoriasis, scleroderma, granuloma pyogenicum, the vasculogenesis of cardiac muscle, plaque neovascularization, crown pleurapophysis, the brain pleurapophysis, arteriovenous malformotion, ischemic limb vasculogenesis, keratopathy, flush, neovascular glaucoma, diabetic retinopathy, retrolental fibrous tissue forms, sacroiliitis, the diabetic neovascularization, macular degeneration, wound healing, stomach ulcer, (Helicobacter) is diseases related for Helicobacter pylori, fracture, keloid, vasculogenesis, hemopoietic, ovulation, pass through, placentation and cat scratch fever.

Another object of the present invention is to provide the composition of the growth that is used for the treatment of or suppresses cancer.

An object of the present invention is to provide and regulate or the production of analogue enztme or the compound of activity, enzyme wherein produces inhibitor of the present invention in vivo or in vitro.

Another purpose of the present invention is to provide inhibitor or counter inhibitor antibody by the human or animal's direct injection inhibitor DNA to this treatment of needs.

An object of the present invention is to provide the method for the specific antibody existence of this inhibitor in check and quantitative analysis body fluid.

Another object of the present invention is to provide check or the method for prognosis of cancer.

Another object of the present invention is to provide for showing and the quantitative analysis composition in the site of inhibitor combination in vivo and in vitro.

Another purpose of the present invention is to provide for check and the biosynthetic composition of quantitative analysis inhibitor.

Another purpose of the present invention is to provide the cancer therapy with minimal side effect.

Another purpose of the present invention is to provide the composition of the inhibitor peptide fragment that contains endothelial cell proliferation inhibitor of the present invention or be connected with cytotoxic agent.

Another object of the present invention is to provide targeted delivery inhibitor dependency composition to the method for privileged site.

Another purpose of the present invention is to provide composition and the method for the gene therapy that is applicable to regulate endothelial cell proliferation such as angiogenesis.

Will understand these purposes of the present invention and other purpose, Characteristics and advantages after reading and appraising following detailed description to disclosed embodiment and appended claim.

Description of drawings

Fig. 1 has described the restraining effect of endothelial cell proliferation, is expressed as the percent change of cell count and the function of the kringle that separates 5 peptide fragment of the human plasminogen that adds cell.

Fig. 2 illustrates the gel electrophoresis analysis that separates from the preparation of the kringle of human plasminogen 5 peptide fragment.Swimming lane 1 is the Kringle 5 that separates; Swimming lane 2 is molecular weight markers.

Fig. 3 illustrates the Kringle 1,2,3 of human plasminogen, the Amino Acid Range in 4 and 5 districts.

Fig. 4 illustrates the anti-endothelial cell proliferation activity of the human plasminogen Kringle 5 that contains and do not contain amino carbonic acid (AMCHA), and it shows that lysine-binding site is not the reason that produces the anti-endothelial cell proliferation activity of Kringle 5.

Fig. 5 illustrates the inhibition of 5 pairs of bovine aortic endothelial cells propagation of restructuring Kringle.

Composition provided by the invention and method be inhibition of endothelial cell proliferation effectively, regulates vasculogenesis, and suppresses unwanted vasculogenesis, the particularly vasculogenesis relevant to tumor growth.The present invention includes a kind of protein endothelial cell proliferation inhibitor, it is characterized in that to derive from approximately 80 amino acid whose sequences of human plasminogen Kringle 5.This aminoacid sequence of inhibitor can slightly change because kind is different.Should understand that the amino acid no in this activity inhibitor molecule is variable, suppress all active close homology aminoacid sequences and all should think and be included in the present invention so have endothelium.

The present invention by the human or animal who suffers from undesirable endothelial cell proliferation is used comprise can be in analyzed in vitro the about composition of Kringle 5 of human plasminogen of inhibition of endothelial cell proliferation, and supplying method and composition are with disease and the process for the treatment of by undesirable and not controlled epithelial cell proliferation such as angiogenesis-mediated.The protein of this separation is preferably at least about 80% purity, more preferably at least about 90% purity, most preferably at least about 95% purity.The present invention is particularly suitable for treating or suppressing the growth of tumour.The human or animal who suffers from front vascularization metastatic tumor is used growth and the amplification that this inhibitor helps to prevent those tumours.

The present invention also comprises the DNA sequence dna of the endothelial cell proliferation inhibitor of encoding, contain the expression vector of the DNA sequence dna of the endothelial cell proliferation inhibitor of encoding, and contain one or more expression vectors cell of (this expression vector contains the DNA sequence dna of this inhibitor of encoding).The present invention further comprises gene therapy, and the DNA sequence dna of this endothelial cell proliferation inhibitor of encoding is whereby introduced the patient to modify inhibitor content in vivo.

The present invention also comprises diagnostic method and test kit, with the endothelial cell proliferation inhibitor in check and mensuration biofluid and tissue, and this inhibitor in position tissue and cell.This diagnostic method and test kit can be the arbitrary structures that those of ordinary skills know.The present invention also comprises the specific antibody of this endothelial cell proliferation inhibitor and part thereof, and the antibody of combination that suppresses the specific antibody of this endothelial cell proliferation inhibitor.These antibody can be polyclonal antibody or monoclonal antibody.The specific antibody of this endothelial cell proliferation inhibitor can be used in diagnostic kit checking existing and content of this inhibitor, and its diagnosable or prognosis is by the cancer of angiogenesis-mediated or outbreak or the recurrence of Other diseases.The specific antibody that also can use endothelial cell proliferation inhibitor to the human or animal is with this human or animal of immunity passively and anti-this inhibitor, so reduce the inhibition of vasculogenesis.

The present invention also comprises diagnostic method and test kit existing and content with the check antibody that endothelial cell proliferation inhibitor is combined in body fluid.This diagnostic method and test kit can be the arbitrary structures that those of ordinary skills know.

The present invention also comprises the counter inhibitor receptor specific antibody, the receptors bind of it and this inhibitor and to the suitable signal of cell conduction, and play stimulant or antagonist.

The present invention also comprises inhibitor peptide fragment and analogue, their available isotopic labelings or with other molecule or protein labeling, be used for by some technical surveys and show this inhibitor binding site, these technology include but not limited to positron emission tomography, radioautography, flow cytometry, radioactive rays receptor binding assay and immunohistochemistry.

These inhibitor peptides and analogue also play stimulant and antagonist action on the inhibitor acceptor, so strengthen or stop the biological activity of this endothelial cell proliferation inhibitor.This class peptide is used to the acceptor molecule that separation energy and this inhibitor are combined specifically.

