CN1309663A

CN1309663A - Anti-angiogenic proteins and methods of use thereof

Info

Publication number: CN1309663A
Application number: CN99808686A
Authority: CN
Inventors: 拉格赫阿姆·凯卢里
Original assignee: Beth Israel Deaconess Medical Center Inc
Current assignee: Beth Israel Deaconess Medical Center Inc
Priority date: 1998-06-17
Filing date: 1999-06-17
Publication date: 2001-08-22
Also published as: AU4575599A; IL140239A0; EP1086129A1; JP2008019271A; JP2002517999A; KR20010053018A; JP2008301823A; WO1999065940A1; KR100682666B1; CA2331332A1; AU753249B2

Abstract

Proteins with anti-angiogenic properties are disclosed, and methods of using those proteins to inhibit angiogenesis.

Description

Anti-angiogenic proteins and application thereof

The application that relates to

The application requires U.S. Provisional Application 60/089,689, and (on June 17th, 1998), and all authority of U.S. Provisional Application 60/126,175 (on March 25th, 1999), their whole technology contents are incorporated in this paper by quoting from herein.

Background of invention

Basement membrane of blood vessel is formed (Timpl, R., 1996, modern cytobiology viewpoint 8:618-24) by macromolecular substance such as collagen protein, ln, heparan sulfate proteoglycan, fibronectin and sense of touch albumen.Collagen protein can promote cytoadherence, transfer, alienation, growth (Paulsson on the function, M., 1992, Crit.Rev.Biochem.Mol.Biol.27:93-127), and play an important role by the endotheliocyte breeding and the behavior fermentation of these functions in angiogenesis, angiogenic action is meant that neovascularity is organized in process (Madri, the J.A.et al. that forms in original vascular tissue, 1986, J.Histochem.Cytochem.34:85-91; Folkman.J., 1972, Ann.Surg.175:409-16).Angiogenic action is the process of a complexity, and it refers to growth, transfer, the breeding of endotheliocyte, and the endotheliocyte alienation that reaches growth becomes tubular structure, and forms the process of film-shaped matrix around the vascular tissue of growing.Angiogenic action also can be in some normal physiological phenomena in addition, as: injury repairing, uterine endometrium process of reconstruction vital role (Folkman, J.et al., 1995, J.Biol.Chem.267:10931-34).Clearly prove now vasculogenesis in process, work (Folkman, J., 1972, Ann.Surg.175:409-16 greater than the growth of several cubic millimeters solid tumor and transfer; Folkman, J., 1995, Nat.Med.1:27-31).Some studies show that, the metabolic inhibitor of collagen protein has the characteristic of angiogenesis inhibitor, confirmed film collagen protein synthesis, and deposition be very crucial theory (Maragoudakis, M.E.et al. to the generation and the growth of vascular tissue, 1994, Kidney Int 43:147-50; Haralabopoulos, G.C.et al., 1994, Lab.Invest.71:575-82).But collagen protein is still unclear in the composition and the accurate status in the vascularization of matrix membrane.

The present invention's general introduction

The present invention relates to the albumen in NC I district of the type protein alpha chain of angiogenesis inhibitor function.The invention particularly relates to Arresten, Canstatin, newfound albumen such as Tumstatin, and their fragment that biological activity (as blood vessel formation against function) arranged, mutant, analogue, homologue and their derivative, polymer (as dimer) and fusion rotein.These albumen have all comprised the C-terminal fragment in type albumen NC1 district.More precisely, Arrestem, Canstatin and Tumstatin are respectively the C-terminal fragments in the proteic α 1 of type, α 2, α 3 chains NC1 district separately.These three kinds of albumen all are monomeric proteins, and they suppress tumor growth in testing in vivo, also suppress the hyperplasia of capillary vessel in the external several experiments that comprise endothelium tubulose growth experiment.

Present invention includes through the Arresten (generally being also referred to as Arrestin) that separates and reorganization generates.Arresten comprises the NC1 district of α 1 chain of IV collagen protein, the activity that angiogenesis inhibitor (hyperplasia) arranged, also comprise and separate its fragment that the angiogenesis inhibitor function is arranged that obtains among the Arresten, fragment polymer, and the polynucleotide (nucleic acid) of coding anti-angiogenic proteins.The present invention also comprises and containing with isolating Arresten in addition, the fragment of its angiogenesis inhibitor, or the two is simultaneously as the composition of bioactive ingredients.Say that from another point of view it is sick as method for cancer to the invention provides in the treatment Mammals reproduction type, and one of characteristics of cancer are angiogenic activities, this method comprises Arresten or its fragment that comprises angiogenesis inhibitor to Mammals.The Arresten of angiogenesis inhibitor and fragment thereof also can be used to prevent the breeding of cellular invasion (transfer) or endotheliocyte.The present invention has also comprised the Arresten and the segmental antibody thereof of isolating anti-angiogenesis activity.

Present invention includes through the Canstatin that separates and reorganization generates.Canstatin comprises the NC1 district of α 2 chains of IV collagen protein, the activity that angiogenesis inhibitor (hyperplasia) arranged, the fragment that the angiogenesis inhibitor function is arranged that also comprises isolating Canstatin, the segmental polymer of isolating Canstatin and angiogenesis inhibitor, and the polynucleotide of coding anti-angiogenic proteins.The present invention also comprises and contains separative Canstatin in addition, the fragment of its angiogenesis inhibitor, or the two is simultaneously as the composition of bioactive ingredients.Say from another point of view, it is sick as method for cancer to the invention provides in the treatment Mammals reproduction type, and one of characteristics of cancer are angiogenic activities, and this method comprises Canstatin or its segmental composition that comprises angiogenesis inhibitor to Mammals.The Canstatin of angiogenesis inhibitor and fragment thereof also can be used to prevent the breeding of cellular invasion (transfer) or endotheliocyte.The present invention has also comprised the Canstatin and the segmental antibody thereof of isolating angiogenesis inhibitor.

The present invention has also comprised through the Tumstatin that separates and reorganization generates.Tumstatin comprises the NC1 district of α 3 chains of IV collagen protein, the activity that angiogenesis inhibitor (hyperplasia) arranged, also comprise the fragment that the angiogenesis inhibitor function is arranged of separating Tumstatin, separate Tumstatin and the segmental polymer of angiogenesis inhibitor, and the polynucleotide of coding anti-angiogenic proteins.The present invention also comprises and containing with Tumstatin in addition, the fragment of its angiogenesis inhibitor, or the two is simultaneously as the composition of bioactive ingredients.Say that from another point of view it is sick as method for cancer to the invention provides in the treatment Mammals reproduction type, and one of characteristics of cancer angiogenic activity, this method comprises Tumstatin or its segmental composition that contains angiogenesis inhibitor to Mammals.The Tumstatin of angiogenesis inhibitor and fragment thereof also can be used to prevent the breeding of cellular invasion (transfer) or endotheliocyte.The present invention also comprises the Tumstatin and the segmental antibody thereof of isolating angiogenesis inhibitor.

Brief description of drawings

Figure 1A, 1B are respectively people's type protein alpha 1 chain nucleotide sequence figure (Figure 1A, SEQ ID NO:1) and amino acid (Figure 1B, SEQ ID NO:2).Marked the position (SEQ ID NO:3) of forward and reverse primer among the figure, (SEQ ID NO:4).

Fig. 2 is Arresten cloning vector pET22b (+) structural diagrams.Forward primer (SEQID NO:3), the reverse primer (SEQ ID NO:4) and the insertion site of Arresten gene also mark.

Fig. 3 A, 3B be respectively Arresten and Endostatin contain useful ³The graphic representation of the endotheliocyte breeding of the thymus pyrimidine of H mark.X-coordinate is respectively Arresten (0～10 mcg/ml), Endostatin (0～10 mcg/ml); Ordinate zou is the quantity of endotheliocyte (CPAE).

Fig. 4 A, 4B, 4C, 4D are that Arresten and Endostatin are to containing usefulness ³The histogram of the influence of the endotheliocyte breeding of H mark thymus pyrimidine.Fig. 4 A, 4B, 4C are respectively that Arresten is that (4A, 4B), 0～10 mcg/ml (4C) is to 786-0, PC-3, the action diagram of three kinds of cells of HPEC for 0～50 mcg/ml.Fig. 4 D is the action diagram of the Endostatin of 0.1～10 mcg/ml to the A-498 cell.

Fig. 5 A, 5B, 5C are cover microphotograms.Shown that (2 mcg/ml, 5B), (20 mcg/ml are 5C) to inducing the action diagram of endotheliocyte transfer impact between people's umbilical region endotheliocyte of chemotactic through FBS for Endostatin for Arresten.5A the contrast figure that does not handle.

Fig. 6 is Fig. 5 result's a histogram.Fig. 6 shows the effect that Arresten (2 mcg/ml, 20 mcg/ml) and Endostatin (2.5 mcg/ml, 20 mcg/ml) shift the ECV-304 endotheliocyte respectively.

Fig. 7 is Arresten forms influence to interior skin pipe effect.The per-cent that tubular tissue forms is shown in the y axle, and the concentration of inhibition such as x axle show.Several groups of experiments are respectively: do not add (contrast ◆) whatever, add BSA (contrast △), add 7S district (contrast X), Arresten (■).

Fig. 8 A, 8B show the effect that Arresten forms interior skin pipe respectively.The concentration of Arresten is 0.8 mcg/ml among the 8B; 8A is contrast.

Fig. 9 A, 9B, 9C, 9D are Arresten and the Endostatin graphic representations to the influence of tumor growth in vivo.9A be depicted as use respectively Arresten (10 mg/kg,) and BSA (+) handles and the mouse interior tumor of contrast (●) from the situation of 700 cubic millimeters of growths; 9B is depicted as and uses 10 mg/kg Arresten () respectively, and the tumour after BSA (+) handles is from 100 cubic millimeters of growth patterns; Be to use 10 mg/kg Arresten () shown in the 9C, Endostatin (▲) handles and contrasts the situation of (●) middle tumour from 100 cubic millimeters of growths; 9D is depicted as with Arresten () and handles the situation of back tumour from 200 cubic millimeters of growths, and (●) is contrast.

Figure 10 A, 10B are respectively people's type protein alpha 2 chain Nucleotide (Fig.10A, SEQ ID NO:5) and amino acid whose sequence chart (Fig.10B, SEQ ID NO:6).Wherein the position of forward primer (SEQ ID NO:7) and reverse primer (SEQ ID NO:8) is also dated.

Figure 11 shows that the collection of illustrative plates of the cloning vector pET22b (+) of Canstatin, wherein the insertion site of forward primer (SEQ ID NO:7) and reverse primer (SEQ ID NO:8) and Canstatin gene is also dated.

(HEK293) (Figure 12 B, 12D) breeding influences figure to the Canstatin (x axle) that Figure 12 A, 12B, 12C, 12D are respectively different concns for 786-0, PC-3 to endotheliocyte (C-PAE) (Figure 12 A, Figure 12 C) and non-endotheliocyte.The detection of cell proliferation thymus pyrimidine (H ³) serve as a mark (and Figure 12 A, 12C) and methylene blue staining (12B 12D) observes.

Figure 13 is a histogram, be expressed as in the per unit area (y axle) respectively with not adding VEGF (not having VEGF or serum), add the cell that VEGF (1%FCS+10 nanograms/milliliter) handles, with 0.01 Canstatin (1%FCS+10 nanograms/milliliter VEGF, 0.01 mcg/ml Canstatin), reach the quantity of the endotheliocyte of the transfer of handling with 1.0 mcg/ml Canstatin (1%FCS+10 nanograms/milliliter VEGF and 1 mcg/ml Canstatin).

Figure 14 is for using BSA () respectively, and the endothelium tubing that Canstatin (■) and α 5NC1 (zero) handle forms the per-cent of quantity and contrast (y axle) formation.The longitudinal axis is represented the square root of the variance of mean value.

Figure 15 A, 15B, 15C, 15D is respectively Canstatin (■), (Figure 15 A is 15B) with 786-0 cell (Figure 15 C, gross tumor volume mark (y axle 15D) to the PC-3 cell for Endostatin (zero) and contrast (), 15A, 15B) and gross tumor volume (cubic millimeter, y axle, 15C, the line graph of influence 15D), X-axis are depicted as the fate after the processing.

Figure 16 A, 16B are respectively the sequence chart of people's type protein alpha 3 chain Nucleotide (Figure 16 A, SEQ ID NO:9) Yu amino acid (Figure 16 B, SEQ ID NO:10).The position and the Tumstatin333 of forward primer (SEQ ID NO:11) and reverse primer (SEQ ID NO:12) have also been indicated among the figure, the starting point of Tumstatin334 gene fragment and terminating point.

Figure 17 is cloning vector pET22b (+) collection of illustrative plates of Tumstatin.It is also dated that the gene of forward primer (SEQ ID NO:11) and reverse primer (SEQ ID NO:12) position and Tumstatin inserts the site in the way.

Figure 18 shows that the monomeric blunt end amino acid position of α 3 (IV) NC1 among the mutant Tumstatin N-53 of Tumstatin.Solid torus is 53 amino-acid residues under the N end-grain cutting from the Tumstatin when producing this mutant.Strong with two sulphur of short-term representative is to represent according to its situation about existing in α 1 (IV) NC1 and α 2 (IV) NC1.

Figure 19 A, 19B, 19C are respectively the H that contains with different concns Tumstatin processing ³The C-PAE cell of mark thymus pyrimidine (Figure 19 A), PC-3 cell (Figure 19 B), the situation of 786-0 cell (Figure 19 C).Every class value all contains three repetitions.

Figure 20 is different concns (x axle) Tumstatin (●), BSA (contrast), 7S district (contrast zero) internally influence of skin pipe formation.

Figure 21 A, 21B be for Tumstatin (●), Endostatin (zero) handle with respect to impinge upon processing not on the same day (x axle) back to the line graph of the influence (y axle, cubic millimeter) of gross tumor volume.Data point with the asterisk mark has significance, its P＜0.05.

Figure 22 is control mice () and handles the growth pattern (y axle) of back tumour with Tumstatin mutant (●) that the x axle is the fate after handling.Data with the asterisk mark have significance, its P＜0.05.

