CN1204343A - Endothelial cell proliferation inhibitor and method of use - Google Patents

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 present invention relates to novel endothelial cell proliferation inhibitor.This inhibitor can suppress the diseases related and adjusting angiogenesis of vasculogenesis.In addition, the present invention relates to measure the diagnositc analysis and the test kit of the inhibition dosage that exists in the biological fluid sample, relate to histological chemistry's test kit 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 the specific antibody stimulant and the antagonist of counter inhibitor acceptor, also relate to the cytotoxic agent that is connected with this inhibitor.

The term of Ying Yonging " vasculogenesis " refers to growth neovascularity in tissue or the organ herein, 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.The endotheliocyte and the perithelial cells that are surrounded by basement membrane form capillary vessel.Vasculogenesis starts from the erosion of the enzyme of endotheliocyte and white corpuscle release to basement membrane.The endotheliocyte that is lining in lumen of vessels then 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 mutually combines and forms the capillary vessel button loop, generates neovascularity.

The vasculogenesis that continues, regulated comes across in a large amount of morbid state, metastases and the 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 the vasculogenesis regulated to be classified as angiogenesis-dependent disease or vasculogenesis is diseases related.

Be set forth in 1971 (Folkman J., Tumor angiogenesis:Therapeutic implications., N.Engl.Jour.Med.285:1182-1186,1971) at first about " tumor growth is an angiogenesis-dependent " this hypothesis.In its simplest statement, set forth: " ' capture ' in case tumour occurs, the increase of the new capillary vessel on the tumour of being focused at must be arranged before then each tumor cell number increases." tumour ' captures ' the preceding blood vessel phase (prevascular phase) that is understood that tumor growth now, 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 notion to comprise:

(1) implant growth of tumor speed in the subcutaneous transparent chambers of mouse before forming neovascularity slowly and be linear, but speed is very fast and near exponential form after forming neovascularity.(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) grow in the isolation that blood vessel do not breed, the tumour in the dabbling organ 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 the intracorneal tumor growth rate of no blood vessel slowly and be linear, but after neovascularization, transfer the growth of exponential form to.(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, no blood vessel, and size is limited to＜1mm ³But in case implanted iris vescular bed, they will become neovascularization and ramp, just reach 16,000 times of their original volumes in 2 weeks.(Gimbrone M.A., Jr. etc., Tumor dormancy in vivo by prevention of neovascnlarization.J.Exp.Med.136:261-276)

(5) when tumour being implanted chick embryo villi chorioallantoic membrane, they are poor growth during＞72 hours no blood vessel phase, and mean diameter is no more than 0.93+0.29mm.Begin to form that tumour enlarges rapidly in 24 hours behind the neovascularity, 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 metastatic tumor shows metastatic tumor size unhomogeneity in the rabbit liver, but shows as the point cut-off of relative homogeneous at the size place that forms blood vessel.The tumour of diameter in 1mm do not have blood vessel usually, but just forms neovascularity greater than this diameter.(Lien W., Deng, The blood supply ofexperimental liver metastases.II.A microcirrculatory 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 the blood vessel is limited to＜1mm.During age in week, the island of 4-10% becomes neovascularization, grows the tumour of big vascularization from these islands at 6-7, and gross tumor volume is bigger more than 1000 times than the volume on the preceding 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 " remarkable or strong " suppress 3 kinds of people's growth of tumor that depend on VEGF as their the unique amboceptor of vasculogenesis (in nude mice).This antibody is in the external growth that does not suppress tumour 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 the growth of secretion bFGF as the mouse tumor of its unique vascularization amboceptor.This antibody is in the external growth that does not suppress tumour 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 the growth of primary tumor and metastatic tumor thereof by the growth that stimulates capillary endotheliocyte in the tumour.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 tumor growth and transfer in vivo, but active much smaller in extracorporeal suppression tumor cell propagation.Little 4 orders of magnitude during its inhibition tumor cell proliferation of the concentration ratio when it suppresses vascular endothelial cell proliferation half maximum value.(Ingber D, Deng, Angioinhibins:Synthetic ahalogues 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 with vasculogenesis.