The present invention also comprises the endothelial cell proliferation inhibitor that is used for the treatment of and studies, inhibitor fragment, the specific antisera of this inhibitor, and the inhibitor receptor agonist and the receptor antagonist that are connected with cytotoxic agent.Further, this inhibitor, its fragment, its specific antisera, inhibitor receptor agonist and inhibitor receptor antagonist acceptable vehicle on bound drug all, and optional slowly-releasing compound or composition such as biodegradable polymkeric substance and matrix, to form therapeutic composition.

The present invention includes the molecular probe of Yeast Nucleic Acid and thymus nucleic acid, transcribing and translating of these two kinds of nucleic acid and this endothelial cell proliferation inhibitor is relevant.These molecular probes are applicable to check and measure the inhibitor biosynthesizing in tissue and cell.

More particularly, the present invention includes for check and treatment by composition and the method for endothelial cell proliferation such as angiogenesis-mediated or associated disease and process.Have Kringel 5 peptide fragment that suppress active separation and comprise approximately 80 amino acid whose sequences:

CMFGNGKGYRGKRATTVTGTPCQDWAAQEPHRHSIFTP

ETNPRAGLEKNYCRNPDGDVGGPWCYTTNPRKLYDYC

DVPQ

C=Cys Y=Tyr D=Asp wherein

M＝Met R＝Arg W＝Trp

F＝Phe T＝Thr H＝His

G＝Gly V＝Val S＝Ser

N＝Asn P＝Pro I＝Ile

K＝Lys Q＝Gln A＝Ala

E＝Glu L＝Leu

This inhibitor can separate from Profibrinolysin such as human plasminogen, and is perhaps synthetic by chemistry or biological method (peptide is synthetic, and the external enzyme-catalyzed change of Profibrinolysin or plasmin is to generate activity inhibitor for cell cultures for example, recombinant gene expression).Recombinant technology comprises that using polymerase chain reaction (PCR) carries out gene amplification from the DNA source, and uses reversed transcriptive enzyme/PCR and carry out gene amplification from the RNA source.

The present invention also comprises: contain a kind of composition of carrier, this carrier contains the DNA sequence dna of the endothelial cell proliferation inhibitor of encoding, and can express this inhibitor when wherein this carrier is present in cell; The composition that comprises a kind of cell, this cell contains a kind of carrier, and wherein this carrier contains the DNA sequence dna of coding this inhibitor or its fragment or analogue, and can express this inhibitor when wherein this carrier is present in cell; And comprise the method for cell being implanted people or inhuman animal, and this cell contains a kind of carrier, and wherein this carrier contains the DNA sequence dna of this inhibitor of encoding, and can express this inhibitor when this carrier is present in cell.

When term " basically similar " or " homology basically " are used for the amino acid of this inhibitor of expression and nucleotide sequence, refer to this aminoacid sequence, be that it has endothelial cell proliferation inhibition activity and about molecular weight, it also has the height sequence homology with certain protein, and described protein has specific N terminal amino acid sequence disclosed herein; Perhaps refer to the to encode nucleotide sequence of endothelial cell proliferation inhibitor, described inhibitor have about molecular weight and with the high homology of something, this material has specific N terminal amino acid sequence disclosed herein.

High homology refers to the amino acid identity at least about 80%, preferably at least about 90% amino acid identity, more preferably at least about 95% amino acid identity.The term that this paper uses " it is active that endothelium suppresses " refers to that certain molecule suppresses the ability of vasculogenesis generally, for example, suppresses the ability of the thin endothelial cell growth of ox hair in culture under fibroblast growth factor exists.

The present invention also comprises the check of inhibitor in body fluid and tissue, is intended to diagnose or the prognosis disease of cancer for example.The check of inhibitor binding site and acceptor during the present invention also comprises cell and organizes.The present invention also comprises treatment or the disease of prevention vasculogenesis and the method for process, this class disease includes but not limited to sacroiliitis and tumour, use following method treat or prevent, namely by stimulating the generation of this inhibitor, and/or by the patient being used the inhibitor of separation, or required sublimed inhibitor, or inhibitor stimulant or antagonist, and/or inhibitor specific antisera or for the antiserum(antisera) of inhibitor specific antisera.Other methods for the treatment of comprises uses this inhibitor, its bioactive fragment, inhibitor analogue, inhibitor specific antisera, or the inhibitor receptor agonist and the antagonist that are connected with cytotoxic agent.

Application specific ground can be used to regulate for example reproduction of angiogenesis-dependent process in conjunction with the passive antibody therapy of the antibody of this inhibitor, grows, and wound healing and tissue repair.In addition, can be applied to stop the ability of endogenous inhibitor specific antisera binding inhibitors for the antiserum(antisera) in the Fab district of inhibitor specific antibody.

The present invention also comprises gene therapy, regulates whereby the gene of this inhibitor of coding in patient body.Transfer or DNA delivery are to the whole bag of tricks of cell with expressing gene product albumen matter, or be called gene therapy, all be disclosed in Gene Transfer into Mammalian Somatic Cellsin vivo, N.Yang, Crit.Rev.Biotechn.12 (4): 335-356 (1992), it is for referencial use that the document is incorporated this paper into.Gene therapy comprises that DNA sequence dna is mixed somatocyte or germ line cell is used for ex vivo or intravital treatment.Gene therapy plays a part such, namely sub stituent because of, increase normal or abnormal gene function and antagonism transmissible disease and other pathology.

The countermeasure of processing these medical care problems with gene therapy comprises: therapeutic strategy, for example identify damaged gene, and then add functioning gene with the function of replacement defect gene or strengthen the gene of small and weak function; Perhaps preventative strategies can for example adds the gene of product albumen matter, and this protein can be treated the patient's condition maybe will make tissue or organ more responsive for treatment plan.An example of preventative strategies can is the nucleotide sequence of this inhibitor of coding can be placed in the patient, so prevent the generation of vasculogenesis; Perhaps can insert the gene that makes tumour cell more responsive to radiation, then this tumour of radiation will increase the lethality to tumour cell.

The present invention has imagined many schemes of transfer inhibitor DNA or inhibitor adjusting sequence.Transfection promoter sequence, rather than common sequence of being combined specifically with this inhibitor, or other can increase the sequence of this inhibitor protein matter output, also is envisioned for the method for gene therapy.An example of this technology sees Transkaryotic Therapies, Inc.of Cambridge, and Massachusetts uses " gene switching " insertion cell that homologous recombination can be opened erythropoietin gene.Referring to Genetic Engineering News, April 15,1994.This " gene switching " can be used to activate this inhibitor (or inhibitor acceptor) in can not the cell of this inhibitor of normal expression (or acceptor of this inhibitor).