Figure 23 is respectively the Arresten (●) of different concns, Canstatin (zero), and 12 kDaArresten fragments (■), 8 kDa Arresten fragments (), 10 kDa Canstatin fragments (▲) are the restraining effect of skin pipe formation internally.

Figure 24 is respectively the Tumstatin333 (●) of different concns, and Tumstatin 334 (zero), BSA (■ contrast), and α 6 (contrast), Tumstatin (▲) (x axle) be the restraining effect (y axle) of skin pipe formation internally.

Detailed description of the present invention

Unwanted Angiogenesis often causes numerous disease, in other words, if possible In growth or the expansion of specific condition, specific time or tissue prevention capillary, Numerous disease or undesirable condition can be prevented or be alleviated. The tissue of matrix membrane depends on the IV type The formation of collagen net, and this collagen network structure may pass through the IV collagen The non-collagen spherula of carbon teminal (NC1 district) forms (Timpl.R., 1996, Curr Opin Cell Biol 8:618-24; Timpl.R.et al., 1981, Eur.J.Biochem.120:203-211). Type Ⅳ collagen albumen is made up of six different gene outcomes, called after α 1, and α 2, and α 3, α 4, and α 5, α 6 (Prockop, D.J.et al., 1995, Annu.Rev.Biochem. 64:403-34). α 1, and the same merit body of α 2 is general just to be present in people's the matrix membrane, and other Four kinds show as conditional distribution (Kalluri, R.et al., 1997, J.Clin.Invest. 99:2470-8).

Be the angiogenic effect by depositing the new capillary that forms organizationally, to tumour Growth is necessary (Folkman, R., et al., 1992, J.Biol.Chem. with shifting 267:10931-4; Folkman, J.1995, Nat.Med.1:27-31; Hanahan, D.et al., 1996, Cell 86:353-64). The tumour of humans and animals is blood vessel not to take place turn into when beginning With, yet when the volume of tumour during greater than several cubic millimeters, vascularization just takes place (Folkman, J.1995.Nat.Med.1:27-31; Hanahan, D.et al., 1996, Cell 86:353-64). Change the adjusted that son need to take place to activate to blood vessel the Angiogenesis phenotype into The lower adjusting sub with the angiogenesis function inhibition (Folkman, J.1995, Nat.Med.1:27-31). Tubulose endothelial growth factors (VEGF) and basic fibroblast growth factor (bFGF) It is the angiogenesis factor of the most generally expressing in the tumour. The tumour of Angiogenesis may be crossed scale Reach one or more angiogenesis factors, can work in coordination with like this growth that promotes tumour. Use acceptor It is to be not enough to stop tumour that antagonist suppresses a kind of angiogenesis factor (such as VEGF) Growth. Recently, some Angiogenesiss suppress son and are found, and some are such as IFN-a, blood Platelet factor-4 (Maione, T.E.et al., 1990, Science 247:77-9), and PEX (Brooks, P.C.et al., 1998, Cell 92:391-400) there is no endogenous with tumour cell Contact, Angiostatin (O ' Reilly, M.S.et al., 1994, Cell 19:315-28) and Endostatin (O ' Reilly, M.S.et al., 1997, Cell 88:277-85) really by tumor group Knit inhibition that produce and Angiogenesis Tumor-assaciated itself. Although endogenous with these Growth and transfer that Angiogenesis suppresses son treatment tumour are quite effective, and are to attract The people's, but some potential problems relevant with the blood vessel formation against function therapy must be considered. Especially must consider carefully be the hysteresis toxicity that causes of chronic angiogenesis treatment and The undesired wound repair that causes in the therapeutic process and blood vessel be hyperplasia again.

The invention describes albumen with blood vessel formation against function characteristic, fragment, analog, Derivative, homologue and mutant, also described with these albumen, fragment, analog, The treatment of the proliferative disease that derivative, homologue and mutant inhibition Angiogenesis cause Method. These albumen include NC1 district or its part of the α chain of type Ⅳ collagen albumen, More specifically, include the α 1 of type Ⅳ collagen albumen, α 2, the list in the NC1 district of α 3 chains Body. These albumen, especially free state can stop the growth of tumour, in vivo at body Also organize the formation of capillary in the experiment of outer models, these experiments have comprised subcutaneous Tube generates experiment.

These albumen also may also comprise the coupling part in NC1 district. α 1, and α 2, and α 3 chains are Preferably, show as experiment that α 4, α 5, and α 6 chains do not have or only have very low anti-blood The activity of pipe nucleus formation. Generally speaking, the albumen that the preferred monomers form exists is as having Evidence shows that the albumen of six aggressiveness forms does not almost have activity or activity very low.

More particularly, the invention describes the albumen of a called after Arresten, it is about 230 amino acid whose albumen, with the nitrogen terminal amino acid of human type albumen NC1 district α 1 chain is identical (Hostikka, S.L.et al., 1998, J.Biol.Chem 263:19488-93).

What this paper had also described is, people's Arresten can express in E.coli by the bacterial expression plasmid as pET22b, and these expression plasmids can pass pericentral siphon and form soluble albumen.The fusion rotein that 29 kDa of 6 Histidines are arranged for carbon teminal behind this protein expression.Surpass 26 kDa, 3 kDa in addition and result from polylinker and histidine mark.When with eukaryotic vector pcDNA3.1, Arresten is expressed as the secretory protein in 293 nephrocytes, and the albumen that produces with 293 nephrocytes does not also have purifying not observe mark at present yet.

The Arresren that E.coli produces can the dose dependent mode suppresses the breeding of the endotheliocyte that bFGF stimulates, its ED ₅₀Be 0.25 mcg/ml, and for kidney cancer cell (786-0), prostate cancer cell (PC-3), the breeding of human prostate endotheliocyte (HPEC) is significantly effect not.Endostatin suppresses the C-PAE cell proliferation, and its ED50 is 0.75 mcg/ml, is 3 times of Arresten, and the A-498 cancer cells is not had restraining effect.

As described previously, Arresten suppresses to show that Arresten brings into play its function by the albumen of cell surface or acceptor to the specificity of endotheliocyte breeding and transfer.Inhibition explanation Arresten to matrix metalloproteinase (MMP) has direct effect in suppressing tumor growth, transfer process, be similar to batimastat (BB-94) and marimastat (BB-2516).

In the present invention, Canstatin is the NC1 district of type protein alpha 2 chains, is used to suppress angiogenic action, can suppress in test the breeding and the transfer of endotheliocyte, and the generation of endothelium pipe.Endotheliocyte breeding and the specificity that shifts are suppressed the function that proof Canstatin has angiogenesis inhibitor, and it may work by the acceptor or the albumen of cell surface.Integral protein is because of they binding abilities at extracellular matrix, and the ability of regulating cell proliferation and transfer to be arranged and be considered to may be the acceptor of cell surface Canstatin.Especially, the avb3 integral protein is exactly the Canstatin acceptor probably, because it is induced in angiogenesis, and multiple bonded ability is arranged.

In the present invention, Tumstatin, i.e. the NC1 district of type protein alpha 3 chains (Timpl, R.et al., 1981, Eur.J.Biochem.120:203-11; Turner, N.et al., 1992, J.Clin.Invest 89:592-601), be used in vivo to regulate in the model with external vasculogenesis and tumor growth the formation of vascular endothelial cell breeding and vascular tissue.The distribution of α 3 (IV) chain only limits to resemble GBM, the matrix membrane of ear nest in some particular substrate films, eye matrix membrane such as preceding lens film, the Descemet film, ovary and testis matrix membrane (Frojdman, K.et al., 1998, Differentiation 83:125-30), alveolar capillary matrix film (Kashtan, C.E., 1998, J.Am.Soc.Nephrol.9:1736-50).Yet, in the kidney vascular system, dimension pipe matrix membrane and subcutis matrix membrane, the but not distribution of these α 3 chains in the dimension pipe matrix membrane of liver (Kashtan, C.E., 1998, J.Am.Soc.Nephrol.9:1736-50).In the callus process, be that the proteic α chain of type (not comprising α 3, α 4 chains) can assemble form new capillary vessel, because α 3, α 4 chains are not the compositions of the vascular system that promptly is pre-existing in of original matrix membrane.Because α 3 (IV) chain is not original integral part of normal people's skin, so with after the Tumstatin processing, the aggegation of collagen protein and vasculogenesis can not change in the callus repair process.

α 3 (IV) chain both can in people's kidney vascular stroma film, express and also can in GBM, express (Kalluri, R.et al., 1997, J.Clin.Invest.99:2470-8).These original vascular systems may participate in the formation such as the renal cell carcinoma of pronephros tumour.Tumstatin combines to disturb new angiopoietic mode by α 3 chains and other α chain, effectively treats former tumor of kidney.Be diagnosed as renal cell carcinoma (Mulders, P.et al., 1997, Cancer Res.57:5189-95) nearly 30,000 people of the U.S. in 1996, and very pessimistic to the prediction case of this disease diffusion.Although radiation therapy and chemotherapy have had progress, patient after receiving treatment, do not rise appreciably the life-span (Mulders, P.et al., 1997, Cancer Res.57:5189-95).Lack the outstanding importance that develops new methods of treatment that embodied of the effective ways for the treatment of renal cell carcinoma.Consider this reality, on several animal model experiments, will control new vascularization recently and obtain comparatively ideal effect (Baillie, C.T.et al., 1995, Br.J.Cancer 72:257-67 as target; Burrows, F.J.et al., 1994, Pharmacol.Ther.64:1 55-74; Thorpe, P.E.et al., 1995, Breast CancerRes.Treat 36:237-51).Tumstatin shows that to the growth that suppresses renal cell carcinoma in the body it has very big potential to the treatment (inhibition) of this class tumor-blood-vessel growth.

Among the present invention, Tumstatin suppresses the breeding of endotheliocyte specifically, and external to PC-3, the breeding of 786-0 tumour cell does not have effect.Although Tumstatin does not suppress the transfer of endotheliocyte, it is in the external formation that obviously suppresses the endothelium pipe.These results show that tumstatin has suppressed the formation of neovascularity by the different step in the inhibition angiogenesis.

In the research, Tumstatin suppresses blood vessel and increases in the matrigel test, suppress PC-3 in mouse xenotransplantation, the 786-0 growth of tumor in vivo.Suppressing growth of tumor with Tumstatin, is optimistic, particularly considers clinical treatment tumour.

Because Tumstatin has the pathogen antigen decision position of Goodpasture disease, and the Goodpasture disease is a kind of autonomous immune disorders (Butkowski that shows as pulmonary apoplexy and acute glomerulonephritis, R.J.et al., 1987, J.Biol.Chem.262:7874-77; Saus, J.et al., 1988, J.Biol.Chem.263:13374-80; Kalluri, R.et al., 1991, J.Biol.Chem.266:24018-24), so short-term or can bring out this disease with Tumstatin treatment vasculogenesis for a long time.Several scientific research groups have attempted the sick autonomous position of epitope in α 3 (IV) NC1 district of Goodpasture positioned, and reported that nitrogen end parts, middle portion, carbon teminal part all have this epitope (Kalluri, R.et al., 1995, J.Am.Soc.Nephrol.6:1178-85; Kalluri, R.et al., 1996, J.Biol.Chem.271:9062-8; Levy, J.B.et al., 1997, J.Am.Soc.Nephrol.8:1698-1705; Quinones, S.et al., 1992, J.Biol.Chem.267:19780-4; Kefalides, N.A.et al., 1993, Kidney Int.43-94-100; Netzer, K.O.et al., 1999, J.Biol.Chem.274:11267-74).Reported recently autonomous antibody only be associated with the kidney survival rate of utilizing reorganization embedding and body with the reacting phenomenon of α 3 (IV) NC1 district nitrogen end (Hellmark, T.et al., 1994, Kidney Int, 55:936-44).The 40 amino acid places that this sick antigen decision bit is positioned at nitrogen end end also are confirmed.The Tumstatin of blunt end is synthetic, and it has lacked 53 amino-acid residues of nitrogen end because of cutting the antigen decision bit, and this molecule has the obvious suppression effect to the growth of 786-0 tumour in the mouse xenotransplantation.In addition, the antibody that extracts from several patients Goodpasture of this molecule discord combines.These presentation of results have lacked the fragment that nitrogen end 53 amino acid whose Tumstatin still contain angiogenesis inhibitor.

The specificity that Tumstatin breeds endotheliocyte suppresses explanation, and it may work by the albumen or the acceptor of cell surface.The endotheliocyte glue that vasculogenesis also depends on by integral protein avb3 mediation connects (Brooks, P.C.et al., 1994, Cell 79:1157-64).Tumstain may be by disturbing procreation endotheliocyte and the interaction between the matrix components and endotheliocyte is squeezed in the eubolism of programmed cell death (Re, F.et al., 1994, J.Cell.Biol.127:537-46).Matrix metalloproteinase (MMP ' s) be considered to as be adjusted in the main enzyme that neovascularity generates in the tumour (Ray, J.M.et al., 1994, Eur.Respir.J.7:2062-72).Proof is recently, and inhibition of MMP-2 (PEX) can hinder growth of tumor (Brooks, P.C.et al., Cell 92:391-400) by suppressing vasculogenesis.Tumstatin may work by the activity that suppresses MMP.

No matter in vivo or external, the result that Tumstatin suppresses vasculogenesis stops growth of tumor.For Tumstatin is introduced clinical treatment, must consider its toxicity and side effect in systematic treating.And the distribution of Tumstatin has limitation, and the fact that does not especially generally contain Tumstatin in the sub-endothelial matrix film shows, and is very little with the side effect of Tumstatin treatment vasculogenesis.In addition, the tumour that exists the Tumstatin explanation effectively to treat in the minority organ with it in the vascular stroma film in certain organs (as kidney) has suitable potential.Certainly, finally it is desirable to utilize the transgenosis means to realize tuimstatin gene expression in vivo (Kashihara, N.et al., 1997, Exp.Nephrol.5:126-31 in the tumor vessel tissue; Maeshima, Y.et al., 1996, J.Am.Soc.Nephrol.7:2219-29; Maeshima, T.et al., 1998, J.Clin.Invest.101:2589-97).