(12) be transferred to Vitrea human retinoblastoma and develop into no blood vessel spherule, although these spheroplasts can be survived and combination ³H-thymidine (when from the eye that enucleates, removing during analyzed in vitro), but be limited to＜1mm ³

When (13) ovarian cancer is transferred to peritonaeum minimum no blood vessel white seed (1-3mm ³).Unless one or more becomes neovascularization, otherwise these grafts seldom the president to bigger.

(14) (Weidner N in the 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, Blumenfcld 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 neovascularity at the beginning and just cause damage to thicken, the danger of transfer increases.(Srivastava A, Deng, Theprognostic significance of tumor vascularity in intermediate thickness (0.76-4.0 mm thick) skin melanoma.Amer.J.Pathol.133:419-423,1988)

(16) in bladder cancer, the urine content of vasculogenesis peptide bFGF is to the situation of disease and degree sensitive indicator more than cytology.(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 tumour exists and also can not grow so.In morbid state, prevent that vasculogenesis from can avoid new capillary blood vessel system to invade and the damage that causes.Therapy at the control angiogenesis can cause eliminating or alleviating this class disease.

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 somatomedin in the pre-neoplastic and prevent the formation of capillary vessel in the tumour, thereby suppresses growth of tumor.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 the regeneration.Suppress that 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

The present invention includes the method that isolating Profibrinolysin Kringel 5 districts suppress the endothelium proliferation activity of using.Have active these isolating Kringle 5 peptide fragment of inhibition and comprise about 80 amino acid whose sequences: CMFGNGKGYRGKRATTVTGTPCQDWAAQEPHRHSIFTPETNPRAGLEKNYCRNPDG DVGGPWCYTTNPRKLYDYCDVPQ is 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

F=Glu L=Leu

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

The present invention also comprises diagnostic method and methods of treatment, is used for checking this inhibiting peptide of body fluid whether to exist, and the significant quantity of this compounds go up to(for) needs treatments is used this peptide with the patient who regulates endothelial cell proliferation or specifically in conjunction with the antibody of this peptide.In addition, this inhibiting peptide can use the compound that alleviates the inhibition effect of this peptide with test with propagation endotheliocyte culture external, promptly screens 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 about 80 amino acid whose peptide fragment of human plasminogen, and this segment is equivalent to start from kringle 5 districts of the amino acid 462 of human plasminogen basically.

Another object of the present invention provides treatment by endothelial cell proliferation, the disease of particularly vasculogenesis mediation and the method for process.

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

Another purpose of the present invention provides treatment by the disease of vasculogenesis mediation and the method and composition of process, and 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 provides the composition of the growth that is used for the treatment of or suppresses cancer.

An object of the present invention is to provide the production or the active compound of adjusting or analogue enztme, 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 the quantitative analysis body fluid.

Another object of the present invention provides the check or the method for prognosis of cancer.

Another object of the present invention provides and is used to show and the quantitative analysis composition in inhibitor bonded site in vivo and in vitro.

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

Another purpose of the present invention provides the cancer therapy with minimal side effect.

Another purpose of the present invention provides 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 provides the method for targeted delivery inhibitor dependency composition to privileged site.

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

Read and appraise following to disclosed embodiment detailed description and appended claim after will understand these purposes of the present invention and other purpose, feature and advantage.

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

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

Fig. 3 illustrates the Kringle 1,2,3,4 of human plasminogen and the amino acid scope in 5 districts.

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

Fig. 5 illustrates the inhibition effect of 5 pairs of ox endothelial cell proliferations of reorganization Kringle.

Composition provided by the invention and method be inhibition of endothelial cell proliferation effectively, regulates blood Pipe generates, and suppresses unwanted Angiogenesis, particularly the blood vessel life relevant with tumor growth Become. The present invention includes a kind of protein endothelial cell proliferation inhibitor, it is characterized in that and to derive from About 80 amino acid whose sequences of human plasminogen Kringle 5. This amino acid order of inhibitor The Lie Keyin kind is different and slightly change. Should understand the amino acid no in this activity inhibitor molecule Be variable, all answer so have all active close homology amino acid sequences of endothelium inhibition Think and be included among the present invention.