The gene transfer method that is used for gene therapy is classified as three major types-Physical (electroporation for example, direct gene transfer and particle bombardment), chemical method (based on the carrier of lipid, or other non-viral carrier) and biological process (deriving from carrier and the acceptor picked-up of virus).For example, can use the non-viral carrier, they comprise the liposome that DNA applies.This liposome/DNA complex can be injected the patient by direct intravenously.It is believed that, this liposome/DNA complex is concentrated in liver, and they are delivered to DNA scavenger cell and Kupffer cell in liver.These cells can be survived for a long time thereby can this DNA that sends of long-term expression.In addition, " naked " DNA of carrier or gene directly can be injected desirable organ, tissue or tumour with the targeted delivery therapeutic DNA.

Gene therapy method is learned and also can be described by sending the site.The basic skills of delivery of gene comprises: the ex vivo transgenosis, and in vivo transgenosis, and outer-gene shifts.In the transgenosis of ex vivo, cell is taken from the patient and is grown in cell culture.DNA is transfected into cell, weighs again patients with implantation after the cell quantity of this transfection enlarges.In outer-gene shifted, the cell of conversion was the cell that grows in culture, as tissue culture cells, rather than derived from specific patient's specific cells.Transfection these " experimental cells ", select the cell of transfection and expand after with their patients with implantations or separately use it for anything else.

Intravital transgenosis comprises the cell of DNA being introduced the patient when these cells are in patient body.Method comprises uses virus-mediated transgenosis, uses non-infectious virus that gene delivery is entered the patient or naked DNA injected patient's site, so this DNA is absorbed with certain percentage of cells, gene product protein is expressed in these cells.In addition, other method described herein (as the application of " particle gun ") can be used to external insertion endothelial cell proliferation inhibitor DNA or inhibitor adjusting sequence.

The chemical process of gene therapy can relate to a kind of compound based on lipid, and not necessarily liposome, transport DNA and pass cytolemma.Lipofection agent or cytofectin, the positive ion based on lipid of being combined with the DNA of bear electricity forms a species complex and can pass cytolemma and DNA is sent into cell interior.Another kind of chemical process is used the endocytosis based on acceptor, and it comprises is combined ligands specific and coating with cell surface receptor, then cytolemma is passed in its transhipment.Part should be transported into cell by whole complex body after DNA is combined again.This ligand gene complex body is injected blood flow, and the target cell that then has this receptor will also be transported this part-DNA complex into this cell in conjunction with this part specifically.

Many gene therapy methods are used virus vector gene are inserted cell.For example, the retroviral vector of already having used change in the method for ex vivo is introduced periphery and tumor-infiltrated lymphocyte, liver cell, epidermic cell, myocyte or other somatocyte with gene.Then the cell of these changes is introduced the patient so that the gene product of the DNA that derives from insertion to be provided.

Virus vector also has been used to by scheme in vivo, gene be inserted cell.In order to instruct tissue-specific exogenous gene expression, can use known is tissue-specific cis-acting regulatory element or promotor.Also can reach this purpose by on-the-spot DNA delivery or virus vector to specific anatomical sites in vivo.For example, in vivo transgenosis to blood vessel is to realize by implanting site selected on arterial wall at external endotheliocyte with transduction.Virus infection is also expressed the peripheral cell of this gene product.Virus vector for example can directly be delivered to intravital site by conduit, then only allow certain district by this virus infection, thus long-term, site-specific genetic expression is provided.Use the in vivo transgenosis of retroviral vector also by altered virus is injected logical organotropic blood vessel and obtained confirmation in mammary tissue and hepatic tissue.

The virus vector that already had been used to gene therapy includes but not limited to: retrovirus, other RNA viruses such as poliovirus or sindbis virus, adenovirus, gland dependency virus, simplexvirus, SV40, vaccine and other DNA virus.Copying deficient mouse retroviral vector is the gene transfer vector of widespread use.The murine leukemia retrovirus comprises and core protein and the compound single stranded RNA of polysaccharase (pol), and the glycoprotein coating (env) that is wrapped into albumen barycenter (gag) and be determined host range surrounds.Retroviral genome structure comprises these gag, pol and env gene, they by 5 ' and 3 ' long terminal repeat (LTR) surround.The retrovirus system utilizes following truth, and namely the virus structural protein supplied with of hypothesis is transconfiguration in package cell line, contain this 5 ' and minimum carrier and the packaging signal of 3 ' LTRs be enough to allow carrier package, infect and be integrated into target cell.The principal advantages that is used for the retroviral vector of transgenosis comprises: the cell in most kinds effectively infects and genetic expression, is integrated into single copy carrier accurately of target cell chromosomal DNA, and the ease for operation of this reverse transcription virus gene group.

Adenovirus comprises line style, double-stranded DNA compound with core protein and that wrapped up by capsid protein.The progress of Molecular Virology has caused utilizing these organic biology can the novel gene sequence be transduceed into the carrier of target cell to produce in vivo.Carrier based on adenovirus will be expressed the gene product peptide high-levelly.Adenovirus carrier even also has efficient infectivity under the virus of low titre.In addition, this virus is fully infective as acellular virion, so needn't inject producer's clone.The potential advantage of another of adenovirus carrier is to realize the in vivo ability of long-term expression heterologous gene.

The mechanical means that DNA sends comprises liposome or other carrier of lipid carrier as being suitable for the film fusion of fusion, mix lipid particle such as the lipofection agent of the DNA of cation lipid, the transfer of the DNA of polylysine mediation, the direct injection of DNA, for example with the micro-injection sexual cell of DNA or somatocyte, the particle that pneumatic dispatch DNA applies for example is used for the gold particle of " particle gun " and inorganic chemistry method such as calcium phosphate transfection.Another kind method, i.e. ligand-mediated gene therapy, comprise DNA and ligands specific compound and form part-DNA conjugate, to instruct this DNA to specific cell or tissue.

Found that plasmid DNA is injected the myocyte can obtain the high cell of transfected percentage, these cells can the continuous expression marker gene.The DNA of plasmid can maybe can not be integrated into the genome of cell.The nonconformity effect meeting of the DNA of transfection allow end differentiation eventually, long-time transfection and expressing gene product albumen matter in non-proliferative tissue, and do not worry that sudden change in cellular genome or Mitochondrial Genome Overview is inserted, disappearance or change.Long-term but needn't permanent therapeutic gene be transferred to specific cell and the therapy of inherited disease can be provided or be used for prevention.Can inject again termly DNA and can not occur sudden change to keep the gene product level in the genome of recipient cell.The nonconformity effect of foreign DNA s can allow to have several different foreign DNA constructions in a cell, and all these constructions are expressed different gene product.