The distribution of α 3 (IV) chain is defined in the matrix membrane of certain organs, considers that it may suppress the aggegation of α chain, and the possible toxicity of Tumstatin is very little.In addition α 3 (IV) chain in the vascular stroma film of kidney, also observe (Kalluri, R.et al., 1997, J.Clin.Invest.99:2470-8), and these blood vessels may participate in the generation of former nephroncus (as renal cell carcinoma).Therefore Tumstatin may be by upsetting α 3 (IV) chain and other α interchain in conjunction with effectively suppressing this class tumour.

In this article, term " blood vessel is had an effect " (vasculogenesis) refers to form new blood vessel in tissue and organ, and relates to the breeding of endotheliocyte.Under normal physiological conditions, the human or animal only just has vasculogenesis in very specific period.For example in injury repairing, fetal development, substance luteum, uterine endometrium and placenta, all can be observed the vasculogenesis phenomenon.Term " endothelium " is meant the flat endotheliocyte of the one deck that rearranges serum chamber, lymphatic vessel, blood vessel.Therefore, " anti-angiogenesis activity " is meant the ability of the inhibition angiogenic growth that a material is had.Vessel growth is the process of a complexity, comprise the matrix membrane location cracking that is positioned under the endotheliocyte, the breeding of corresponding epidermic cell, and be transferred to and form the position of blood vessel, these cells are formed the neovascularity film, the termination of endotheliocyte breeding and adventitial cell and other are supported the insertion of the cell of new vessel wall." anti-angiogenesis activity " refers to disturb a step wherein, a few step or Overall Steps in this article, and neovascularization is suppressed.

Anti-angiogenesis activity may comprise that also endothelium suppresses active, subcutaneously suppress the active material that is often referred to and have the ability that suppresses vasculogenesis, suppress the growth or the transfer of the capillary endothelial cell of ox when in substratum, having the inoblast factor, angiogenesis factor or other somatomedin." somatomedin " is the material that a class promotes cell growth, breeding or synthesis capability." vasculogenesis correlation factor " refers to or suppresses or promote the factor of vasculogenesis.One example of blood vessel generation correlation factor is exactly a blood vessel generation somatomedin, and as basic fibroblast growth factor, it is the factor of impelling of vasculogenesis.Another example of blood vessel generation correlation factor is that angiogenic action suppresses son, for example: angiostatin (referring to U.S.Pat.No.5,801,012; U.S.Pat.No.5,837,682; U.S.Pat.No.5,733,876; U.S.Pat.No.5,776,704; U.S.Pat.No.5,639,725; U.S.Pat.No.5,792,845; WO 96/35774; WO 95/29242; WO 97/23500) or endostatin (WO 97/15666).

" identical substantially biological activity " is meant a material aspect angiogenesis inhibitor, to Arresten, Canstatin, Tumstatin similar biological activity arranged in standard test." standard test " includes but not limited to, be used for detecting the measuring and calculating anti-angiogenesis activity in molecular biology, the method for cell cycle stagnation and programmed cell death.These experiments include but not limited to: the endotheliocyte breeding, endotheliocyte shifts, cell cycle is analyzed, the endothelium pipe forms, the detection of programmed cell death, just procedural cell detection or AnnexinV-FITC experiment, chorioallantoic membrane (CAM) experiment, and the inhibition of kidney tumor growth is tested in the mouse.These methods see following giving an example for details.

In this article, Arresten is also writing Arrestin, comprise fragment, mutant, homologue, the analogue of Arresten, the equipotential mutation of aminoacid sequence, the equipotential mutation of the Arresten that comes from other animal, and fragment in addition, mutant, homologue, analogue, aminoacid sequence.

In this article, Canstatin comprises fragment, mutant, homologue, the analogue of Canstatin, the equipotential mutation of aminoacid sequence, the equipotential mutation of the Canstatin that comes from other animal, and fragment in addition, mutant, homologue, analogue, aminoacid sequence.

In this article, Tumstatin comprises fragment, mutant, homologue, the analogue of Tumstatin, the equipotential mutation of aminoacid sequence, the equipotential mutation of the Tumstatin that comes from other animal, and fragment in addition, mutant, homologue, analogue, aminoacid sequence.

It should be understood that the intent of the present invention is to comprise that endothelial tissue suppresses active Arresten, Canstatin, all derivatives of Tumstatin.That is to say has the arbitrary substance that suppresses angiogenic activity usually, and the capillary vessel growth or the transfer that suppress ox under fibroblast growth factor, angiogenic growth correlation factor or other the known somatomedin existence condition are for example arranged in substratum.The present invention includes complete Arresten, Canstatin or Tumstatin albumen, reach these proteic derivatives and bioactive protein fragments is arranged.These have the amino acid replacement that has that Arresten, Canstatin, the active albumen of Tumstatin has, and have plenty of that glycosyl or other molecule are additional to be connected in the amino acid group.

The present invention has also described Arresten, Canstatin, the fragment of Tumstatin, mutant, homologue, analogue.The fragment of Arresten, Canstatin, Tumstatin be meant form to go up less than complete Arresten, Canstatin,, Tumstatin and comprise at least 25 continuous Arresten, Canstatin, the amino acid whose polypeptide of Tumstatin.These molecules may also comprise or not comprise additional aminoacid sequence in clone's process, and for example, the sequence of amino-acid residue is consistent with clone's connector sequence complete or part.No matter the mutant that the present invention includes is with or without additional amino-acid residue, must identical biologic activity be arranged with the isometric albumen of natural or original polypeptide.

A fragment wherein, called after Tumstatin N-53, being found in standard test with complete Tumstatin has identical anti-angiogenesis activity.Tumstatin N-53 has comprised that original complete Tumstatin albumen removes the part of 53 amino-acid residues of nitrogen end.The anti-angiogenesis activity that high level is also arranged in the experiment that other mutant of putting down in writing among the present invention writes down herein.These fragments such as Tumstatin 333, Tumstatin 334,12 kDaArresten fragments, 8 kDa Arresten fragments, 10 kDa Canstatin fragments, their ED50 value is respectively 75,20,50,50,80 nanograms/milliliter.And complete Arresten, Canstatin, Tumstatin are respectively 400,400,550 nanograms/milliliter.Tumstatin333 comprises the 2nd to 125 amino acid of SEQ ID NO:10, and Tumstatin 334 comprises the 126th to 245 amino acid of SEQ ID NO:10.

The mutant of Arresten, Canstatin, Tumstatin is meant the polypeptide that any variation is arranged on original amino acid acid sequence.These changes may form under field conditions (factors), also may be by manually obtaining, as chemical mutation, genetic engineering, hybridization or the exchange of other form genetic material of chemical energy (X ray) or other form.These mutant comprise base change, disappearance, insertion, inversion, be shifted, duplicate etc.The mutant forms of Arresten, Canstatin, Tumstatin is compared with original Arresten, Canstatin, Tumstatin polypeptide, its anti-angiogenesis activity can show as to be increased or reduces, these mutant also may comprise or be not included in additional amino-acid residue in clone's process, for example, with the part or all of identical amino-acid residue or the sequence of connector sequence.

Mutant/the fragment of anti-angiogenic proteins can produce by the PCR clone among the present invention.The fragment of called after Tumstatin 333 and Tumstatin 334 generates with this method, and the former total length Tumstatin albumen of the specific activity of its angiogenesis inhibitor is taller, as the example 18 of bottom, shown in Figure 23,24.For obtaining this fragment, the PCR primer must be from the known array design so that every pair of primer all can expand known fragment down from whole protein gene.Fragment after the amplification further is cloned on the suitable expression vector, and as carrier pET22b, the activity of expressed proteic angiogenesis inhibitor is to detect as described in the experiment of back.

The mutant of anti-angiogenic proteins (fragment) can also obtain by the elastin enzymic digestion of pseudomonas (Pseudomonas) among the present invention, resemble Mariyama, M.et al. (1992, J.Biol.Chem.267:1253-8) and the example 24 of back said like that.Obtained 12 kDa in this way, the Canstatin mutant of 8 kDa and 10 kDa, all these three kinds of mutant are more taller than original complete albumen on anti-angiogenesis activity.

The analogue of Arresten, Canstatin or Tumstatin is meant and complete molecule or its fragment, quite similar non-natural molecule on the equipotential mutation structure, and with former Arresten, Canstatin, Tumstatin have identical or higher biological activity.These analogues include the derivative of the derivative (chemical derivative for example defined above) of bioactive Arresten, Canstatin, Tumstatin and their fragment, mutant, homologue, equipotential mutation, and these derivatives are and the effect that coactivation mutually or antagonism are not arranged through Arresten, Canstatin, Tumstatin polypeptide, fragment, mutant, homologue, the equipotential mutation of modifying.

The equipotential mutation of Arresten, Canstatin or Tumstatin is meant with respect to Arresten, Canstatin or the spontaneous polypeptide that produces variation of Tumstatin standard polypeptid acid sequence.The equipotential mutation of the polynucleotide of polypeptide such as coding Arresten, Canstatin or Tumstatin is meant and contains the polynucleotide that sequence variation has taken place with the polynucleotide of the polypeptide of coding corresponding Arresten, Canstatin or Tumstatin, therefore, the equipotential sudden change of the polynucleotide of coding Arresten, Canstatin or the Tumstatin polypeptide equipotential mutain of Arresten, Canstatin or Tumstatin polypeptide of also encoding.

Also possible is, a certain given polypeptide may be single Arresten, Canstatin, Tumstatin fragment, mutant, analogue, equipotential mutation, also may be two or more in them, for example, the analogue of a certain polypeptide possibility existing Arresten, Canstatin, Tumstatin has its mutant again.Can in the laboratory, synthesize fully than original short Arresten, Canstatin, Tumstatin molecule as one.If this fragment is being undergone mutation through known method, Arresten, Canstatin, the Tumstatin fragment that this molecule not only had been but also be its mutant.After and for example producing a mutant, after prove that again it is the equipotential form of Arresten, Canstatin, Tumstatin in some Mammals, that this mutant also is the equipotential mutation simultaneously.Also will comprise the combination of these fragments, mutant, equipotential mutation, analogue among the present invention.

Attempt an example, the Tumstatin that produces with the escherichia coli cloning expression that mentions in the example 18 is a monomer.It still is a fusion rotein or chimeric protein, because in the process with the escherichia coli cloning expression, can add multi-link header sequence of external source and histidine mark on expressed proteins.The same Tumstatin fragment Tumstatin N-53 that describes in example 18 is the deletion mutant of intact proteins Tumstatin, also can add an appended sequence in the process with the escherichia coli cloning expression, so it also is fusion or the chimeric protein of complete Tumstatin.The inferior monomer of Tumstatin N-53 is connected (being dimer, tripolymer) will produce the proteic poly fusion of a Tumstatin or embedding and mutant fragment.

Comprised the albumen that similar aminoacid sequence is arranged with Arresten, Canstatin, Tumstatin among the present invention, or and the nucleic acid of coding Arresten, Canstatin, Tumstatin the polynucleotide of similar sequences is arranged." sequence about the same " refer to nucleic acid or polypeptide compare with standard sequence cause rare 70% identical; " similar especially " is at least 80% identical; " more similar " is 90%; " very similar " is 95%, and " the most similar " is at least 97%.The length of comparative sequences is generally the shortest to be 75 Nucleotide or 25 amino acid; Comparatively ideal 150 Nucleotide, 50 amino acid of being at least; Optimal is 243-264 Nucleotide, 81-88 amino acid.Polypeptide is meant amino acid whose molecular chain herein, is not the concrete length of special finger product.Therefore, peptide, oligopeptides, albumen include in the polypeptide scope.Polypeptide also comprises modified polypeptides after expressing, for example glycosylation, ethanoylization, phosphorylation or the like.

" sequence is identical " refers to that herein the similarity of subunit's sequence between 2 multimeric molecules compares, as 2 nucleic acid or 2 polypeptide.When being the same monomer subunit on a certain subunit position in the molecule (being Serine as 2 a certain positions of polypeptide chain), they are just identical on this position so.The similarity of two sequences is to match mutually or the direct embodiment of the quantity of same position, is identical if 2 peptide chains or compound sequence have half, and two sequences just have 50% similarity so; If all identical on 90% the position then have a sequence similarity of 90%.For example, sequence R ₂R ₅R ₇R ₁₀R ₆R ₃And R ₉R ₈R ₁R ₁₀R ₆R ₃, in 6 positions 3 identical, so similarity is 50% between the two; And R ₂R ₅R ₇R ₁₀R ₆R ₃And R ₈R ₁R ₁₀R ₆R ₃, in 5 positions 3 identical, the similarity between the two is 60%.And the similarity between the sequence be on the position identical or the coupling a kind of direct embodiment.Therefore, if a certain position of correlated series or part lack in a certain specific amino acids, the part of that disappearance just is not counted in the sequence similarity.As R ₂R ₅R ₇R ₁₀R ₆R ₃And R ₂R ₅R ₇R ₁₀R ₃, in 6 positions 5 identical, the two similarity is 83.3%.

Similarity is measured through sequence analysis software commonly used, as BLASTN or BLASTP (http: ∥ www.ncbi.nlm.nih.gov/BLAST/).(http: ∥ www.ncbi.nlm.nih.gov/gorf/b12.html) matching parameter is as follows between two sequences: coupling is 1, and not matching is-2, and opening 5 is arranged, and big breach 2 is arranged during to nucleotide analysis with BLASTN.During to sequential analysis of protein, coupling is 0 with BLASTP, and not matching is 0, and it is 11 that opening is arranged, and has big

" sequence homology " of two sequences is meant conservative substituting only taken place between the two that other is all identical.Concerning peptide sequence, conservative substituting comprises: the amino acid on the specific position (is for example had some characteristics of common: hydrophobicity, electric charge, other conformation or chemical property by similar other aminoacid replacement between the amino acid, replace bright peace acid as Xie Ansuan, aspargine replaces Methionin); Or do not change influence take place on bioactive conformation of polypeptide or the folding position that one or more non-conserved amino acids replace, disappearance or insert.The example of " conservative property replacement " comprises that a non-polar residue (hydrophobicity) is as replacing mutually between Isoleucine, Xie Ansuan, leucine or methionine(Met); Replace mutually between polarity residual (wetting ability) base: between leucine and Methionin, between glutaminase and the aspargine, between Threonine and the Serine; Phase trans-substitution between one alkaline residue such as Methionin, arginine, Histidine; Trans-substitution mutually between acidic residues such as aspartic acid and L-glutamic acid; Or the original amino acid of amino acid replacement after the chemical action modification; Prerequisite is that polypeptide must have strong biologic activity.Two have the sequence of homology to be called " sequence homologue ".