The present invention comprises energy by the human or animal who suffers from undesirable endothelial cell proliferation is used The composition of the about Kringle 5 of the human plasminogen of inhibition of endothelial cell proliferation in analyzed in vitro, And supplying method and composition are to treat by undesirable and not controlled epithelial cell proliferation such as blood Pipe generates disease and the process of mediation. 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 especially suitable Growth in treatment or inhibition tumour. The human or animal who suffers from front vascularization metastatic tumor is used this Inhibitor helps to prevent growth and the amplification of those tumours.

The present invention also comprises the dna sequence dna of the endothelial cell proliferation inhibitor of encoding, and contains in the coding The expression vector of the dna sequence dna of chrotoplast antiblastic, and contain one or more expression The cell of carrier (this expression vector contains the dna sequence dna of this inhibitor of encoding). The present invention advances one Step comprises gene therapy, and the dna sequence dna of this endothelial cell proliferation inhibitor of will encoding is whereby introduced The patient is to modify inhibitor content in vivo.

The present invention also comprises diagnostic method and kit, with check and mensuration biofluid and tissue In endothelial cell proliferation inhibitor, and this inhibitor in position tissue and the cell. This examining Disconnected method and kit can be the arbitrary structures that those of ordinary skills know. The present invention also Comprise the specific antibody of this endothelial cell proliferation inhibitor and part thereof, and suppress this endothelium The antibody of the combination of the specific antibody of inhibition of cell proliferation. These antibody can be polyclones Antibody or monoclonal antibody. The specific antibody of this endothelial cell proliferation inhibitor can be used for diagnosis To check existing and content of this inhibitor, it is diagnosable or prognosis is living by blood vessel in the kit Become the cancer of mediation or outbreak or the recurrence of Other diseases. Also can use endothelial cell to the human or animal The specific antibody of antiblastic is with this human or animal of immunity passively and anti-this inhibitor, in It is the inhibition that reduces Angiogenesis.

The present invention comprises that also diagnostic method and kit press down with endothelial cell proliferation in check and the body fluid The existence of the antibody of preparation combination and content thereof. This diagnostic method and kit can be this area The arbitrary structures that those of ordinary skill is known.

The present invention also comprises the CI receptor specific antibody, the acceptor knot of it and this inhibitor Merging is conducted suitable signal to cell, and plays excitant or antagonist.

The present invention also comprises inhibitor fragments of peptides and analog, their available taggings or With other molecule or protein labeling, be used for by some technical checkings and this inhibitor knot of demonstration The co-bit point, these technology include but not limited to positron emission tomography, autoradiography, stream Formula cytometry, radioactive ray receptor binding assay and immunohistochemistry.

These inhibitor peptides and analog also play excitant on the inhibitor acceptor and antagonist is done With, so strengthen or stop the biologically active of this endothelial cell proliferation inhibitor. This class peptide is used The acceptor molecule of being combined specifically in separation energy and this inhibitor.

The present invention also comprises the endothelial cell proliferation inhibitor that is used for the treatment of and studies, the inhibitor sheet Section, the specific antisera of this inhibitor, and the inhibitor acceptor that is connected with cytotoxic agent Excitant and receptor antagonist. Further, this inhibitor, its fragment, it special Property antiserum, inhibitor receptor agonist and inhibitor receptor antagonist all can connect on the bound drug The excipient that is subjected to, and optional slowly-releasing compound or composition such as biodegradable polymer and Matrix is to form therapeutic combination.

The present invention includes the molecular probe of ribonucleic acid and DNA, these two kinds of nucleic acid with This endothelial cell proliferation inhibitor transcribe with translate relevant. These molecular probes are applicable to check With the inhibitor biosynthesis of measuring in tissue and the cell.

More particularly, the present invention includes for checking and treating by endothelial cell proliferation such as blood vessel Generate disease mediation or associated and composition and the method for process. Have and suppress active Kringel 5 fragments of peptides that separate comprise about 80 amino acid whose sequences: CMFGNGKGYRGKRATTVTGTPCQDWAAQEPHRHSIFTP ETNPRAGLEKNYCRNPDGDVGGPWCYTTNPRKLYDYC DVPQ Wherein C=Cys Y=Tyr D=Asp 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 plasminogen such as human plasminogen, perhaps by chemistry or biological method (for example cell is cultivated, recombinant gene expression, and peptide is synthetic, and plasminogen or fibrinolysin External enzyme-catalyzed change is to generate activity inhibitor) synthetic. Recombinant technique comprises the application polymerase chain Reaction (PCR) is carried out gene magnification from the DNA source, and uses reverse transcriptase/PCR from RNA Gene magnification is carried out in the source.