At first the gene transfer method of particle mediation is used to the conversion of plant tissue.Answer alpha bombardment device or " particle gun ", the high-density particles (for example gold or tungsten) that the power of generation applies DNA accelerates to high-speed thereby can penetrate target organ, tissue or cell.Particle bombardment can be used to vitro system, perhaps use ex vivo or intravital method DNA is introduced cell, tissue or organ.

The electroporation applied current of transgenosis makes cell or tissue responsive for the transgenosis of electroporation mediation.The permeability that the brief electrical pulse of using certain field intensity increases film makes DNA molecular can penetrate cell.This technology can be used in vitro system, perhaps use ex vivo or intravital method DNA is introduced cell, tissue or organ.

In vivo carrier mediated transgenosis can be used to foreign DNA is transfected into cell.This carrier-DNA complex body can be introduced easily body fluid or blood flow then by locus specificity instruct target organ or tissue to the body.Can use liposome and polycation, as polylysine, lipofection agent or cytofectin.Can develop cell-specific or organ specific liposome, so the foreign DNA that is carried by liposome will be absorbed by target cell.The immunoliposome that injection is directed on some cell specific acceptor can be used as DNA is inserted the facilitated method of the cell that carries this receptor.Another carrier system that has been employed is asialoglycoprotein/polylysine conjugate system, and it is suitable for carrying DNA to liver cell in transgenosis in vivo.

Having turned seven DNA also can be compound with other type of carrier, so this DNA is carried into recipient cell, then retains in kytoplasm or caryoplasm.DNA can be protein-coupled and directly carried into nucleus with carrier core in the vesicle complex body of through engineering approaches specifically.

The generegulation of inhibitor of the present invention can realize like this, namely by using the compound of being combined with the gene of this inhibitor, or with the control region of this gene-correlation, or its corresponding rna transcription thing speed of transcribing or translate with modification.In addition, can be applied to the patient so that intravital inhibitor source to be provided with the cell of DNA sequence dna transfection of this inhibitor of coding.For example, cell can be with the carrier transfection of the nucleotide sequence that contains this inhibitor of encoding.

Term " carrier " expression that this paper uses can contain specific nucleotide sequence or associated carrier, and its effect is that this specific nucleotide sequence is transported into cell.The example of carrier comprises plasmid and infective micro-organisms such as virus, or non-virus carrier such as part-DNA conjugate, liposome, lipid-DNA complex.May wish comprising that the recombinant DNA molecules of endothelial cell proliferation inhibitor DNA sequence dna is connected to form the expression vector that can express this inhibitor effectively with expression control sequenc.The cell of transfection can be the cell of the healthy tissues that derives from the patient, patient's illing tissue, can be perhaps non-patient's cell.

For example, can use from the tumour cell of patient's excision and can express the carrier transfection of inhibitor protein matter of the present invention, then return and introduce the patient.Tumour cell after transfection produces inhibitor in the patient amount can suppress the growth of tumour.The patient can be people or inhuman animal.In addition, inhibitor DNA can not need directly to be injected the patient by means of carrier.Specifically, inhibitor DNA can be injected into skin, muscle or blood.

Inhibitor is expressed and can be continued a very long time or can regularly use inhibitor DNA to keep inhibitor protein matter level required in cell, tissue or organ or biofluid.

Although do not want to be subjected to the constraint of following hypothesis, but think when tumour becomes angiogenic, it discharges one or more peptides (such as aFGF, bFGF, VEGF, IL-8, GM-CSF etc.) that generates blood vessel, they act near primary tumor target endothelium partly from EV direction, and do not circulate (perhaps shorter transformation period of circulation).The amount that these peptides that generate blood vessel must produce should be enough to overcome the effect of endotheliocyte inhibitor (inhibitor of vasculogenesis), so that primary tumor continues its quantity of amplification.In case this primary tumor well-grown, it just continues to discharge the endotheliocyte inhibitor to the recycle system.According to this hypothesis, these inhibitor are the target capillary endothelium from endovascular directive effect in metastatic tumor at distance primary tumor So Far Away, and continues circulation.Therefore, when remote metastatic tumor may begin to activate vasculogenesis just, the inhibitor that near the capillary endothelium it is entered suppressed.

like application class, technology realizes that the production of about endothelial cell proliferation inhibitor of the present invention can use the recombinant DNA technology that comprises the following steps to complete, namely identify and purifying is as described herein and by the inhibitor of accompanying drawing illustration (1), (2) determine the N terminal amino acid sequence of sublimed inhibitor, (3) by synthetic 5 ' and 3 ' the DNA Oligonucleolide primers that produces this inhibitor sequence, (4) use this inhibitor gene sequence of polymeric enzymatic amplification, the sequence that (5) will increase is inserted suitable carrier such as expression vector, the carrier that (6) will contain gene inserts microorganism or other expression system that can express this inhibitor gene, and (7) separate the inhibitor of this recombinant production.Suitable carrier comprises virus, bacterium and (for example yeast) eucaryon expression vector.As above technology has more completely been described in laboratory manual, this handbook such as " Molecular Cloning:A LaboratoryManual " Second Edition by Sambrook etc., Cold Spring Harbor Press, 1989, it is for referencial use that it is merged in this paper.

Yet the another kind of method of producing this inhibitor or its bioactive fragment is method of peptide synthesis.The aminoacid sequence of this inhibitor for example can be measured by the automated peptide sequence measurement.In addition, in case gene or the DNA sequence dna of coding inhibitor for example separated by aforesaid method, this DNA sequence dna just can use well known in the art manually or the automatic sequencing method measure.Nucleotide sequence provides the information of relevant aminoacid sequence conversely.

In case know the aminoacid sequence of this peptide, just can by technology synthetic peptide fragment well known in the art, for example pass through " Solid Phase Peptide Synthesis:A PracticalApproach " E.Atherton and R.C.Sheppard, IRL Press, Oxford, England.Can synthesize a lot of fragments then connect together them and form larger fragment.Can be used on also that the specific location aminoacid replacement prepares these synthetic peptide fragments so that external or test excited activity and antagonistic activity in vivo.Can use with the peptide fragment of organizing high-affinity to be combined and separate receptors bind thing inhibitor on affinity column.