Concerning polypeptide, the homology sequence analysis software (as the sequence analysis software bag of Univ Wisconsin-Madison USA's biotechnology center heredity computer group, No. 1710, the big ways for education, Madison, WI 53705) measure.Analysis of protein software analysis similarity sequence be by give to substitute, disappearance or other modifies its degree of appointment and more similar sequence is finished.Conservative property substitutes and refers generally to substituting of following each group: glycine, L-Ala; Xie Ansuan, Isoleucine, leucine; Aspartic acid, L-glutamic acid, aspargine, glutaminase; Serine, Threonine; Methionin, arginine; Phenylalanine, tyrosine.

The chemical derivative that has also comprised Arresten, Canstatin, Tumstatin among the present invention." chemical derivative " refers to a polypeptide, and it has the chemically modified residue derivative that generates behind one or more residues and the functioning side radical reaction.These derivative residues comprise that its free amino group forms the molecule of amine through deriving: salt acidic group, p-toluenesulfonyl, benzyloxy carboxyl, fourth oxygen carboxyl, chloracetyl, formyl radical etc.Free carboxyl group generates salt after deriving, the ester of methyl or ethyl ester or other form or hydrazine class.Free hydroxyl can form O-acyl or O-alkyl derivative.The nitrogen imidazolyl of Histidine can generate N-to the phenmethyl Histidine.Chemical derivative also comprises the peptide that contains one or more 20 amino acid whose natural derivatives of routine, substitutes proline(Pro) as the 4-oxyproline; The 5-oxylysine substitutes Methionin; 3-Methyl histidine replaces Histidine; Homoserine is for Serine; Ornithine replaces Methionin.

The present invention also comprises and contains anti-angiogenic proteins, their fragment, mutant, homologue, analogue, the fusion rotein and the chimeric protein of equipotential mutation.Fusion rotein or chimeric protein can produce in recombinant expressed and clone's process, this albumen may contain additional amino acid or with the part or all of identical aminoacid sequence of connector.For example the Arresten among the present invention (sees example 2) when expression in escherichia coli, contains the carrier sequence that is added on this albumen, comprising histidine mark.In this article, term " fusion rotein or embedding and albumen " is included in this change on original aminoacid sequence basis.This change (seeing example 11 and 18 respectively) is also arranged among Canstatin and the Tumstatin.A certain fusion rotein or embedding and albumen can be single proteic polymer, for example repetitions of anti-angiogenic proteins; Fusion rotein or embedding and albumen also can be made of (for example, several anti-angiogenic proteins) several albumen.Fusion or embedding and albumen can be formed (as angiostatin and endostatin, or bioactive angiostatin and endostatin being arranged) by the linker of 2 or a plurality of known anti-angiogenic proteins; It also can be the combination (combination that endostatin and Urogastron EGF or RGD peptide chain are formed) that the anti-angiogenic proteins and the target factor are formed; Or the combination formed of anti-angiogenic proteins and immunoglobulin (Ig) (particularly removing the combination that the IgG after the Fc part forms) as endostatin and IgG.Fusion rotein and embedding and albumen also can comprise the combination that anti-angiogenic proteins, their fragment, mutant, homologue, analogue, equipotential mutation and other anti-angiogenic proteins are formed, as endostatin or angiostatin.Other anti-angiogenic proteins may also comprise restin and apomigren; The fragment of (PCT/US98/26058, it is instructed by being incorporated in this paper in this citation) and endostatin (PCT/US98/26057, it is instructed by being incorporated in this paper in this citation).Herein, term " fusion rotein " and " embedding and albumen " also comprise supplementary component, as are used to send the supplementary component of chemotherapeutics, and the nucleic acid of the chemotherapeutics of just encoding links to each other with the gene of coding anti-angiogenic proteins.Fusion rotein or embedding and albumen also comprise a kind of polymer of anti-angiogenic proteins, for example dimer or tripolymer.These fusion roteins or embedding and albumen can connect together by the modification after the translation, but are also produced by reorganization fully.

The present invention also comprises and contains Arresten, Canstatin, Tumstatin, their fragment, mutant, homologue, analogue, the multimeric protein of equipotential mutation." polymer " referred to comprise 2 or a plurality of replisome of a protein protomer.Proteic subunit can be the albumen (as Arresten dimerization or polymer) among the present invention, or fragment, mutant, homologue, analogue, equipotential mutation (for example Tumstatin mutant or fragment, as Tumstatin 333) repeat 2 times or more times.A polymer also can be the peptide chain of a fusion rotein or embedding and albumen (as: a multiple Tumstatin mutant may combine with multi-link sequence) and/or one or more anti-angiogenic proteins, this peptide chain exist with monomeric form or with the multiple form exist (as an albumen can by 2 or a plurality of polymer form).

The present invention has also comprised the composition of the polynucleotide that contains separative one or more coding Arresten, Canstatin, Tumstatin, and carrier and the host cell that contains this polynucleotide, and produce Arresten, Canstatin, Tumstatin, the method for their fragment, mutant, homologue, analogue, equipotential mutation.In this article, term " carrier " refers to the material that can insert or clone some nucleic acid therein, and its function is that this section nucleic acid that will insert is transferred in the host cell.The duplicating and/or expressing of the nucleic acid that can also bring like this is transferred.Some such carriers include from the nucleic acid of the virus of plasmid, phage, plant and animal material or insertion; The carrier that has is the carrier of non-viral, as part-nucleic acid binding substances, liposome, or fat-nucleic acid complex.May be for it is desirable to, but the nucleic acid molecule place of working of shifting is connected on the expression control sequenc, generate a kind of expression vector that can express this exogenous nucleic acid.This tranfer system of nucleic acid is called " conversion ", refers to exogenous nucleic acid sequences to be inserted in the host cell, no matter and inserted mode.For example: directly absorption, transduction or hybridization etc.Exogenous nucleic acid can exist with unconformable form, as a plasmid or other form; Also can be incorporated in the host genome." but place of working connection " showed and decided to connect by the mode that can exercise its predetermined function between component.For example: to an encoding sequence, the mode of connection that refers to them can obtain the expression of encoding sequence under the condition compatible with control sequence with a control sequence " but place of working connection "." encoding sequence " refers to that one section nucleotide sequence can be transcribed into mRNA earlier under suitable regulating and controlling sequence regulation and control, translate into polypeptide afterwards.The scope of encoding sequence is by the translation stop codon decision of the translation initiation codon and the 3 ' end of 5 ' end.This sequence length can be a nature, also can import or add nucleic acid fragment in a known manner.One encoding sequence may be including but not limited to the nucleotide sequence of mRNA, cDNA, reorganization.

There is the carrier of nucleic acid to select to wherein cloning, promptly in eucaryon or prokaryotic organism, expresses and select according to it.Two carriers that can clone and express arresten, canstatin, tumstatin gene are arranged, and they are pET22b and pET28 (a) carrier (the Novagen company that comes from Wisconsin, USA Madison city); With the pPICZ α A carrier (the InVitrogen company that comes from U.S.'s California Diego California) after the modification.These two kinds of carriers can make albumen express (referring to PCT/US98/25892, it is instructed by all being incorporated in this paper in this citation) respectively in bacterium and yeast.

After a nucleic acid fragment is cloned on the suitable carrier, just can transform an into proper host cell.Term " host cell " refers to accept contain the segmental recipient cell of exogenous nucleic acid by what carrier carried.But the host cell prokaryotic cell prokaryocyte is eukaryotic cell also, as Mammals, plant or insect; It can be unicellular, also can be cellulous combination (as at substratum or tissue culture medium (TCM) or a tissue, organ).Host cell can be extracted by normal many cells organ, also can have in the pathological tissue of many cells organ to extract (as Mammals).In this article, host cell not only refers to transform the initiating cell of exogenous nucleic acid, also refers to the progeny cell that contains exogenous nucleic acid of this cell.

The subsidiary nucleic acid connexon that connects a coding peptide chain on the gene of the coding anti-angiogenic proteins that is separated in one embodiment.Such connexon all is well known in the art, and for example, connexon comprises at least one at least one additional amino acid whose codon of encoding.The length of general connexon is 1 to 20 or 30 amino acid.The connexon of nucleic acid is also translated with the gene of coding anti-angiogenic proteins, and the result produces an anti-angiogenic proteins at amino or the subsidiary at least amino-acid residue of C-terminal.The important point is the activity that additional amino acid does not have angiogenesis inhibitor.

After being inserted into purpose nucleic acid in the carrier, carrier is transformed in the suitable protokaryon bacterial strain, and be fit to have under the conditions suitable of the bioactive protein expression of anti-angiogenic hyperplasia, producing has bioactive anti-angiogenic proteins, mutant, derivative, fragment or fusion rotein.In one embodiment, the present invention includes the polynucleotide of coding anti-angiogenic proteins is cloned into carrier pET22b, pET17b or pET28a, be transformed in the bacterium then.Bacterial host strains produces anti-angiogenic proteins then.The amount that anti-angiogenic proteins produces in general every liter of nutrient solution is 10～20mg or more.

In another embodiment among the present invention, eukaryotic vector comprises the yeast vector after the modification.Contain a multiple clone site in the pPICZ α plasmid of using in a kind of method.Insert a histidine mark fragment in the multiple clone site.In addition, carrier also can insert a Nde I site or other restriction enzyme site that is fit to.These sites are well known in the art.The anti-angiogenic proteins that contains histidine mark that produces in this embodiment contains 1 or more, is typically 5-20 Histidine.Mark must not influence proteic anti-angiogenesis activity.

Produce Arresten, a kind of method of Canstatin or Tumstatin is respectively to SEQID NO:1, the polynucleotide of SEQ ID NO:5 or SEQ ID NO:9 increases, the clone advances expression vector respectively, as pET22b, pET 28 (a), pPICZ α A or other expression vector, the carrier of the external source polynucleotide that will contain then transforms in the host cell of the genetic expression that into can make coded polypeptide and goes, under the suitable condition that is fit to protein expression, cultivate host cell, from culture, extract and purifying then.Produce the general method of anti-angiogenic proteins, the method that particularly produces Arresten, Canstatin, Tumstatin is introduced in the example of back.Arresten, Canstatin, Tumstatin also can produce expression product in transgenic animal, for example, as the transgenosis milk cow, a component of the milk of sheep or pig), or in transgenic plant expression product, for example in corn, combine with starch molecule.

Arresten, Canstatin, Tumstatin also may be produced by the method for tradition as chemosynthesis.It is well known to those skilled in the art being used to make up proteic synthetic method of the present invention.If synthetic Arresten, Canstatin, Tumstatin protein sequence have same one, two, three structure and/or conformation with Arresten, Canstatin, the Tumstatin that reorganization produces, then they can have same biological activity, comprise the biological activity of angiogenesis inhibitor.Therefore, synthetic Arresten, Canstatin or Tumstatin protein sequence can be used as separated Arresten, Canstatin or the proteic substitute of Tumstatin that reorganization produces, be used for screening to the treatment compound, and the immunological method that is used for developing antibody.

Arresten, Canstatin and Tumstatin albumen are that effectively this point is proved conclusively in standard test, and provides in the example below preventing aspect the vasculogenesis.Arresten, Canstatin, Tumstatin do not suppress the growth of other cell type, as non-endotheliocyte.

The nucleic acid of coding Arresten, Canstatin or Tumstatin can be by cloning in separated DNA or the cDNA library.In this article, if nucleic acid and polypeptide are called as " isolating ", then refer to nucleic acid or polypeptide with respect to freely separate (biogenetic derivation refer to nucleic acid mixture or cell) from their biogenetic derivation, they can be through further processing.The nucleic acid of " separation " or polypeptide comprise polypeptide or the nucleic acid that obtains with method described herein, similarity method or other usability methods, also comprise nucleic acid or polypeptide behind the abundant purifying, nucleic acid or polypeptide with chemosynthesis, in conjunction with the nucleic acid and the polypeptide of chemistry and biological method generation, and the nucleic acid and the polypeptide of the reorganization that is separated to generation.Therefore, the polypeptide of " separation " refers to not and other albumen, the albumen that glycosyl, fat or cell binding substances combine." isolating nucleic acid " refers to not can be immediately and the covalently bound nucleic acid of its source biological gene group, and under state of nature, their are covalent attachment immediately.Therefore, isolating nucleic acid refers to general carry intravital nucleic acid (virus of self-replicating or plasmid) with being attached to, or a nucleic acid that is independent of other nucleic acid, for example, and with the nucleic acid fragment that is independent of other nucleic acid that produces behind traditional chemical method or the restriction enzyme digestion.

Polynucleotide among the present invention can be used for designing the probe that separates other anti-angiogenic proteins with albumen.United States Patent (USP) the 5th, 837 provides some good methods in No. 490 (licensing to people such as Jacobs), and all instructions of the document are all by being incorporated in this paper in this citation.The design of oligonucleotide probe should be followed following parameter: will comprise that (a) unknown radix amount (N) is few as far as possible; (b) its T _mValue is (supposing that to each A or T be 2 ℃, is 4 ℃ to each G or C) about 80 ℃.

The most handy g-of oligonucleotide ³²P-ATP (specific activity is 6000 Ci/mmol) and T4 polynueleotide kinase, and adopt the technology that is generally used for labeled oligonucleotide to come mark.Also can use other marking method.The most handy gel permeation chromatography of unconjugated probe or other method of having set up are removed.The quantity that radioactive substance is inserted in the probe is calculated with scintillometer.Probe activity behind the mark is preferably 4 * 10 ⁶Dpm/pmol.The bacterial cultures that contains full-length clone colony preferably after thawing, is inoculated into the aseptic 25ml that contains with 100 μ l and adds shaking in the bottle of 100mg/ml LB (meat soup) nutrient solution.Culture is preferably in 37 ℃ and is cultured to saturatedly, dilutes in aseptic LB nutrient solution afterwards.The experiment of the five equilibrium of diluent is preferably in dull and stereotyped going up and determines its extent of dilution and quantity, make its final 37 ℃ of incubated overnight after, generate 5000 single bacterium colonies on penbritin 100 mcg/ml, agar 1.5%, 150 millimeters petri flat boards of LB solid containing.Also can adopt the method for the single bacterium colony of other known generation.