The present invention also comprises: contain a kind of composition of carrier, this carrier contains the coding endothelial cell The dna sequence dna of antiblastic can be expressed this inhibition when wherein this carrier is present in the cell Agent; The composition that comprises a kind of cell, this cell contains a kind of carrier, and wherein this carrier contains The encode dna sequence dna of this inhibitor or its fragment or analog, and wherein this carrier is present in carefully Can express this inhibitor in the time of among the born of the same parents; And comprise the side that cell is implanted people or inhuman animal Method, this cell contains a kind of carrier, and wherein this carrier contains the dna sequence dna of this inhibitor of encoding, When being present in the cell, this carrier can express this inhibitor.

Term " basically similar " or " basically homology " are used for this inhibitor of expression When amino acid and nucleotide sequence, refer to this amino acid sequence, namely it has endothelial cell proliferation and presses down Active and the about molecular weight of system, it also has the height sequence homology with certain protein, institute State protein and have specific N terminal amino acid sequence disclosed herein; The endothelium that perhaps refers to encode is thin The nucleotide sequence of born of the same parents' antiblastic, described inhibitor have about molecular weight and and something High homology, 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% ammonia The base acid homology is more preferably at least about 95% amino acid identity. The term that this paper uses " in It is active that skin suppresses " refer to that certain molecule suppresses the ability of Angiogenesis generally, for example, becoming fiber There is the lower ability that suppresses the thin endothelial cell growth of ox hair in the culture in Porcine HGF.

The present invention also comprises the check of inhibitor in body fluid and the tissue, for example is intended to diagnosis or prognosis The disease of cancer. The inspection of inhibitor binding site and acceptor during the present invention also comprises cell and organizes Test. The present invention also comprises treatment or the disease of prevention Angiogenesis and the method for process, this class disease Disease includes but not limited to arthritis and tumour, uses following method to 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 excitant or antagonist, and/or the inhibitor spy Opposite sex antiserum or for the antiserum of inhibitor specific antisera. Other methods for the treatment of bag Draw together and use this inhibitor, its bioactive fragment, inhibitor analogue, the inhibitor specificity is anti-Serum, or the inhibitor receptor agonist and the antagonist that are connected with cytotoxic agent.

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

The present invention also comprises gene therapy, regulates whereby the base of this inhibitor of coding in patient body Cause. Transfer or DNA delivery are to the whole bag of tricks of cell with expressing gene product albumen matter, or title Be gene therapy, all be disclosed in Gene Transfer into Mammalian Somatic Cells In vivo, N.Yang, Crit.Rev.Biotechn.12 (4): 335-356 (1992), the document is also It is for referencial use to enter this paper. Gene therapy comprises mixes body cell with dna sequence dna or germ line cell is used for Treatment ex vivo or intravital. Gene therapy plays a part such, namely sub stituent because of, Increase normal or unusual gene function, and antagonism infectious disease and other pathology.

The countermeasure of processing these medical care problems with gene therapy comprises: therapeutic strategy, for example identify Damaged gene adds then functioning gene with the function of replacement defect gene or strengthens small and weak merit The gene of energy; Perhaps preventative strategies for example adds the gene of product albumen matter, and this protein can The treatment patient's condition maybe will make tissue or organ more responsive for therapeutic scheme. One of preventative strategies Example is the nucleotide sequence of this inhibitor of coding can be placed the patient, so anti-hemostatic tube is given birth to The generation that becomes; Perhaps can insert the gene that makes tumour cell more responsive to radiation, radiation should then Tumour will increase the lethality to tumour cell.