This inhibitor is effective in cure in the disease for the treatment of vasculogenesis for example or process, this vasculogenesis be by the endothelial cell proliferation mediation or relate to endothelial cell proliferation.The present invention includes the method for the disease for the treatment of angiogenesis-mediated, it uses the inhibitor of significant quantity, or its bioactive fragment, or the molectron that generally has the inhibitor fragment of anti-angiogenesis activity, or inhibitor stimulant and antagonist.The disease of angiogenesis-mediated includes but not limited to: solid tumor; Blood is given birth to tumour such as leukemia; Metastases; Innocent tumour such as vascular tumor, auditory nerve knurl, neurofibroma, trachoma, and granuloma pyogenicum; Rheumatoid arthritis; Psoriasis; The eye vasculogenesis is sick, diabetic retinopathy for example, and the prematureness retinopathy, macular degeneration, the corneal transplantation rejection, neovascular glaucoma, retrolental fibrous tissue forms, flush; Osler-Webber syndrome; Angiogenesis of cardiac muscle; Plaque neovascularization; Telangiectasis; The haemophiliac joint; Hemangiofibroma; And wound granulation.

This inhibitor is applicable to treat the excessive stimulation of endotheliocyte or the disease of abnormal stimulation.This class disease includes but not limited to: intestinal adhesion, atherosclerosis, scleroderma and hypertrophic scar are keloid.This inhibitor can be made the birth control agent by preventing that the required vascularization of embryo's implantation from using.This inhibitor is applicable to treat disease such as cat scratch disease (Rochele minalia quintosa) and the ulcer (ulcers) (helicobacter pylori (Helicobacterpylori)) take vasculogenesis as pathological examination.

The synthetic peptide fragment of this inhibitor serves many purposes.With this peptide of receptors bind by radio-labeling and be used for adopting demonstration and the quantitative analysis binding site of autoradiographic technique and film combination technology, wherein be subjected to physical efficiency high degree of specificity and high affinity ground in conjunction with this inhibitor.

In addition, the inhibitor of short-lived isotope mark or its peptide fragment can show receptor binding site in vivo, and its application positron emission tomography or other modern radioautography are in order to determine the position of tumour with the inhibitor binding site.

Cytotoxic agent (as ricin) is connected with this inhibitor and high-affinity peptide fragment thereof, thereby provides a kind of destruction to be combined with the instrument of the cell of this inhibitor.These cells are found in a lot of positions, include but not limited to micrometastasis knurl and primary tumor.The peptide that is connected with cytotoxic agent is poured in the mode that is intended to farthest to be delivered to desired area.For example, can complete by the intubate that the intubate of inserting vascular is supplied with target site or directly inserted target and send.This class reagent can also be in check mode by being sent with the osmotic pump of infusion cannula coupling.The molectron of inhibitor antagonist can use to increase the vascularization of tissue together with the stimulator of vasculogenesis.This treatment plan provides the effective ways of eliminating metastatic carcinoma.

This inhibitor can be combined with other composition and the method for the treatment of disease.For example, then tumour available operation of being combined with this inhibitor usually, radiation or embolic chemotherapy treatment can then use this inhibitor with the latent period that extends the micrometastasis knurl and the growth of stablizing and suppress any remaining primary tumor to the patient.In addition, this inhibitor, its fragment, inhibitor specific antisera, inhibitor receptor agonist and antagonist or its molectron, with pharmaceutically acceptable vehicle, and optional sustained-release matrix such as biodegradable combination of polymers and form therapeutic composition.

Sustained-release matrix used herein is the matrix that a kind of raw material by being generally polymkeric substance consists of, and they can or pass through solvency action and degrade by enzymically hydrolyse or acidolysis/alkaline hydrolysis.In case in the insertion body, this matrix just is subjected to the effect of enzyme and body fluid.This sustained-release matrix is preferably selected from biocompatibility raw material such as liposome, polylactide (poly(lactic acid)), PGA (polymkeric substance of oxyacetic acid), poly-(rac-Lactide-glycollide) (PLGA), polyanhydride, poly-(ortho acid) ester, polypeptide, hyaluronic acid, collagen, chondroitin sulfate, carboxylic acid, lipid acid, phosphatide, polysaccharide, nucleic acid, polyamino acid, amino acid such as phenylalanine, tyrosine, Isoleucine, polynucleotide, polyvinyl propylene, Polyvinylpyrolidone (PVP) and polysiloxane.Preferred biodegradable matrix is one of following matrix: polylactide, PGA or poly-(rac-Lactide-glycollide) (PLGA).

Vasculogenesis of the present invention-adjustment for the treatment of composition can be solid, liquid or aerosol, and can be arbitrarily known route of administration is used.The example of solid therapeutic composition comprises ball, emulsifiable paste and implantable dose unit.But the ball oral administration, the therapeutic emulsifiable paste can topical application.Implantable dose unit can topical application, for example use at tumor locus, perhaps implantable it in order to discharge Therapeutic angiogenesis-adjusting composition at whole body, for example implant subcutaneous.The example of liquid composition comprises: be suitable for the temper of subcutaneous injection, intravenous injection, intra-arterial injection, and be suitable for temper local and that intraocular is used.The example of aerosol temper comprises the inhalation temper that lung is used.

Inhibitor protein matter of the present invention also can be used to produce has specific antibody to this inhibitor and acceptor thereof.This antibody-like can be polyclonal antibody or monoclonal antibody.These can be used in diagnostic method that those of ordinary skills know and test kit content with this inhibitor content or inhibitor acceptor in check or quantitative analysis body fluid or tissue in conjunction with the antibody of this inhibitor or inhibitor acceptor specifically.These test-results can be used for diagnosing or predict generation or the recurrence of the disease of cancer and other angiogenesis-mediated.

This inhibitor also can be used for developing diagnostic method and test kit can be in conjunction with the antibody of this inhibitor with check and quantitative analysis.These test kits should be able to be checked the inhibitor specific antibody of circulation.With the patient of the counter inhibitor antibody of this class circulation is probably long, kinds of tumors and cancer are arranged, and cancer recurs possibly after treatment or catabasis.The Fab fragment of these antibody can be used as antigen to produce the counter inhibitor specificity Fab-fragment antiserum(antisera) that can be used for counter inhibitor antibody.This method can reduce by counter inhibitor antibody to the removing of circulation inhibitor, so effectively improve the content of circulation inhibitor.

Another aspect of the present invention is the method that stops the effect of excessive endogenous inhibitor.This can be specific antibody to undesirable inhibitor in system and come passively that immune human or animal realizes by using.This methods for the treatment of is passed through and placentation in the abnormal ovulation for the treatment of, and blood vessel generation aspect can be very important.This just provides useful tool for the effect of removing transfer process of check inhibitor.The Fab fragment of inhibitor specific antibody contains the binding site of inhibitor.Use technology well known by persons skilled in the art and separate this fragment from the inhibitor specific antibody.The Fab fragment of inhibitor specific antisera is used as antigen to cause the production of anti-Fab fragment serum.This antiserum(antisera) of the anti-specific Fab fragment of this inhibitor of perfusion can prevent the combination of this inhibitor and inhibitor antibody.Thereby by in and endogenous counter inhibitor antibody reach result for the treatment of by stoping inhibitor to be combined with the Fab of counter inhibitor fragment.The pure effect of this treatment is the ability that enhanced endogenesis circulation inhibitor arrives target cell, so reduce the diffusion of metastatic tumor.