Use with colony lift to nitrocellulose filter cracking, sex change, oven dry then with the colony hybridization of standard.Highly rigorous condition at least 1 * SSC at 65 ℃, or 1 * SSC and 50% methane amide be at 42 ℃, and the rigorous condition of moderate should be at least 4 * SSC at 65 ℃, or 4 * SSC and 50% methane amide are at 42 ℃, low rigorous condition should be at least 4 * SSC at 50 ℃, or 6 * SSC and 50% methane amide are at 40 ℃.

Then, film is containing 6 * SSC, (20 * stock solution be 175.3gNaCl/L, it is 7 that the 88.5g trisodium citrate is transferred pH with NaOH), 0.5%SDS, 100 mcg/ml cerevisiae dnas are in the damping fluid of 10mM EDTA (approximately 10ml/150mm filter membrane), 65 ℃, slight vibration was hybridized 1 hour.Preferably subsequently with concentration more than or equal to 1 * 10 ⁶Dpm/ml adds probe in the hybridization system, and Hybond membrane is at 65 ℃ then, and slight vibration hybridization is down spent the night.Use the damping fluid of 500ml 2 * SSC+0.5%SDS at room temperature to wash film then, do not shake, the damping fluid of preferably using 500ml 2 * SSC+0.5%SDS subsequently is jog 15 minutes at room temperature.The washing for the third time that can adopt or not adopt 65 ℃ of 0.1 * SSC+0.5%SDS damping fluids to wash 30 minutes to 1 hour.Preferably with the film drying, the enough time of freely developing the color under X ray is so that observe its positive findings then.Also can be with other known hybridizing method.Then positive bacterium colony is chosen, cultivated, put forward plasmid DNA with ordinary method.Then, the clone can use restriction enzyme digestion again, and hybridization or dna sequencing are determined.

Present invention includes in mammalian tissues the method that suppresses vasculogenesis with Arresten, Canstatin and Tumstatin or their fragment that biologic activity is arranged, analogue, homologue, derivative or mutant.Particularly, the present invention includes the illness for the treatment of the vasculogenesis mediation with a kind of effective dose or a multiple anti-angiogenic proteins or its bioactive fragment, bioactive fragment combination, activator, antagonist.The anti-angiogenic proteins of effective dose is meant the vasculogenesis that is enough to suppress to cause disease, thus and the dosage of all or part of subduction of disease.Subduction to the illness of vasculogenesis mediation can weaken by observing the symptoms to determine, as minimizing on the gross tumor volume or inhibition growth of tumor.In this article, term " effective dose " refers to that also each has the total amount of biologically active components in the composition, this amount can be to show the effect favourable to patient, promptly treat, cure, prevent or therapeutic efficiency, healing are slowed down, increased to the related medical condition, to the control of disease or alleviate, and the method dosage that produces above-mentioned effect.When being used in combination, this term refers to the summation of each active principle that produces curative effect, no matter and be in conjunction with administration, administration or administration simultaneously successively.The disease of vasculogenesis mediation comprises (but being not limited to) cancer, the solid knurl, blood is given birth to cancer knurl (leukemia), tumour diffusion (transfer), innocent tumour is (as vascular tumor, the sound sensation neuroma, neurofibroma, trachoma, the meat odontoma of suppurating), rheumatoid arthritis, psoriasis, the vision vasculogenesis disease (as: visual deterioration of diabetes, retinopathy, retinopathy of prematurity, spot degraded, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia beans shape fibrous tissue, rubeosis), the Oslerwebber syndromes, myocardial infarction (vasculogenesis), spot neovascularization (vasculogenesis), capillary blood volume expansion (hemophiltac joints).Blood morbidity, hemangiofibroma, the wound granulation.Anti-angiogenic proteins is useful to treatment endotheliocyte disease too much or improper stimulation.These diseases include, but is not limited to the adhesion of intestines, Crohn ' s disease, atherosclerosis, scleroderma, loose scar (as sick keloid).Anti-angiogenic proteins can be as the birth control agent that prevents vascularization when embryo transfer.Anti-angiogenic proteins has effect with vasculogenesis as physiology result's disease such as cat cut disease (Rochele ininalin quintosa) and ulcer (Heliobacter pylori) to treatment.The anti-angiogenic proteins grafting vessel narrow and fat (expanding) that also can be used for preventing to dialyse as stoping in the obesity tissue capillary vessel to form to prevent its with excessivelying.Anti-angiogenic proteins also can be used for treated tissue (being non-transfer) disease." cancer " refers to the growth of knurl, and hyperplasia or hyperplasia growth or cell development abnormal state of affairs also comprise the solid knurl, non-solid knurl, and abnormal cell proliferation is as leukemia.In this article, " cancer " also refers to depend on the knurl and the tumour of vasculogenesis, and promptly tumour is come blood supply because of quantity and density that its growth (volume and/or quality hyperplasia) needs to increase blood vessel." disappear " and refer to the minimizing of measuring gross tumor volume and quality with method method well known in the art.

Yet the increase of vasculogenesis needs sometimes, as wound healing, or in behind cardiac infarction, organizing, the antibody of anti-angiogenic proteins or antiserum(antisera) can be used to stop fixed, and therefore original anti-angiogenic albumen and process thereof increase the generation of neovascularity tissue and suppress Telatrophy.

Anti-angiogenic proteins can combine the treatment disease with other material, method.For example, tumour can be treated with traditional surgical operation, ray, chemotherapeutic immunity method and in conjunction with anti-angiogenic proteins, then, continues anti-angiogenic proteins is delivered medicine to patient, the resting stage that prolongs micrometastasis is suppressed the growth of remaining primary tumo(u)r with stable.Anti-angiogenic proteins or its fragment, antiserum(antisera), receptor stimulant, receptor antagonist, or their binding substances, also can with other anti-angiogenic compounds, anti-angiogenic proteins (as tubulation Zhang Shu, endostatin), fragment, antiserum(antisera), receptor stimulant, the combination of acceptor antibiont.In addition, anti-angiogenic proteins or their fragment, antiserum(antisera), receptor stimulant, receptor antagonist or their binding substances, can combine with acceptable vehicle on the medicament, also optionally release matrix (as the polymkeric substance of biological degradability) is kept in combination, thereby forms medicinal compositions.Composition of the present invention can also comprise other anti-angiogenic proteins or compound, as endostatin or angiostatin, and their mutant, fragment and analogue.These compositions can also further comprise other reagent, and these other reagent may be promoted protein-active or auxiliary its therapeutic activity, or is used for the treatment of purpose, for example chemotherapy or radioreagent.These additional factors and/or reagent can be included in the composition, produce with protein of the present invention and suppress the combined effect that the mammalian tissues medium vessels generates, or it is minimum that side effect is dropped to.In addition, can be together to adopt wherein with other therapeutics with the administering mode that contains proteic composition among the present invention, for example, together adopt with chemotherapy or radiotherapy.

The present invention includes and suppress vasculogenesis in the Mammals with containing proteic composition contact tissue of the present invention." contact " not only refers to topical application, also refers to the mode of sending to of going in the cell in composition introducing tissue or the tissue.

The present invention also comprises using and regularly discharges or keep release delivery system.These systems can not or be ideal very in operation very at need, and for example patient is because of older or stadium is long and in poor health, or when the pros and cons analysis revealed need be controlled treatment.

In this article, keeping and discharge matrix and refer to the matrix is made up of certain material, generally is polymer, and it can be by enzyme liberating, or by sour, basic hydrolysis or dissolved.In the time of in matrix body is inserted into body, it is had an effect with enzyme and body fluid.The type of keeping discharges matrix and preferably is selected from biocompatible substance, as liposome, PLA salt, poly glycolide (polymkeric substance of glycolic acid), poly(lactic acid) is glycolide (interpolymer of lactic acid and glycolic acid) altogether, the poly acid anhydride, multi-polyester (neighbour), polyprotein, hyaluronic enzyme, collagen protein, chondroitin sulfate, carboxylic acid, lipid acid, phosphatide, saccharan, nucleic acid, polyamino acid, amino acid, as the propyl benzene propylhomoserin, tyrosine, Isoleucine, polymerized nucleoside, poly ethylene, propylene, polyethylene pyrrole purine alkane ketone and tripoli.Biodegradable matrix is poly(lactic acid) preferably, and poly glyco1ide or PLA are total to-glycolide.

It can be solid that vasculogenesis among the present invention is regulated composition, liquid or aerosol, also available known any administration.The example of solids composition has pill, creme and implantable medicament unit.Pill can be oral, and creme can be at the body surface topical.Implantable medicament unit can maybe can carry out system to the vasculogenesis instrumentality and discharge, as subcutaneous implantation focus administration the position of tumour (for example).The example of fluid composition comprises and is suitable for hypodermic prescription, intravenous prescription, the prescription of intra-arterial injection, and the prescription of body surface and intraocular treatment.Aerocolloidal example has the suction prescription of pair lung administration.

Albumen and the protein fragments that anti-angiogenesis activity is arranged described above can be made with compound method well known in the art, and the albumen or the protein fragments of isolating or substantially pure can be accepted administration in the prescription on pharmacology.These prescriptions can be used the conventional route administration.In general, binding substances can pass through body surface, and brain is through skin, in (intracranial), the brain inner room of subcutaneous, abdominal cavity, encephalic, the brain, intravaginal, intrauterine, and oral cavity, rectum or parenteral approach (as intravenous, dorsal column, subcutaneous or muscle) administration.In addition, anti-angiogenic proteins can combine with the biodegradable polymer, makes compound keep release, and polymer moves to be implanted in to be needed to discharge near the position of medicine, for example, and at tumor sites, or in the site implantation that can realize that slow system discharges.Miniature osmotic pump also can be used to control the transporting of high density anti-angiogenic proteins that is delivered to target ground by tubule, and the point of destination refers to that the tumour of transforming growth or other support blood vessel.Biodegradable polymer and their application also have description, and as detailed description (J.Neurosurg.74:441-446) is arranged in the article of Brem et al (1991), the document is by being incorporated in this paper in this citation.

The composition that contains polypeptide of the present invention can intravenously administrable, as injecting by unitary dose.In this article, term " unitary dose " refers to be suitable for the discrete unitary dose of physical aspect of object when referring to therapeutic composition of the present invention, and per unit contains the predetermined amount that combines the active substance of the required result of treatment of back generation with thinner such as carrier.

The administration of composition of the present invention comprises: intravenous injection, and intramuscular, in intraperitoneal, the thoracic cavity, subcutaneous and joint injection and input.The composition that is used for parenteral injection comprises acceptable aseptic aqueous solution or non-aqueous solution on the pharmacology, dispersion, and suspension, and emulsion, and add aseptic injection liquid or disperse sterile powder in the thing.The water or the nonaqueous carrier that are fit to, thinner, solvent, or carrier (for example comprises water, ethanol, polyvalent alcohol, glycerol, propylene glycol, polyoxyethylene glycol etc.), carboxymethyl cellulose, and their corresponding mixture, the organic ester of vegetables oil (as sweet oil) and injectivity such as ethyl oleic acid etc.Should keep the fluidity that is fit to, for example, wrap quilt, in dispersion agent, keep required granular size with surfactant with lecithin etc.Can also comprise adjuvant in these plants, as sanitas, wetting agent, emulsifying agent and diffusant.For preventing action of microorganisms, can in composition, comprise the medicament of multiple antibacterium or fungi, as to the carboxyl benzoic ether, ammonification alcohol, phenol Sorbic Acid etc.When needed, also can add some grades and ooze material, as sugar, NaCl etc.For the medicament that makes injection absorbs for a long time, can add the aluminium salt and the gelatin of single-stearic acid, thus can delayed absorption.Injectable storage form is to be total to-glycolide with PLA, and biological degradability polymers such as adjacent ester of poly and poly acid anhydrides are made the microcapsule basal granule with medicine.Can control drug release by controlling medicine and polymeric ratio and used polymeric type.Also available liposome of injected material or the microemulsion stored wrap up medicine, and liposome and microemulsion can be compatible with tissue.The prescription (agent) of injection can be sterilized, and filters as using the bacterium filtering membrane, or mixes with aseptic solids composition, and these solids compositions can be dissolved or dispersed in other aseptic injectable media before use.

Therapeutic composition among the present invention can comprise acceptable salt on the pharmacology, for example organic or inorganic hydrochlorate." acceptable salt on the pharmacology " herein refers to be used for the human or animal tissues nontoxicity in the medical science standard range, does not have allergy, and the salt of a rational harm ratio is reacted and had to ametaboly.Can be known in the art by the salt that is subjected on the pharmacology, as S.M.Berge et al. acceptable salt on the detailed pharmacology of having described in J.Pharmaceutrcal Sciences (pharmaceutical science magazine) (1977) the 66:1 et seq, the content of the document is by being incorporated in this paper in this citation.Acceptable salt comprises the additive salt (forming with the free amino group of polypeptide) of acid on the pharmacology, i.e. the salt that forms with mineral acid, for example, example hydrochloric acid or phosphoric acid, or organic acid such as acetate, tartrate, amygdalic acid etc.The salt that forms with free carboxyl group also can be generated by mineral alkali, for example, and the oxyhydroxide of sodium, potassium, ammonia, calcium, iron, or organic bases such as different third ammonia, 2-ethanol ammonia, Histidine, PROCAINE HCL, PHARMA GRADE etc.Salt also may produce when the last isolation and purification of compound of the present invention, or generates with free alkali and suitable organic acid reaction in addition.Representational acid salt includes, but is not limited to acetate, adipate, alginate, Citrate trianion, aspartate, benzoate, benzene sulfonate, hydrosulfate, butyrates, the camphor amino acid salt, digluconate, glycerophosphate, Hemisulphate, heptose hydrochlorate, hexose hydrochlorate, fumarate, hydrochloride, hydrobromate, hydriodate, Alpha-hydroxy metilsulfate (isethionate), lactic acid salt, maleate, metilsulfate, nicotinate, 2-naphthalene vitriol, oxalate, pamoate, pectate, pesulfate, 3-Propiophenone hydrochlorate, picrate, pivalate, pyruvate salt, succinate, tartrate, thiocyanate-, phosphoric acid salt, glutaminate, supercarbonate is to toluene vitriol and undecane hydrochlorate.The group that contains basic nitrogen can form the level Four thing with some groups, and these genes have lower alkyl halogenide such as methyl, ethyl, propyl group, the chlorate of butyl, Bromide, iodized salt; Dialkyl sulfate is as dimethyl, diethyl, dibutyl and diamyl vitriol; Long-chain halogenide is as decyl, lauryl, myristyl and stearyl chlorination thing, bromide, iodide; Aralkyl chimney thing is as phenmethyl, styroyl.Thereby can obtain the aqueous solution or ester solution or suspension.The example that can form the mineral acid of the acceptable acid salt class of pharmacology has: spirit of salt, Hydrogen bromide, sulfuric acid, phosphoric acid; Organic acid has: oxalic acid, toxilic acid, succsinic acid, citric acid.