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

The gene transfer method that is used for gene therapy is classified as three major types-Physical (electroporation for example Method, direct gene transfer and particle bombardment), chemical method (based on the carrier of lipid, or other The non-viral carrier) and bioanalysis (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 intravenous. It is believed that this liposome/dna complex exists Be concentrated in the liver, they are delivered to DNA macrophage and Kupffer cell in liver. These are thin Born of the same parents can survive for a long time thereby can this DNA that sends of long-term expression. In addition, can be with carrier or gene " naked " DNA directly inject 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 Comprise: the ex vivo transgenosis, in vivo transgenosis and outer-gene shift. Coming In the transgenosis in body, cell is taken from the patient and is grown in cell culture. With DNA Be transfected into cell, weigh again patients with implantation after the cell quantity of this transfection enlarges. Turn at outer-gene In moving, the cell of conversion is the cell that grows in the culture, such as tissue culture cells, and not It is the specific cells that derives from specific patient. Transfection these " experimental cells ", the selection transfection Cell and expand after with their patients with implantations or use it for anything else in addition.

Intravital transgenosis comprises when these cells are in patient body introduces the patient with DNA Cell. Method comprises uses virus-mediated transgenosis, uses non-infectious virus with base Inject patient's site because sending into the patient or with naked DNA, so this DNA is with certain thin Born of the same parents' percentage is absorbed, and gene outcome protein is expressed in these cells. In addition, this paper Other method (such as the application of " particle gun ") of describing can be used to external insertion endothelial cell Antiblastic DNA or inhibitor are regulated sequence.

The chemical method of gene therapy can relate to a kind of compound based on lipid, not necessarily fat Plastid transports DNA and passes cell membrane. Lipofection agent or cytofectin are with the bear electricity The cation based on lipid of DNA combination, form that a species complex can pass cell membrane and incite somebody to action DNA sends into cell interior. Another kind of chemical method is used the encytosis based on acceptor, its bag Draw together ligands specific is combined with cell surface receptor and coating, again cell is passed in its transhipment Film. Part should be transported into cell by whole complex after DNA is combined again. With this ligand gene Complex injects blood flow, and the target cell that has then this receptor will be specifically in conjunction with this part also This part-dna complex is transported into this cell.

Many gene therapy methods are used viral vectors gene are inserted cell. For example, from The retroviral vector of already having used change in the method in the body with gene introduce periphery and Tumor-infiltrated lymphocyte, liver cell, epidermal cell, myocyte or other body cell. So After the cell of these changes is introduced the patient so that the gene outcome of the DNA that derives from insertion to be provided.

Viral vectors 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 that tissue-specific cis acting is transferred Joint element or promoter. Also can be extremely in vivo specific by on-the-spot DNA delivery or viral vectors Anatomical sites and reach this purpose. For example, in vivo transgenosis to blood vessel is by inciting somebody to action external The endothelial cell of transduction is implanted site selected on the arterial wall and is realized. Virus infections is also expressed The peripheral cell of this gene outcome. Viral vectors for example can directly be delivered to live body by conduit In the site, so only allow certain district by this virus infections, thereby provide long-term, the site is special The gene expression of the opposite sex. The in vivo transgenosis of application retroviral vector is also passed through will Altered virus is injected logical organotropic blood vessel and has been obtained card in breast tissue and hepatic tissue Real.

The viral vectors that already had been used to gene therapy includes but not limited to: retrovirus, its Its RNA virus is such as poliovirus or sindbis virus, adenovirus, the gland venereal disease of being correlated with Poison, herpesviral, SV40, vaccine and other dna virus. Copy deficient mouse reverse transcription Viral vectors is the gene transfer vector of extensive use. The murine leukemia retrovirus comprise with The compound single stranded RNA of core protein and polymerase (pol) is wrapped into albumen barycenter (gag) and quilt The glycoprotein coating (env) of determining host range surrounds. Retroviral genome structure comprises These gag, pol and env gene, they by 5 ' and 3 ' LTR (LTR) surround. The retrovirus system utilizes following truth, i.e. the virus structural protein of hypothesis supply is thin in packing Be transconfiguration in the born of the same parents system, then contain this 5 ' and minimum carrier and the packaging signal of 3 ' LTRs be enough to permit Permitted carrier package, infected and be integrated into target cell. The retroviral vector that is used for transgenosis Principal advantages comprise: in the cell of most kinds, effectively infect and gene expression, integrate Enter accurately single copy carrier of target cell chromosomal DNA, and this reverse transcription virus gene group Ease for operation.