Should understand, the present invention is intended to comprise to have any derivative that endothelial cell proliferation suppresses the inhibitor of activity.The present invention includes the derivative of whole inhibitor protein matter, inhibitor protein matter and the bioactive fragment of inhibitor protein matter.They comprise having the protein that suppresses active, and protein is connected with sugar or other molecule with the aminoacid replacement thing or on the aminoacid functional base.The present invention also comprises the gene of this inhibitor of coding and inhibitor acceptor, and by the protein of these genetic expressions.

Above-mentioned have that the protein that suppresses active can separate with protein fragments and substantially the protein of purifying and the form of protein fragments provide, they are in the pharmaceutically acceptable temper of using accord method modulation known to persons of ordinary skill in the art.These temper available standards approach are used.Usually, this based composition can be used by following approach: local, in skin, endoperitoneal, encephalic, ICV, brain, intravaginal, in utero, per os, per-rectum or parenteral (for example intravenous, intravertebral, subcutaneous or intramuscular) approach.In addition, this inhibitor can be mixed biodegradable polymkeric substance and make it this compound of slowly-releasing, this base polymer is implanted to be needed near the place of drug delivery, for example at tumor locus, perhaps implant can this inhibitor of whole body slowly-releasing the place.Also can use the infiltration micropump controlled this inhibitor of sending high density is provided, namely be delivered to interested position by intubate and for example directly send into the transforming growth cancer or send blood vessel into this tumour.The biodegradable polymkeric substance of this class and application examples thereof be as being described in detail in Brem etc., J.Neurosurg.74:441-446 (1991), and it is for referencial use that the document is incorporated this paper in full into.

Inhibitor dosage of the present invention will depend on subject morbid state or situation and other clinical factor such as human or animal's body weight and healthy state, also have the route of administration of this compound.As for the treatment human or animal, can use approximately this inhibitor of 0.5mg/kg to 500mg/kg.Depend on the transformation period of this inhibitor in particular animal or people, the frequency of administration of this inhibitor can be between every day, several be extremely weekly.Should understand, application facet of the present invention comprises that people and animal doctor use.The inventive method expectation single administration and repeatedly using can be side by side or within lasting for some time.

This inhibitor temper comprises those that are suitable for following route of administration, it is per os, per-rectum, through (the comprising in intravitreous or the eye-chamber) of eye, intranasal, local (comprise cheek with hypogloeeis), in utero, (comprise subcutaneous, endoperitoneal, intramuscular, intravenous, intracutaneous, encephalic, endotracheal and peridural) vagina or parenteral uses.This inhibitor temper can be expediently unit dosage form and can prepare by usual drug technique.This class technology comprises the step that activeconstituents is combined with pharmaceutical carrier or vehicle.Substantially, can prepare like this this class temper, be about to activeconstituents and liquid carrier or solid-state carrier in small, broken bits or with both equably, closely be combined, then if required these goods are shaped.

The temper that suitable parenteral is used comprises: water-based and aseptic parenteral solution non-water, the solute that acceptor blood that it can contain oxidation inhibitor, buffer reagent, fungistat and can make this temper and expection etc. oozes; And water-based and sterile suspensions non-water, it can comprise suspension agent and thickening material.This class temper can be contained in unitary dose or multi-dose container, and for example ampoule and the bottle of sealing, can store under cryodesiccated (freeze-drying) condition, just only need add aseptic liquid carrier, for example water for injection before application.The solution of on-the-spot injection or suspension can be from sterilized powder, particle and the sheet preparations of aforesaid kind.

Preferred unitary dose temper comprises per daily dose or unit, day sub-doses or its suitable part of using composition.Should understand, except the top composition of mentioning specially, temper of the present invention also can comprise other habitual reagent of temper of the relevant described type in this area.Cytotoxic agent can be chosen wantonly and mix or otherwise binding inhibitors protein or its biological functionality peptide fragment, to provide double treatment to the patient.

Vasculogenesis inhibiting peptide of the present invention can synthesize in the microchemistry equipment of standard, and available HPLC and mass spectroscopy detection purity.Method of peptide synthesis, HPLC method of purification and mass spectroscopy are well known by persons skilled in the art.Inhibitor peptide and inhibitor acceptor peptide also can be produced in recombination bacillus coli (E.coli) or yeast expression system, then use the column chromatography purifying.

The different peptide fragment that can synthesize this complete inhibitor molecules are for the several applications that includes but not limited to following aspect: as antigen for the exploitation specific antisera, as at the activated stimulant of inhibitor binding site and antagonist, as being connected with cytotoxic agent or being used for peptide in conjunction with cytotoxic agent for the directed cell that kills and wounds in conjunction with this inhibitor.The aminoacid sequence that comprises these peptides is selected and is easy in conjunction with antiserum(antisera) in the position of this molecule outside area based on them.The aminoterminal of this inhibitor and carboxyl terminal, and the middle region of this molecule is presented at respectively between fragment to be synthesized.

Utilize protein sequence database such as GenBank, Brookhaven Protein, SWISS-PROT and PIR that these peptide sequences and known array are compared to determine possible sequence homology.This information helps to eliminate the sequence that shows as with the sequence homology of other numberator height, so strengthen antiserum(antisera), stimulant and the specific potential of antagonist camber of this inhibitor of exploitation.

But the application standard method is with inhibitor and derivative peptide and other molecule coupling of inhibitor.The aminoterminal of this inhibitor and carboxyl terminal all contain tyrosine residues and lysine residue, all available several different methods is carried out isotopic labeling and heterotope mark, for example use conventional techniques (tyrosine residues-chloramine-T, idodine (iodogen), lactoperoxidase; Lysine residue-Bolton-Hunter reagent) carry out radio-labeling.Those skilled in the art know these coupling technologies.Also can add the fragment that does not possess these residues to promote the mark to the reactive amino on peptide and hydroxyl in tyrosine or Methionin.The selection of coupling technology is based on obtainable function base on amino acid, and these function bases include but not limited to amino, sulfydryl (sulfhydral), carboxyl, acid amides, phenol and imidazoles.The all ingredients that carries out coupling comprises glutaraldehyde, diazotizing p-diaminodiphenyl, carbodiimide and p-benzoquinones etc.