In this article, the term different with other of term " acceptable on the pharmacology ", " can tolerate on the physiology " is when being used for composition, carrier, thinner and vehicle etc., can replace use mutually, it refers to such material, be that the slightest negative physiological response appears in these materials to the Mammals administration time, as nauseating, dizzy, regurgitation etc.The medicinal compositions that preparation contains dissolving and active ingredient dispersed is to know in this area, and is not restricted to prescription.Generally speaking, such composition is prepared to injectable formulation, as solution or suspension, yet, also can be prepared into the solid that is dissolved in liquid before use and forms solution or suspension.Preparation also can be emulsified.

Activeconstituents can mix in vehicle, and vehicle must be acceptable and compatible with activeconstituents on the pharmacology, and its usage quantity will be suitable for various methods of treatment described herein.Suitable vehicle comprises: water, salt solution, D-glucose, glycerine, ethanol or their mixture.In addition, if desired, can contain a small amount of ancillary component (adjuvant) in the composition as wetting agent or emulsifying agent, pH buffer reagent etc. are to improve the effect of activeconstituents.

Anti-angiogenic proteins among the present invention also can be among prodrug be contained in composition.In this article, term " prodrug " refers to can transform rapidly in vivo the material that generates parental generationization and thing, such as, the hydrolytic action by enzyme in blood generates.T.Higuchi and V.Stella are at " with prodrug as new transfer system " (Prodrugs as NovelDelivery Systems, Vol 14 of the ACS Symposium Series) and Edward B.Roche ed., at " bioreversible carrier in the medicinal design " (Bioreversible Cariersin Drug Design, united states drug association and Permeagon press, 1987) have a detailed description and introduce in two literary compositions, the instruction of these two pieces of documents is all by being incorporated in this paper in this citation.In this article, term " prodrug of accepting on the pharmacology " refers to, (1) prodrug of compound of the present invention, they are within normal medical judgment standard range, be suitable for use in human or animal tissues and nontoxicity, reactions such as allergy, irritability have the dangerous ratio of suitable interests and can play desired effects; (2) the zwitterion form of parental generation compound, if possible.

The dosage of anti-angiogenic proteins of the present invention is decided according to sick state or treatment condition or clinical other factors such as human or animal's body weight and condition and route of administration etc.For treatment people or merit thing, albumen dosage is generally 10 to 20 mg/kg body weight.In combination therapy, when using, can suitably reduce dosage as uniting with the albumen among the present invention and radiotherapy, chemotherapy or immunotherapy, for example, 0.1～0.2 mg/kg body weight.Controlled the intravital transformation period of animal or human according to anti-angiogenic proteins, anti-angiogenic proteins changes between weekly administration within a certain period of time every day several times.Having a bit must be clear, and the albumen among the present invention is suitable equally for people and animal doctor.Method among the present invention comprises single administration and multiple dosing, comprises simultaneously and administration within a certain period of time.In addition, anti-angiogenic proteins can be used in combination with the treatment of other form, as chemotherapy, and radiotherapy, immunotherapy.

The medicine of anti-angiogenic proteins comprise be suitable for oral, rectum, eyes (comprise vitreum in), in the nose, body surface (comprising the hypogloeeis), intrauterine, vagina, or parenteral (comprise subcutaneous, intraperitoneal, intramuscular, vein, intracutaneous, in encephalic, the tracheae, dura mater) administration.The medicament of anti-angiogenic proteins can conveniently be prepared with also available conventional medicament of unit dosage form method.These methods comprise the step that activeconstituents and pharmaceutical carrier or vehicle are combined together.In general, medicaments preparation comprises equably and closely both combine with activeconstituents and liquid vehicle or thin solid carrier or they, then, if desired, makes product moulding.

The medicament that is suitable for administered parenterally comprises aseptic injectable solution water-based or nonaqueous, they can comprise antioxidant, damping fluid, fungistat, reach isoosmotic solvency with target recipient blood, and the water-based or the nonaqueous sterile suspensions that comprise suspension agent and intensifier.Medicament can be packed in container unitary dose or multiple doses, for example, seals close ampoule or tubule, can be stored under the lyophilisation condition, before use, only need add aseptic liquid carrier, as adds entry and be used for injection.Interim injection solution and suspended substance can be with aseptic medicinal powder described above, and particle and tablet are made.

When the albumen of the present invention of significant quantity adopts when oral, anti-angiogenic proteins of the present invention should be tablet, capsule, medicinal powder, solution or pill form.When using tablet, can contain solid carrier in addition in the medicinal compositions, as gelatin or adjuvant.It is 5～95% that tablet, capsule and medicinal powder contain the proteic amount of the present invention, preferably contains 25～90%.When with the liquid form administration, can add liquid phase carrier, as water, Vaseline, animal oil or vegetables oil (as: peanut oil, inorganic oil, soybean oil or sesame oil) or synthetic oil.The medicinal compositions of liquid form can further contain normal saline solution, glucose or other saccharide solution, or alcohol (as ethylene glycol, propylene glycol, or polyoxyethylene glycol).When with the liquor administration, medicinal compositions contains the albumen of the present invention of month 0.5～90% weight, preferably contains 1～50% albumen of the present invention.

When the albumen of the present invention of significant quantity was used for vein, skin or subcutaneous injection, albumen of the present invention should be non-pyrogeneous substance, the absorbable liquid form of parenteral.Have suitable pH, etc. ooze, the preparation of the acceptable protein solution of parenteral of stability etc., be known in the art.Preferably being suitable for vein, skin or hypodermic medicinal compositions should be outside albumen of the present invention, also comprise etc. and to ooze carrier such as chloride injection thing, the Ringer injected material, the Dextrose injection, Dextrose and NaCl injection, lactic acid Ringer injection, or other carrier known in the art.Medicinal compositions among the present invention also can contain the stablizer of knowing in this area, sanitas, damping fluid, antioxidant or other additive.

In medicinal compositions of the present invention, proteic content of the present invention will be according to the degree of the disease of required treatment and character, and the character of the treatment once accepted of patient etc. and deciding.Finally decide every patient's albumen consumption by the doctor.Just the time spent, the doctor uses the protein for treatment patient of the present invention of few dosage, and observes its reaction.Can strengthen dose in right amount then, up to when this medicine when patient reaches best curative effect on one's body, dosage no longer increases.

Different according to the degree of disease and character and patient's specific reaction, intravenous injection treatment time of medicinal compositions of the present invention is with difference.The time length of the each intravenously administrable continuously of every kind of albumen of the present invention should be in 12-24 hour scope.Finally judge the suitable persistent intravenous injection time of used medicinal compositions of the present invention by the doctor.

The medicament of preferred unitary dose is a dosage or unitary dose every day, every day sub-doses, or the part of therapeutic dose.It should be understood that except that above-mentioned composition medicament of the present invention also can comprise the composition of commonly used other of this concrete medicament.In addition, cytotoxic substance can also be joined in anti-angiogenic proteins or its bioactive fragment, or be used in combination with them, thereby can play double-deck curative effect patient in other mode.

Formula of medicine of the present invention also is applicable to veterinary applications.Particularly domestic animal and purebred horse are the desirable objects of protein for treatment among the present invention except that the people.

Cytotoxic agent such as ricin can be linked on anti-angiogenic proteins or its fragment, can be used as the destruction instrument that anti-angiogenic proteins is taken place the cell of connection.These cells can be found at a lot of positions, include but not limited to micrometastasis enzyme and primary tumo(u)r.The albumen of linking on the cytotoxin is transported to the purpose position in a kind of mode more efficiently.For example, link on the fragment of a high affinity can be by tubular transport in the tissue of supporting target site or directly be transported to the purpose site for ricin.These materials pass through the input tubule by osmotic pump in the mode that can control.Anti-angiogenic proteins increases tissue and vasculogenesis with activation that can work in coordination with after antagonist combines as blood vessel formation against function.This therapeutic modality provides a kind of effective way of destroying metastatic cancer.

Other methods of treatment comprises to be linked anti-angiogenic proteins, fragment, analogue, antiserum(antisera) or their receptor activation thing, receptor antagonist on the cytotoxin albumen.It should be understood that anti-angiogenic proteins can derive from the people and also can derive from animal.Anti-angiogenic proteins is can be by chemical reaction synthetic or with recombinant technology and expression system generation.Anti-angiogenic proteins also available enzyme is cut the protein that type albumen produces anti-angiogenesis activity.It also can be cut type albumen by the compound of similar cell endonuclease activity and form.The generation of anti-angiogenic proteins can also be modified by influencing the active compound of nickase.

The present invention also comprises gene therapy, be about to encode anti-angiogenic proteins, fragment, mutant, or in the gene of their the fusion rotein importing patient body, and regulate.Numerous methods that DNA is changed in the cell and carries out the proteic expression of gene product, be also referred to as gene therapy methods, at " animal somatic cell is intragentic to be changed over to " [Gene Transfes intoMammalian Somatic Cells in vivo, N.Yang (1992), Crit.Rev.Biotechn.12 (4): 335-336] in have a detailed description, the content of the document is by being incorporated in this paper in this citation.Gene therapy comprises dna sequence dna imported carries out in the body or external methods of treatment in somatocyte or the embryonal system cell.Gene therapy is by replacing the function of the normal or abnormal gene of generegulation, thus opposing infectious virus or other cause of diseases.

The strategy for the treatment of these diseases with gene therapy comprises that the therapeutic strategy comprises as determining defective gene, inserts a gene with function then, perhaps replaces the function of defective gene, perhaps the function of enhancing gene; Also comprise preventative strategy, as increase a gene, its expressed proteins this disease of verifying has curative effect, and perhaps it can make tissue or organ responsive more to a kind of therapy.As an embodiment of preventative strategy, a kind of gene of one or more anti-angiogenic proteins of encoding is placed in patient's body, just can prevent the generation of vasculogenesis; Perhaps a kind of tumour cell that can make is placed in the body the responsive more gene of radiation, make and to kill more tumour cell at the radiation of tumour.

The present invention has considered the scheme of the regulating and controlling sequence of many transfer DNAs or anti-angiogenic proteins.The transfection promoter sequence, perhaps a kind of gene relevant specifically with anti-angiogenic proteins, perhaps those sequences that can increase the expression amount of anti-angiogenic proteins are also paid attention to as gene therapy methods.The embodiment of this technology is at transfection chromaticness therapy (the Transkaryotic Therapies of company in Cambridge, Massachusetts, Inc., Cambridge, Mass) implemented, they use homologous recombination technique to insert a gene switching, the expression that can open erythropoietin gene in cell." genetic engineering news " (Genetic Engineering News) referring on April 15th, 1994.Do not have in the cell of normal expression at those anti-angiogenic proteins (perhaps their acceptor), this gene switching can be used for activating their expression.

The gene transfer method of gene therapy can be classified as three major types: physics method (as electroporation, direct gene changes method and partickle bombardment method over to); Chemical process (as the lipid carrier method, perhaps other non-virus carrier method); And biological method (as viral derivative vector and acceptor absorption process).For example, can use non-virus carrier, DNA liposome wherein.This liposome/dna complex can be injected directly in the patient body by intravenous injection.It is believed that this liposome/dna complex can assemble at liver, DNA is transferred in scavenger cell and the Kupffer cell there.These cells are long-lived, therefore can make the gene that is transferred obtain secular expression.In addition, carrier or naked DNA can be injected directly into the organ that needs, and in tissue or the tumour, make curative DNA be sent to the target place.

Gene therapy method also can be classified by the position that gene is sent to.The basic skills of sending to gene comprises stripped (ex vivo) gene transfer method, (in vivo) gene transfer method and external (in vitro) gene transfer method in the body.The ex vivo gene transfer method is to extract cell on one's body from patient, carries out cell cultures.Then with the DNA transfection in cell, enlarging the number of transfected cell, again with in the Transplanted cells ex vivo.The outer-gene transfer method is, transfected cell is to cultivate in the cell culture medium, such as tissue culture cells, and it is not to take from special patient special cell on one's body.After these " laboratory cells " were transfected, through screening, enlarged culturing was used or other purposes as transplanting to patient.

The vivo gene transfer method need import to DNA in the intravital cell of patient.Such method comprises with virus-mediated transgenosis, utilize noninfective virus vector with transgenosis in the patient body, perhaps directly naked DNA is expelled to the intravital a certain position of patient, these DNA are absorbed by the cell of some amount, and make gene product protein obtain expressing therein.In addition, additive method described herein as particle bombardment, also can be used for inserting in vivo DNA or modulability sequence, with the expression of control anti-angiogenic proteins.

The chemical process of gene therapy need be with the lipid material, and not necessarily liposome is helped DNA and passed through cytolemma.Lipofectin or cytofectin based on the band positive ion of lipid can combine with electronegative DNA, form a kind of mixture, can pass through cytolemma, and DNA is entered in the cell.Another chemical process is the endocytosis that utilizes based on acceptor, this need be attached to specifically can with cell surface receptor bonded part on, it is wrapped up, cytolemma is passed through in transhipment.Part combines with DNA, and whole mixture is transferred to the cell the inside.Part/gene composite is injected in the blood flow, has the specific binding partner of cell meeting of this acceptor then, and part/DNA mixture is transferred in the cell.

Many gene therapies utilize virus vector that gene is imported in the cell.For example, retroviral vector through transforming has obtained utilizing in the method for exsomatizing, gene is imported to periphery lymphocyte and tumour infiltration lymphocyte, liver cell, epithelial cell, myocyte, or in other somatocyte, then, these cells of being transformed are failed back in the patient body, provided to be inserted into the expression of gene product.