Adenovirus comprises compound and line style, double-stranded DNA that wrapped up by capsid protein with core 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 under the virus of low titre, also have efficient infectivity.In addition, this virus is infective fully 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 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, promptly 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 the non-proliferative tissue, and do not worry that sudden change in cellular genome or the 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 DNA termly again and can in the genome of recipient cell, not occur sudden change to keep the gene product level.The nonconformity effect of foreign DNA s can allow to have several different foreign DNA constructions in the cell, and all these constructions are expressed the gene product that has nothing in common with each other.

The gene transfer method of particle mediation at first is used to transform 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 the transgenosis sensitivity of cell or tissue for the 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 the 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 to acceptor specific on some cell can be used as the facilitated method of DNA being inserted 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 the transgenosis in vivo.

The DNA of transfection also can be compound with other type of carrier, so this DNA is carried into recipient cell, retains in then in kytoplasm or the 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, promptly by using the gene bonded compound with 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, the dna sequence dna cells transfected with this inhibitor of coding can be applied to the patient so that intravital inhibitor source to be provided.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 the recombinant DNA molecules that comprises the endothelial cell proliferation inhibitor dna sequence dna is connected the expression vector that can express this inhibitor to form effectively with expression control sequenc.Cells transfected can be the cell of the healthy tissues that derives from the patient, patient's illing tissue, perhaps can right and wrong patient cell.

For example, the tumour cell available energy of extracing from the patient is expressed the carrier transfection of inhibitor protein matter of the present invention, returns and introduces the patient.Tumour cell after the transfection produces inhibitor in the patient amount can suppress growth of tumor.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 the biofluid.

Though do not want to be subjected to the constraint of following hypothesis, but think when tumour becomes angiogenic, it discharges one or more peptides (for example aFGF, bFGF, VEGF, IL-8, GM-CSF etc.) that generates blood vessel, they act near the primary tumor target endothelium partly from EV direction, and do not circulate (perhaps short 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 at the target capillary endothelium of distance primary tumor So Far Away from endovascular directive effect in metastatic tumor, and the continuation circulation.Therefore, when remote metastatic tumor may begin to activate vasculogenesis just, the inhibitor that near the capillary endothelium it is entered suppressed.

Use similar techniques and realize 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 finish, promptly 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 of this inhibitor sequence ' and the 3 ' DNA Oligonucleolide primers that produces, (4) use this inhibitor gene sequence of polymeric enzymatic amplification, (5) sequence that will increase is inserted appropriate carriers such as expression vector, (6) carrier that will contain gene inserts microorganism or other expression system that can express this inhibitor gene, and (7) separate the inhibitor of this recombinant production.Appropriate carriers comprises virus, bacterium and (for example yeast) eucaryon expression vector.As above technology has more completely been described in the laboratory manual, this handbook is " Molecular Cloning:A LaboratoryManual " Second Edition by Sambrook etc. for example, 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 a 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 the 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 for example pass through " Solid Phase Peptide Synthesis:A PracticalApproach " E.Atherton and R.C.Sheppard, IRL Press by technology synthetic peptide fragment well known in the art, Oxford, England.Can synthesize a lot of fragments connects together them then and forms bigger 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 organizing high-affinity bonded peptide fragment and on affinity column, separate receptors bind thing inhibitor.

This inhibitor is effective in cure in 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 of treatment vasculogenesis mediation, it uses the inhibitor of significant quantity, or its bioactive fragment, or generally has the segmental molectron of inhibitor of anti-angiogenesis activity, or inhibitor stimulant and antagonist.The disease of vasculogenesis mediation 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; Eye vasculogenesis disease, diabetic retinopathy for example, 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 the excessive stimulation of treatment 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 that it is disease such as the cat scratch disease (Rochele minalia quintosa) and the ulcer (ulcers) (helicobacter pylori (Helicobacterpylori)) of pathological examination that platform is treated with the vasculogenesis.