Inhibitor peptide and isotropic substance, enzyme, carrier protein, cytotoxic agent, fluorescence molecule, chemoluminescence agent, luminescent biological agent and other compound chemistry coupling are supplied multiple application.The efficient of linked reaction can be measured with the different methods that is suitable for concrete reaction.For example, use the Na of chloramine-T and high specific acitivity ¹²⁵I realizes using ¹²⁵The radio-labeling of I to the inhibitor peptide.Stop this reaction with Sodium Pyrosulfite, with mixture desalination on disposable post.The peptide of wash-out mark and collect fraction from the post.Adopt equal sample from each fraction and measure radioactive activity with the γ calculating instrument again.Press the method with unreacted Na ¹²⁵I separates from the inhibitor peptide of mark.The peptide fraction that storage has high specific activity activity is for example analyzed it and the sero-fast binding ability of inhibitor for subsequent applications.

The Another application that peptide is puted together is to produce polyclonal antiserum.For example, using glutaraldehyde will contain the inhibitor peptide of lysine residue and be connected with the bovine serum albumin of purifying.By measuring the efficient of determining this reaction of mixing of radiolabeled peptides.Separate unreacted glutaraldehyde and peptide by dialysis.Store this conjugate for subsequent applications.

Peptide fragment and the inhibitor acceptor that can produce for this inhibitor, inhibitor analogue, this inhibitor are specific antiserum(antisera).After synthetic and purifying, uses prior art generation mono-clonal well known by persons skilled in the art and polyclonal antiserum at peptide.For example, can produce polyclonal antiserum in rabbit, sheep, goat or other animal.To be combined with adjuvant mixture with inhibitor peptide or inhibitor itself that carrier molecule such as bovine serum albumin are puted together, emulsification, subcutaneous injection back, neck, flank and a plurality of positions of palmula sometimes.In regular interval as every 2 to 4 all booster shots.Per injection approximately after 7～10 days, is obtained blood sample by venipuncture, for example use after dilatation in one's ear along vein.Placing under 4 ℃ spends the night solidifies blood sample, then at 4 ℃ and approximately under 2400Xg centrifugal approximately 30 minutes.Take out serum, be divided into equal sample, storing under 4 ℃ for storing for follow-up analysis immediately or under-20～-90 ℃.

Analysis derives from all serum samples of producing polyclonal antiserum or derives from manufacture order clone's sero-fast middle level sample (media samples) to measure antigen titration.Determine titre by several methods, for example, use dot blotting and density analysis method; Also the peptide of application of radiation mark-antibody complex body precipitator method, wherein use a-protein, secondary antiserum(antisera), cold ethanol or gac-dextran, then measures active with the γ calculating instrument.The antiserum(antisera) of high titre is also used commercially available affinity column purifying.Make inhibitor peptide and gel coupling in affinity column.The antiserum(antisera) sample flow is crossed this post, and counter inhibitor antibody keeps being combined with this post.Then these antibody of wash-out are collected and estimate titre and specificity.

Test the inhibitor specific antisera of high titre to determine following performance: a) the high specific combination of antigen and the best antiserum(antisera) extent of dilution of minimum non-specific binding, b) in the standard replacement curve in conjunction with the ability of the inhibitor peptide of increasing amount, c) to the potential cross reactivity of the protein of relevant peptide and related specy, d) detect in the extract of blood plasma, urine, tissue and the ability of the inhibitor peptide in cell culture medium.

Measure the test kit of inhibitor and inhibitor acceptor, also belong to part of the present invention.Further check this antiserum(antisera), namely have the highest titre and specificity and can detect in the extract of blood plasma, urine, tissue and the inhibitor peptide in cell culture medium, with confirm to be easy to use test kit rapidly, reliable, sensitive and measure specifically and locate inhibitor.These assay kits include but not limited to following technology: competitive and noncompetitive analysis, radioimmunoassay, noclilucence and chemiluminescence analysis, fluorometric assay is analyzed, sandwich assay, immunoradiometric assay(IRMA), dot blotting, the enzyme connection that comprises ELISA is analyzed, Microplate test, for bar or the measuring staff of the antibody coating of fast monitored urine or blood, and immunocytochemical method.For every kind of test kit, determined scope, sensitivity, tolerance range, reliability, specificity and the circulation ratio of its analysis.Change in having determined in the displacement curve of standard or activity curve to analyze on 20%, 50% and 80% point and between analyzing.

Be usually used in studying with clinical in an example of assay kit be radioimmunoassay (RIA) test kit.Hereinafter set forth inhibitor RIA.After successfully radioiodination and purifying inhibitor or inhibitor peptide, will have the test tube that the antiserum(antisera) of high titre adds the radioactive activity that contains relative constant basis under several extent of dilution, suitable 10 in buffering system for example, 000cpm.Other test tube contains buffer reagent or preimmune serum to measure non-specific binding., add a-protein and rotate test tube after 24 hours in insulation under 4 ℃, at room temperature be incubated 90 minutes, then at 4 ℃ and the about antibody complex body of centrifugal antigen combination with the precipitation isolabeling under 2000-2500Xg.The sucking-off supernatant liquor is with the radioactive activity in γ calculating instrument counting centrifugal film.After the deduction non-specific binding, further identify in conjunction with the about antiserum(antisera) extent of dilution of 10%～40% mark peptide.

Then, add by the peptide with known quantity and contain radiolabeled peptide and sero-fast test tube and estimate be used to the dilution range of cultivating sero-fast inhibitor peptide (about 0.1pg～10ng).Insulation for example after 24～48 hours, adds a-protein again, and test tube is centrifugal, and the sucking-off supernatant liquor is also counted radioactive activity in centrifugal film.Provide typical curve with the combination of the radiolabeled inhibitor peptide of unlabelled inhibitor peptide (standard substance) displacement.Add analysis tube to identify the sero-fast specificity of this inhibitor other inhibitor peptide fragment of several concentration, the inhibitor that derives from different plant species and homology peptide.

The extract for preparing various tissues, these tissues include but not limited to culture, placenta, uterus and other tissue such as brain, liver and intestines nascent and secondary tumour, Lewis lung cancer, inhibitor production cell.After tissue extract freeze-drying or traditional vacuum evaporation concentration, add the analysis buffer reagent, different equal samples is placed in the RIA test tube.Inhibitor is produced the displacement curve that the extract of cell forms and is similar to typical curve, but the extract that does not produce the tissue of inhibitor can not be replaced radiolabeled inhibitor from inhibitor.In addition, the extract that derives from animal urine, blood plasma and the celiolymph of suffering from Lewis lung cancer is added analysis tube with the amount that increases progressively.Similar displacement curve shows the effectiveness aspect the inhibitor of this inhibitor analytical method in measuring tissue and body fluid.