In the transgenosis scheme in vivo, also utilized virus vector to realize transgenosis in cell.For realizing the tissue specific expression of foreign gene, can utilize to have tissue-specific cis-regulating element or promotor.In addition, also can utilize original position DNA to shift or have tissue-specific virus vector realizes this goal.For example, for transgenosis is arrived in the intravital blood vessel cell, the endotheliocyte of vitro conversion can be implanted the position of selecting on the arterial blood tube wall.Virus has also infected peripheral cell, and these cells have also given expression to gene product.Use conduit virus vector directly can be imported to a certain position of live body, make virus can only infect specific scope like this, reach secular, site-specific genetic expression.In vivo in the transgenosis, on mammalian tissues and liver organization, also utilize retroviral vector by transformed virus injection is advanced in the blood vessel, thereby gene is imported to organ.

Virus vector as gene therapy includes but not limited to, retrovirus, other RNA viruses such as poliovirus (poliovirus), or Xin Bisi (Sindbis) virus, adenovirus, adeno associated virus (adeno-associated virus), simplexvirus, SV40 virus, cowpox, and other dna virus.The mouse retrovirus of replication defect type is a gene transfer vector the most commonly used.The retroviral composition of murine leukemia comprises the single stranded RNA composition, is compounded with core protein and polysaccharase, and a kind of gag albumen packing is arranged outward, is wrapped up by glycoprotein env outward, and it determines host range.Retroviral genome comprises gag, pol, env gene and 5 ' and 3 ' long terminal repeat LTR.The retrovirus system is based on the following fact: if there is auxiliary package cell line that virus structural protein is provided, the minimum virus that so only contains 5 ' and 3 ' LTR sequence and packaging signal be enough to realize virus packing, infect and be incorporated into process in the target cell.Retrovirus as the basic advantage of gene transfer vector is, in the cell of most of type, can both infect effectively, gene can be expressed effectively, only there is the gene of a copy to be integrated on the purpose cyto-chromatin DNA, and the genomic operation ratio of reverse transcription is easier to carry out.

Adenovirus comprises a linear dsdna, and core protein is wrapped up by coat protein outward.The progress of Molecular Virology makes and can utilize these microorganisms to develop carrier, so that in vivo new genetic sequence is transferred in the purpose cell.Carrier based on adenovirus can make gene obtain high-caliber expression.Adenovirus carrier has very high infectivity, even if also like this with the very low virus of titre.Therefore and as acellular virion, adenovirus is to have infectively fully, there is no need to inject production clone.Another strong advantage is that adenovirus carrier can make foreign gene obtain secular expression in vivo.

The mechanical means of transgenosis comprises that merging lipid vesicle such as liposome or other is used for the vesica that film merges, the lipid granule such as the lipofectin that contain the DNA of positively charged lipid, the DNA of (polylysine-mediated) of poly-lysine mediation shifts, the direct injection of DNA, as DNA being injected sperm or somatocyte by microinjection, shift the DNA particle that wraps up by pneumatic action, as the gold grain that uses in the particle bombardment, mineral compound method such as calcium phosphate transfection method.The transgenosis of particle mediation is used in plant tissue transforms at first.Adopt the partickle bombardment device, or particle bombardment, produce a power DNA particle (as gold grain or tungsten particle) that density is very big and accelerate to very big speed, make it can penetrate organ, tissue or cell.The partickle bombardment method can be used in vitro system, also can use in the technology in stripped or body, with DNA transfered cell, tissue or organ.Another kind method is ligand-mediated gene therapy, and it need link to each other DNA with specific part, form part-DNA mixture, thereby by part DNA is imported in the specific cell or tissue.

Have been found that plasmid DNA is expelled in the muscle cell that the muscle cell that has very high per-cent is transformed, and keep the expression of marker gene.Plasmid DNA perhaps can, the chromogene group that perhaps can not be incorporated into cell gets on.The DNA of nonconformity type transforms and makes and can finally keep the expression of long period in the tissue that break up, non-proliferative, and needn't worry can cause producing the sudden change of inserting, delete or make a variation in cell or Mitochondrial Genome Overview.Secular, but not necessarily permanent, being transferred in the specific cell of therapeutic gene, can be to some genetic diseases effective in cure or prophylactic effect.DNA can regularly inject repeatedly, has certain level to keep gene expression product, but can not produce sudden change in the genome of recipient cell.Not with exogenous origin gene integrator in cellular genome, can allow to have simultaneously several different foreign DNAs to exist, express several different gene products simultaneously.

The transgenosis sensitivity that the electroporation gene transfer method uses an electric current to make that tissue or cell mediate electroporation.The short electricimpulse of a given intensity can increase the permeability of cytolemma, to such an extent as to dna molecular can penetrate and enter cell.This technology can be used in vitro system, or with technology in stripped or the body DNA is imported in cell, tissue or the organ.

Carrier mediated vivo gene transfer can be used for foreign DNA is transferred in the cell.The carrier-DNA complex body can be directed to body fluid or blood flow very easily, tissue-specific then target approach organ or tissue.Liposome and polycation as poly-lysine, lipid transformant (lipofectin) or cell transformation body (cytofectin), can use.Liposome can make pair cell or tissue and have specificly, and the foreign DNA that carries of liposome just can directly be absorbed by target cell like this.It is a kind of shortcut that foreign DNA is inserted into the target cell that contains this acceptor that injection has specific immunoliposome to the specific acceptor of some cell surface.Another already used carrier system is 5 asialoglycoproteins (asialoglycoprotein)/poly-lysine coupling system, and it is applied in the vivo gene transfer DNA be imported to liver cell.

The DNA that is transformed also can link to each other with the carrier of other type, so that they are imported in the recipient cell, and it can be resided in tenuigenin or the cell caryoplasm.DNA can link to each other with carrier core albumen, and this nucleoprotein is special engineering particle composites, directly DNA is directed in the nucleus.

The gene regulating of anti-angiogenic proteins can by increase can with the gene bonded compound of coding anti-angiogenic proteins, the perhaps zone of control and gene-correlation, or control the speed that its corresponding rna transcription product transcribes or translate with adjusting and realize.And the DNA cell transformed of the anti-angiogenic proteins that is encoded can fail back in patient's body, originates to provide in this proteic body.For example, with carrying the carrier transformant of angiogenesis inhibitor gene, can be cell from patient's healthy tissues on one's body by cell transformed, patient's diseased region cell, cell that also can the right and wrong patient.

For example, can fail back again in patient's body then with expressing the proteic carrier transfection of the present invention from patient's tumour cell on one's body.Transfected tumour cell produces the protein expression of certain level in patient's body, can suppress growth of tumor.The patient can be the people, also can be animal.Cell also can be by non-carrier transfection, or with the method for physics known in the art, chemistry such as electroporation, ion duct method, or particle bombardment.And DNA can pass through the auxiliary of carrier, and is injected directly in patient's body.Particularly be expelled to subcutaneous, in muscle or the blood.

By transfection anti-angiogenic proteins gene to the intravital gene therapy scheme of patient can be by integrating the anti-angiogenic proteins gene integration in patient's cellular genome or Mitochondrial Genome Overview, also can allowing it be present in tenuigenin or the cell caryoplasm with independently reproducible or not reproducible dna structure form.Can keep a long time to the expression of anti-angiogenic proteins, also can make the level of protein in cell, tissue, organ or blood be maintained at needed level by constantly injecting gene repeatedly.

In addition, the invention still further relates to antibody and antiserum(antisera), they can be used to detect new anti-angiogenic proteins, also can be used for appearance or corresponding active diagnosis, prediction and the treatment that lacks the disease that is feature with relevant anti-angiogenesis activity.Such antibody and antiserum(antisera) also can be used to raise the vasculogenesis that needs adjusting in needs, for example in the cardiac muscular tissue after myocardial infarction, proteic antibody of the present invention or antiserum(antisera) can be used for blocking partial, natural anti-angiogenic proteins and generative process, increase the formation of new blood vessel, suppress cardiac muscular tissue's atrophy.

These antibody and antiserum(antisera) can combine with acceptable composition on the pharmacology and carrier, form diagnosis, prediction or medicine for treatment thing.Here " antibody " and " antibody molecule " are meant the immunocompetent part of having of a group immunoglobulin molecules and/or immunoglobulin (Ig), just contain the molecule of antibody combining site or paratope.

The antibody therapy of the passiveness that use can be specifically carried out in conjunction with the antibody of anti-angiogenic proteins can be regulated the physiological process that depends on vasculogenesis, as reproduction, growth, wound healing, tissue repair.And adding can also be blocked endogenic at this proteic antiserum(antisera) and this proteic combination with the monoclonal antibody district bonded antiserum(antisera) of anti-angiogenic proteins.

Anti-angiogenic proteins of the present invention can also be used for preparation these supressors and acceptor are had specific antibody.Antibody can be that polyclonal antibody also can be a monoclonal antibody.These can be used to diagnosis and test kit with anti-angiogenic proteins bonded antibody or their acceptor specifically, and all to be that those skilled in the art is known be used for detecting or the quantitative technology of body fluid or tissue anti-angiogenic proteins or its acceptor for they.The result of these detections can be used for diagnosing or predicting the generation or the recurrence of the tumour and the disease of other vasculogenesis mediation.

The present invention also comprises anti-angiogenic proteins, its antibody and the composition be made up of these albumen and/or their antibody with the vasculogenesis is being the diagnosis of disease of feature or the use in the pre-diagnosis.In this article, term " pre-diagnostic method " speech is meant the method that can make a prediction to a kind of advancing of disease of human or animal, and this disease especially depends on vasculogenesis.Term " diagnostic method " speech is meant can clearly determine human or animal's the existence that depends on vasculogenesis class disease or the method for its kind.

Anti-angiogenic proteins can be used to detect and quantitatively can be in conjunction with diagnostic method that changes protein antibodies and test kit.These test kits can detect the antibody of anti-angiogenic proteins in the blood circulation, and there is the diffusion that shows by under the existence of tumor in situ excretory anti-angiogenic proteins in these albumen.There is the patient of anti-angiogenic proteins more likely to develop into many tumours or cancer in this blood, after the recovery of treatment or short-term, also more likely recurs.The Fab segment of these anti-proteic antibody can be used as antigen be used for preparation anti--albumen Fab segment antiserum(antisera), during they can be used for and the antibody of anti-angiogenic proteins.This method can reduce by anti--protein antibodies cause to the proteic removing of round-robin.Thereby improve the level of anti-angiogenic proteins in the blood circulation effectively.

The present invention also comprises the separation and Extraction to the special acceptor of anti-angiogenic proteins.The albumen segment that tissue is had a high binding affinity can be come the acceptor of separation and Extraction anti-angiogenic proteins with affinity column.These acceptors of separation and purification are towards the basic step of illustrating the anti-angiogenic proteins mechanism of action.Separate acceptor with identify its agonist and antagonist will help to develop the last approach of these receptor actives-bioactive of regulating-medicine.Separate these acceptors and allow to the designing nucleic acid probe, determine the position and the synthetic situation of acceptor with in situ hybridization or solution hybridization technology.Further, can separate the gene of acceptor, be inserted into an expression vector, transformant, such as patient's tumor cell are to strengthen cell, tissue or tumour in conjunction with anti-angiogenic proteins and the ability that suppresses the local vascular generation.

Anti-angiogenic proteins is used to develop affinity column, with the acceptor of the anti-angiogenic proteins in the separating tumor cell culture.The acceptor that separation and purification obtains carries out amino acid sequence analysis.Utilize sequence information, can identify, separate the gene of the acceptor that obtains encoding.Then, cloned genes is inserted in the expression vector.These technology all are the technology of knowing very much in this area.The accounting sequence of coding acceptor is transformed in the tumour cell, goes out acceptor, can improve the response capacity of these cells, thereby reduce the speed of growth of metastatic tumor for endogenic or ectogenic anti-angiogenic proteins by the tumor cells expression that transforms.

Vasculogenesis arrestin of the present invention can be synthetic with the microchemistry equipment of standard, and its purity is checked with FPLC and mass spectroscopy.Protein synthesis, FPLC and mass spectral method all are the technology of normal well-known very in this area.Anti-angiogenic proteins and their receptor protein are expressed in recombination bacillus coli or yeast expression system, use the column chromatography method purifying.

The different segments that can synthesize anti-angiogenic proteins, be used for various uses, for example (but being not limited to) is used to prepare the antigen of specific antisera, activator or inhibitor as the anti-angiogenic proteins combining site, or, be used to kill the cell that combines anti-angiogenic proteins in conjunction with cytotoxic agent.

Synthetic anti-angiogenic proteins segment can be used for various uses.With radio-labeling the anti-angiogenic proteins acceptor is had the albumen segment of high-affinity, be used for the demonstration and the quantitative analysis of radioactive automatic developing and film combination technology.This is applied as diagnosis and provides an important instrument with research.The signal conduction mechanism that helps to probe into acceptor about the knowledge of receptor-binding characteristic.

Can use standard method with anti-angiogenic proteins and by their deutero-albumen and other molecule coupling.The aminoterminal of anti-angiogenic proteins and carboxyl terminal all contain tyrosine and lysine residue, with radioactivity or on-radiation technical mark.For example carry out radio-labeling (tyrosine-chloramine-T, radioiodine iodogen, lactoperoxidase with conventional art; Lysine residue-Bolton-Hunter reagent).These coupling technologies all are the routine techniques of knowing very much in the art.Perhaps, tyrosine or Methionin are joined in the protein that does not have this residue, to help carrying out radio-labeling at these proteic aminoterminals and carboxyl terminal.Coupling technology is based on available functional group on some amino-acid residues as (but being not limited to) amino, sulfydryl, carboxyl, phenyl, imidazolyl.The various reagent that influence these linked reactions comprise glutaraldehyde, diazonium benzidine (diazotized benzidine), carbodiimide (carbodiimide) and adjacent benzoquinones (p-benzoquinone).