The synthetic peptide fragment of this inhibitor serves many purposes.With this peptide of receptors bind by radio-labeling and be used to adopt the 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 so that 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 finish 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 infusion cannula link coupled osmotic pump.The molectron of inhibitor antagonist can be used to increase the vascularization of tissue with the stimulator of vasculogenesis.This treatment plan provides the effective ways of eliminating metastatic carcinoma.

This inhibitor can be used in combination with other composition and the method for treatment disease.For example, tumour common available and this inhibitor bonded operation, radiation or embolic chemotherapy treatment can then be used this inhibitor with latent period that prolongs the micrometastasis knurl and the growth of stablizing and suppress any remaining primary tumor to the patient then.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 a kind of matrix that is made of the raw material that is generally polymkeric substance, 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)), poly-glycollide (polymkeric substance of oxyacetic acid), poly-(rac-Lactide-glycollide) (multipolymer of lactic acid and oxyacetic acid), 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, poly-glycollide or poly-(rac-Lactide-glycollide) (multipolymer of lactic acid and oxyacetic acid).

Vasculogenesis of the present invention-adjustment of treatment composition can be solid, liquid or aerosol, and can any known route of administration use.The example of solid therapeutic composition comprises ball, emulsifiable paste and implantable dose unit.But the ball dosage forms for oral administration, the therapeutic emulsifiable paste can topical application.Implantable dose unit can topical application, for example uses at tumor locus, perhaps implantable it so that discharge therapeutic vasculogenesis-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 the test kit content with this inhibitor content or inhibitor acceptor in check or quantitative analysis body fluid or the tissue in conjunction with the antibody of this inhibitor or inhibitor acceptor specifically.These test-results can be used for diagnosing or predicting the generation or the recurrence of the cancer and the disease of other vasculogenesis mediation.

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 round-robin inhibitor specific antibody.The patient who has this class round-robin counter inhibitor antibody grows probably kinds of tumors and cancer is 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 improve the content of circulation inhibitor effectively.

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 the system and come immune passively human or animal to realize by using.This methods of treatment pass through and placentation, and blood vessel generation aspect can be very important in the unusual ovulation of treatment.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.The anti-segmental this antiserum(antisera) of the specific Fab of this inhibitor of perfusion can prevent combining of this inhibitor and inhibitor antibody.Thereby by in and endogenous counter inhibitor antibody reach result of treatment by stoping inhibitor to combine with the Fab fragment of counter inhibitor.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 that the present invention is intended to comprise to have any derivative that endothelial cell proliferation suppresses active inhibitor.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 active protein of inhibition, and protein has the aminoacid replacement thing or be connected with sugar or other molecule 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 suppress active protein and protein fragments can protein isolating and purifying substantially and the form of protein fragments provide, they are in the synthetic pharmaceutically acceptable temper of the application known accord method of those of ordinary skills.These temper available standards approach are used.Usually, this based composition can be used by following approach: partial, 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 that medicine sends, 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, promptly be delivered to interested position and for example directly send into the transforming growth cancer or send blood vessel into this tumour by intubate.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 this inhibitor of about 0.5mg/kg to 500mg/kg.Depend on the transformation period of this inhibitor in particular animals or people, the frequency of administration of this inhibitor can be between every day, several be extremely weekly.Should understand that 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 in 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, partial (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 unit dosage form expediently and can prepare by usual drug technique.This class technology comprises activeconstituents and pharmaceutical carrier or vehicle bonded step.Substantially, can prepare this class temper like this, be about to activeconstituents and liquid carrier or solid-state carrier in small, broken bits or with the two equably, closely combine, the words that need then shape these goods.

The temper that suitable parenteral is used comprises: water-based and aseptic parenteral solution non-water, and it can contain oxidation inhibitor, buffer reagent, fungistat and can make the isoosmotic solute of acceptor blood of this temper and expection; 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 Mi Feng ampoule and bottle can be stored under cryodesiccated (freeze dried) condition, just only needs to 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 that 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, use the column chromatography purifying again.