The tissue extract that contains inhibitor is also analyzed equal sample by reversed-phase HPLC and is identified.Collect elutriated fraction, dry in SpeedVac, analyze with this inhibitor RIA again after rebuilding in the RIA buffer reagent.The immunoreactive maximum of inhibitor is arranged in the fraction corresponding to the wash-out position of inhibitor.

This assay kit furnish an explanation book, antiserum(antisera), inhibitor or inhibitor peptide, and might radiolabeled inhibitor and/or be used for the inhibitor of precipitation combination-inhibitor antibody complex body reagent.This test kit is applicable to measure in biofluid and suffers from and do not suffer from inhibitor in animal and human's tissue extract of tumour.

Another kind of test kit is used to the inhibitor in position tissue and cell.This inhibitor immunohistochemistry test kit book that furnishes an explanation, the inhibitor antiserum(antisera), and with fluorescence molecule such as fluorescein isothiocyanate or be used for showing possible blockade serum and the secondary antiserum(antisera) that elementary sero-fast some other reagent is connected.Those skilled in the art know immunohistochemistry technology.This inhibitor immunohistochemistry test kit permission applied optics microscopy and electron microscopy are come the inhibitor in position tissue section and culturing cell.It can be used for studies and clinical application.For example, tumour carried out examination of living tissue or gather tumour and carry out tissue slice with inspection inhibitor production site with slicing machine.This information is applicable to the diagnostic use of test-and-treat cancer aspect and possible treatment is used.The another kind of method in demonstration inhibitor biosynthesizing site comprises that radio-labeling is used for the nucleic acid of on-the-spot hybridization to survey the inhibitor messenger RNA(mRNA).Equally, this inhibitor acceptor can locate with immunohistochemistry technology, demonstration and quantitative analysis.

Embodiment

Further explain the present invention by following embodiment, but can not think that these embodiment are limited scope of the present invention by any way.Otherwise, should understand clearly that those skilled in the art can draw various other embodiment of the present invention, improved form and equivalents after reading this specification sheets.

Embodiment 1

With the active source of the endothelial cell proliferation inhibitor of the Kringle 5 of description of test human plasminogen: must Kringle 5 fragments with suitable enzymic digestion human plasminogen.Separate the standard method purifying that uses protein well known to those skilled in the art and peptide purification after this peptide fragment.The purity of Kringle 5 after separation as shown in Figure 2, this illustrates in gel electrophoresis the result of this peptide formulations of operation.

Analyze: by DNA in the thin endotheliocyte of ox hair synthetic ([methyl- ³H] the thymidine fusion) inhibition and analyze endothelial cell inhibiting activity.The capillary endotheliocyte is grown on the 48 hole microtiter plates that gelatin applies with the bovine adrenal preparation is rear.

The Kringle 5 that the difference amount has been separated adds culturing cell, then measures the variation of cell count.The percent change of cell count is got the graphic representation of Fig. 1 as the function described point of the amount that adds kringle 5 in culture.

Fig. 3 illustrates the comparison between similar 80 amino acid whose sequences in the

Kringle

1,2,3 that derives from human plasminogen, 4 and 5 districts.

Claims

1. the method for vitro inhibition endothelial cell proliferation, it comprises uses to the capillary endotheliocyte protein that the aminoacid sequence by Profibrinolysin molecule Kringle 5 peptides of significant quantity forms.

2. the process of claim 1 wherein that this protein comprises 80 amino acid.

3. the process of claim 1 wherein that the molecular weight of this protein is 14kD.

4. the Profibrinolysin that the process of claim 1 wherein is human plasminogen.

5. the process of claim 1 wherein that this protein is comprised of aminoacid sequence CMFGNGKGYRGKRATTVTGTPCQDWAAQEPHRHSIFTPETNPRAGLEKNYCRNPDG DVGGPWCYTTNPRKLYDYCDVPQ.

6. the process of claim 1 wherein that this protein is comprised of aminoacid sequence CMFGNGKGYRGKRATTVTGTPCQDWAAQEPHRHSIFTPETNPRAGLEKNYCRNPDG DVGGPWCYTTNPRKLYDYCDVPQC.

7. the protein that is comprised of the aminoacid sequence of Profibrinolysin molecule Kringle 5 peptides is for the preparation of the purposes in the medicine of the undesirable capillary endothelial cell proliferation in the individuality that suppresses to suffer from undesirable capillary endothelial cell proliferation.

8. the purposes of claim 7, wherein this protein comprises 80 amino acid.

9. the purposes of claim 7, wherein the molecular weight of this protein is 14kD.

10. the purposes of claim 7, Profibrinolysin wherein is human plasminogen.

11. the purposes of claim 7, wherein this protein is comprised of aminoacid sequence CMFGNGKGYRGKRATTVTGTPCQDWAAQEPHRHSIFTPETNPRAGLEKNYCRNPDG DVGGPWCYTTNPRKLYDYCDVPQC.

12. the purposes of claim 7, wherein this protein is comprised of aminoacid sequence CMFGNGKGYRGKRATTVTGTPCQDWAAQEPHRHSIFTPETNPRAGLEKNYCRNPDG DVGGPWCYTTNPRKLYDYCDVPQ.

13. the purposes of claim 7, individuality wherein refers to the people.

14. the purposes of claim 7, individuality wherein suffers from the disease of angiogenesis-mediated.

15. the purposes of claim 14, wherein the disease of angiogenesis-mediated refers to cancer.

16. the purposes of claim 15, cancer wherein is solid tumor.

17. a pharmaceutical composition that is used for inhibition of endothelial cell proliferation, it comprises a kind of protein of endothelial cell proliferation amount of suppression, and described protein is comprised of the aminoacid sequence of Profibrinolysin molecule Kringle 5 peptides.

18. the pharmaceutical composition of claim 17, wherein this protein comprises 80 amino acid.

19. the pharmaceutical composition of claim 17, wherein the molecular weight of this protein is 14kD.

20. the pharmaceutical composition of claim 17, Profibrinolysin wherein is human plasminogen.

21. the pharmaceutical composition of claim 17, wherein this protein is comprised of aminoacid sequence CMFGNGKGYRGKRATTVTGTPCQDWAAQEPHRHSIFTPETNPRAGLEKNYCRNPDG DVGGPWCYTTNPRKLYDYCDVPQC.

22. the pharmaceutical composition of claim 17, wherein this protein is comprised of aminoacid sequence CMFGNGKGYRGKRATTVTGTPCQDWAAQEPHRHSIFTPETNPRAGLEKNYCRNPDG DVGGPWCYTTNPRKLYDYCDVPQ.