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 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 in the position of this molecule outside area based on them and is easy in conjunction with antiserum(antisera).The aminoterminal of this inhibitor and carboxyl terminal, and the middle region of this molecule is presented at respectively between the 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 inhibitor deutero-peptide and other molecule coupling.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) carries out radio-labeling.Those skilled in the art know these coupling technologies.Tyrosine or Methionin adding can not possessed the fragment of these residues to promote the mark to reactive amino on the peptide and hydroxyl yet.The selection of coupling technology is based on obtainable functional group on the amino acid, and these functional groups include but not limited to amino, sulfydryl (sulfhydral), carboxyl, acid amides, phenol and imidazoles.Carry out the link coupled all ingredients and comprise glutaraldehyde, diazotizing p-diaminodiphenyl, carbodiimide and right-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 ¹²⁵I is to the radio-labeling of 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 the five equilibrium sample from each fraction and measure radioactive activity with the γ calculating instrument again.This method of pressing is with unreacted Na ¹²⁵I is separated 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.

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

Analysis derives from all serum samples of producing polyclonal antiserum or derives from manufacture order clones 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 antibody complex body precipitator method of application of radiation mark are wherein used a-protein, secondary antiserum(antisera), cold ethanol or gac-dextran, then measure active with the γ calculating instrument.The antiserum(antisera) of high titre is also used commercially available affinity column purifying.In affinity column, make inhibitor peptide and gel coupling.The antiserum(antisera) sample flow is crossed this post, and counter inhibitor antibody keeps combining with this post.These antibody of wash-out are collected and estimation titre and specificity then.

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

Measure the test kit of inhibitor and inhibitor acceptor, also belong to part of the present invention.Further check this antiserum(antisera), promptly have the highest titre and specificity and can detect in the extract of blood plasma, urine, tissue and the inhibitor peptide in the cell culture medium, with confirm to be easy to use test kit rapidly, reliable, sensitive and measure and locate inhibitor specifically.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, the microtiter plate method, for the bar or the measuring staff of the antibody coating that fast monitored is urinated or blood is used, 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 the activity curve to analyze on 20%, 50% and 80% the 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, under several extent of dilution, will have the test tube that the antiserum(antisera) of high titre adds the radioactive activity that contains relative constant basis, suitable 10 in the buffering system for example, 000cpm.Other test tube contains buffer reagent or preimmune serum to measure non-specific binding.4 ℃ down insulation add a-protein and also rotate test tube after 24 hours, at room temperature be incubated 90 minutes, centrifugal to precipitate the antigen bonded antibody complex body of isolabeling under 4 ℃ and about 2000-2500Xg again.The sucking-off supernatant liquor is with the radioactive activity in the γ calculating instrument counting centrifugal film.After the deduction non-specific binding, further identify antiserum(antisera) extent of dilution in conjunction with about 10%～40% mark peptide.

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

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 and analyze buffer reagent, different five equilibrium samples is placed 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 by branch samples such as reversed-phase HPLC analyses and identified.Collect elutriated fraction, dry in SpeedVac, analyze with this inhibitor RIA again after in the RIA buffer reagent, rebuilding.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 to precipitate bonded inhibitor-inhibitor antibody complex body reagent.This test kit is applicable to be measured in the 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 the cell.This inhibitor immunohistochemistry test kit book that furnishes an explanation, the inhibitor antiserum(antisera), and with fluorescence molecule such as fluorescein isothiocyanate or with possible blockade serum and the secondary antiserum(antisera) that are used to show 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 the culturing cell.It can be used for research and clinical application.For example, tumour is carried out examination of living tissue or gather tumour and carry out tissue slice to check the inhibitor production site with slicing machine.This information is applicable to and detects and the diagnostic use of treatment cancer aspect and possible treatment application.The another kind of method that shows 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.

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.The standard method purifying that separates utilization protein well known to those skilled in the art and peptide purification behind this peptide fragment.The purity of Kringle 5 after the 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 bovine adrenal preparation back.

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

Fig. 3 illustrates the comparison between similar 80 amino acid whose sequences in the Kringle 1,2,3,4 that derives from human plasminogen and 5 districts.

Claims (1)

1. compound, it comprises:

Molecular weight is about 14kD, has the protein of a N terminal amino acid sequence GPVGAGEPKCPLMVKVLDAV, and wherein said protein has the ability of inhibition of endothelial cell proliferation in analyzed in vitro.

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