In various application, with chemical method with anti-angiogenic proteins and isotropic substance, enzyme, carrier proteins, cytotoxin reagent, fluorescence molecule, chemical illuminating reagent, bioluminescent reagents and the coupling mutually of various other compounds.For specific reaction, the efficient of linked reaction is determined with various different technologies.Proteic radio-labeling for example of the present invention uses chloramine-T (Chloramine T) and the NaI with high specific ¹²⁵, reaction stops with sodium metabisulfite (Sodium metabisulfite), and reaction mixture is with disposable pillar desalination.The albumen that is labeled is come out by wash-out, collects component.From component, shift out sample aliquot, measure radioactive intensity with gamma counter.In this way, responseless NaI ¹²⁵From albumen, separated.Have the radioactive protein ingredient of high specific and store for future use, as be used to analyze anti-angiogenic proteins in conjunction with sero-fast ability.

In addition,, use tomography X technology or other modern radiological imaging technology to observe the combining site of acceptor in vivo, determine the position of tumour with this with short isotopic labeling anti-angiogenic proteins of transformation period.

Carry out the aminoacid replacement of system in these synthetic protein, obtain the anti-angiogenic proteins acceptor is had the protein agonist or the inhibitor of high-affinity, they can improve or reduce the combination to acceptor.These agonists are used for suppressing the growth of small metastatic tumor, thus the diffusion of restriction cancer.The inhibitor of anti-angiogenic proteins uses under the insufficient situation of vasculogenesis, with the restraining effect of blocking-up anti-angiogenic proteins, improves vasculogenesis.For example, that the diabetic subject is improved wound healing is effective in cure for this therapy.

The present invention is further illustrated with following embodiment, but these embodiment and do not mean that and can limit the scope of the invention.On the contrary, what also should be expressly understood that is, for various other embodiments can also be arranged, or amendment scheme and any other embodiment that is equal to, as long as these schemes become after those skilled in the art have read content as herein described and are clear, and do not deviate from the scope of the claim of essence of the present invention and/or appendix.Embodiment

Embodiment 1: the separation of natural A rresten

Can obtain milligram Arresten of level from people's placenta and amnion tissue.Separate Arresten or other similar proteic method had report (as, Langeveld, J.P. etc., 1988, J.Biol.Chem.263:10481-10488; Saus, J. etc., 1988, J.Biol.Chem.263:13374-13380; Gungwar S. etc., 1990, J.Biol.Chem.265:5466-5469; Gungwar S. etc., 1991, J.Biol.Chem.266:15318-15324; Kahsai, T.Z. etc., 1997, J.Biol.Chem.272:17023-17032).Neilson etc. to the reorganization Arresten production also have report (1993, J.Biol.Chem.268:8402-8406).This albumen also can be expressed (for example, the method for describing with Hohenester E. etc., 1998, EMBO J.17:1656-1664) in 293 kidney cells.Arresten also can separate by the method for Pihlajaniemi T. etc. and obtains (1985, J.Biol.Chem 260:7681-7687).

Fig. 1 has shown the nucleotide sequence (SEQ IDNO:1) and the aminoacid sequence (SEQ ID NO:2) of type albumen NC1 structural domain α chain.Top mark the proteic general initial and end position of Arresten.Arresten has consisted essentially of the NC1 structural domain of type protein alpha chain, may also have the joining region, and it just is 12 aminoacid sequences before the NC1 structural domain.

From people's placenta separating natural Arresten, use anion-exchange chromatography with the bacterium Collagenase, the overanxious chromatography of gel, HPLC and affinity chromatography carry out separation and purification (GunwarS. etc., 1991, J.Biol.Chem.266:15318-24; Weber S. etc., 1984, Eur.J.Biochem.139:401-410).Type albumen from people's placenta is the HPLC purifying, with C-18 drainage column (Pharmacia, N.J. Piscataway).The albumen fine gradient of second (32%-39%) wash-out.A main peak is arranged, and one little bimodal.SDS-PAGE analyzes and finds that there are two bands at first peak, and second peak do not have protein band.Immunoblotting is also found not detect albumen at second peak, and first peak proves Arresten.

Embodiment 2: the Arresten that produces reorganization with intestinal bacteria

From α 1 NC1 (IV) pDS carrier pcr amplification Arresten gene (NeilsonE.G. etc., 1993, J.Biol.Chem.268:8402-8405), use forward primer 5 ' CGG GAT CCT TCT GTT GAT CAC GGC TTC-3 ' (SEQ ID NO:3) and reverse primer 5 '-CCC AAG CTT TGT TCT TCT CAT ACA GAC-3 ' (SEQ ID NO:4).The cDNA segment that obtains is cut with BamH I and Hind III enzyme, is connected on pET22b (+) carrier that enzyme has cut in advance (Novagen company, Wisconsin Madison).Fig. 2 represents the building process of plasmid.Arresten is in the downstream of pelB leader sequence, and is in the same reading frame with it, the Arresten of expression is secreted in the pericentral siphon chamber, with the soluble proteins formal representation.Albumen adds the preceding paragraph carrier sequence in addition, and one section amino acid MDIGINSD (SEQ ID NO:13) encodes.Sequence 3 ' end histidine-tagged with poly (His Tag) links to each other.Additional carrier sequence encoding amino acid sequence KLAAALE (SEQ ID NO:14) between 3 ' and the His-Tag of cDNA.The positive colony of these two chains is carried out sequencing to be identified.

The plasmid that will contain coding Arresten gene is Transformed E .Coli.HMS174 (Novagen company, Wisconsin, USA Madison) at first, transforms BL21 cell (Novagen company, Wisconsin, USA Madison) then and expresses.Inoculum with incubated overnight is inoculated in the 500 ml LB substratum.Continue to cultivate about 4 hours, make nutrient solution OD ₆₀₀Reach 0.6.Add IPTG (final concentration is 1-2mM) then, inducible protein is expressed.After inducing two hours, the 5000g centrifugal collecting cell is used the 6M guanidine, 0.1M NaH ₂PO ₄, the damping fluid suspension cell of 0.01M Tris-HCl (pH8.0), cracking.The cell ultrasonic disruption that suspends, 12, centrifugal 30 minutes of 000g.Supernatant is crossed the Ni-NTA agarose pillar (Qiagen company, German Hilden) 4-6 time of a 5ml, and flow velocity is 2ml/min.The albumen of the non-specific binding 8M urea that contains 10mM and 25mM imidazoles, 0.1M NaH ₂PO ₄, the eluant solution of 0.01M Tris-HCl (pH8.0) is removed.Then with increasing imidazole concentration (50mM, 125mM, 8M urea 250mM), 0.1M NaH ₂PO ₄, the eluant solution Arresten albumen of 0.01M Tris-HCl (pH8.0).The albumen that wash-out obtains dialyses twice in 4 ℃ to PBS, has small part albumen can precipitate in dialysis procedure.Collect the albumen of dialysing, centrifugal with about 3500g, precipitation separation and supernatant part.The protein concentration of each component is with BCA analytical method (PierceChenical company limited, the U.S., Illinois Rockford) and quantitatively the SDS-PAGE analytical method is definite.The precipitation Partial Protein accounts for 22%, 78% and reclaims in solution.Gained total protein output is about 10mg/l.

The albumen of escherichia coli expression is mainly with the proteic isolated in form of solvability, and the SDS-PAGE electrophoresis shows at 29 kDa places a monomer band.3 extra kDa carry out immunodetection from poly connexon and histidine-tagged (His-Tag) sequence with Arresten and 6His-Tag antibody.

Embodiment 3: at 293 embryo's kidney cell expressing Arresten

With the pDS plasmid that contains α 1 (IV) NC1 gene is template amplification Arresten, and signal peptide sequence is added to (Invitrogen company, California, USA San Diego) on the carrier for expression of eukaryon pcDNA3.1 in same reading frame.5 ' the end of the signal peptide of α 1 (IV) chain 5 ' end being cloned into NC I territory is in order to allow protein excretion in substratum.The recombinant plasmid that contains the Arresten gene checks order with the flank primer.Do not have wrong cDNA to be further purified, be used for external Translation Study to confirm protein expression.With the CaCl method, transform 293 cells with the plasmid and the control plasmid that contain Arresten, screen the positive colony (Life Technologies/Gibco BRL, Maryland, USA Gaithersburg) that is transformed with Geneticin (geneticin).In the presence of microbiotic, with the processing of cell, till not having tangible necrocytosis through 3 weeks.Then the clone is spread in the T225 culturing bottle and cultivate.Collect supernatant, concentrate (Amicon) with the Amicon concentrating instrument and handle.The supernatant that concentrated SDS-PAGE electrophoresis, the expression of immunoblotting and elisa assay Arresten.Detecting with ELISA has very strong combination in the supernatant.The main band of SDS-PAGE analysis revealed is a single band at 30 kDa places.Contain Arresten the supernatant antibody affinity chromatography separation and purification special to Arresten (Gunwar etc., 1991, J.Biol.Chem.266:15318-24).Obtain a main peak, contain the monomer that molecular weight is about 30 kDa, Arresten antibody is had immunoreactivity.Every liter of nutrient solution can obtain albumen 1-2mg reorganization Arresten albumen approximately.

Embodiment 4:Arresten suppresses the propagation of endotheliocyte

The C-PAE cell is cultured to cell and converges in the DMEM substratum that contains 10% foetal calf serum (FCS), keep in touch and suppressed 48 hours.Control cells is 786-0 (kidney) cell, PC-3 cell, HPEC cell and A-498 (kidney) cell.Cell, is collected (Life Technologies/Gibco BRL, Maryland, USA Gaithersburg) in 37 ℃ of insulations digestion in 5 minutes with trypsinase.The cell suspending liquid that will contain DMEM 12500 cells, 1%FCS is added on the plate in 24 holes that the fibronectin with 10 μ g/ml wrapped up.Containing 5%CO ₂, the following 37 ℃ of insulations of the condition of humidity 95% 24 hours.Remove substratum, replace the bFGF (R﹠amp that contains 0.5%FCS and 3ng/ml; D system company, Minn. Minnesota) DMEM.There is not the stimulated control cell not add bFGF.Cell is handled with the Arresten or the endostatin of various concentration (0.01 μ g/ml to 50 μ g/ml).All holes all add the 3H-thymus pyrimidine of 1 microcurie when handling.24 as a child after, remove substratum, aperture is washed with PBS.With 1N NaOH cell is washed, be added in the flicker tubule of the Scinti Verse solution (Fisher Scientific company, Pennsylvania, America Pittsburgh) that 4ml arranged.The thymus pyrimidine that absorbs is measured with scintillometer.The result is shown in Fig. 3 A and 3B, and their expressions absorb with the Arresten of different concns or the C-PAE cell of endostatin processing ³The situation of H-thymus pyrimidine.Fig. 4 A, 4B, 4C and 4D represent the cellular control unit situation handled with Arresten or endostatin, and Arrestaten is to 786-0 cell (Fig. 4 A), PC-3 cell (Fig. 4 B), or the effect that HPEC cell (Fig. 4 C) has is less.Endotstin is less to the effect (Fig. 4 D) of A-498 cell.All groups of Fig. 3 and Fig. 4 are all represented the data of three samples.

Embodiment 5:Arresten suppresses endothelial cell migration

Arresten and endostatin are with people's umbilical cord endotheliocyte (ECV-304 cell to the retarding effect of FBS inductive chemotaxis, ATCC1998-CRL, ATCC (American Type Culture Collection, No. 10801, Manassas city, the U.S. VA state university street 20110-2209) uses Boyden analytical method (the Neuro-Probe company limited of Maryland, USA Jhon Cabin) to measure.The ECV304 cell is grow overnight in the M199 substratum that contains 10% FBS and 5 ng/ml DilC18 (3) living body fluorescent staining agents (Ore. EugeneMolecular Probes company limited).Behind the tryptic digestion, wash with the M199 substratum that contains 0.5%FBS, diluting cells in the upper chamber in each hole, adds (or not adding) Arresten or endostatin (2-40 μ g/ml) with 60000 cell inoculations simultaneously.Lower chamber adds the M199 contain 2% FBS and tends to agent as chemistry, and it is that 8 μ m poly carbonic ether filter membranes (Poretics company, California, USA Livermore) separate that the chamber of containing cell and chemistry tend between the agent with the aperture.37 ℃ at 5%CO ₂, insulation is 4.5 hours under the condition of humidity 95%.Remove the cell that does not have migration, wash the upper strata cell with the PBS damping fluid, filtering membrane is scraped with plastic blade, is soaked among the PBS that contains 4% formalin, places on the lanternslide.With a superpower fluorescence source, use digital SenSys ^TMCamera utilizes PMIS software processes (RoperScientific/Photometerics, State of Arizona, US Tue cson) can record many pictures that independence is similar.Fig. 5 A, 5B, 5C show the Arresten of 2 μ g/ml the same with 20 μ g/ml effectively.Cell counting is carried out with software OPTIMAS 6.0 (MediaCybemetics company, New York Rochester), and the result shows the result who is illustrated in the Photomicrograph to be seen with the diagrammatic form as shown in Figure 6.

Embodiment 6:Arresten suppresses the formation of interior cutaneous vessel

In order to test to the angiopoietic inhibition of endothelium, in every hole of 24 orifice plates, add 320 μ l matrigel (Collaborative Biomedical Products company, Massachusetts, United States Bedford), allow its polymerization (Grant, D.S. etc., 1994, Pathol.Res.Pract.190:854-63).About 25,000 mouse aortic endothelial cells are suspended with EGM-2 substratum (Clonetics Corporation, California, USA San Diego), and added with antibiotic is not added in each hole.Cell is handled with the Arresten or the BSA that increase concentration gradually, aseptic PBS or 7S structural domain.All analyze all do three parallel.Cell is observed with CK2 Olympus microscope (CK2 Olympus microscope) (3.3 times on eyepiece, 10 times on object lens) 37 ℃ of insulations 24-48 hour.The TMAX film (Kodak) that applies with 400DK is taken pictures then.Cell with stop bath dyeing (Sigma Chemical Company, St. Louis), is taken pictures.Clap ten times, count all blood vessel numbers, calculating mean value.The result as shown in Figure 7, the result shows, and compares, Arresten has suppressed vascularization.By the visible representative better person of vasculogenesis of Fig. 8 A.Fig. 8 B represents to handle with the Arresten of 0.8 μ g/ml, and the MAE cell seldom or the person's (100X demonstration) that do not have the vasculogenesis.

Also carrying out intravital Matrigel with the C57/BL6 mouse analyzes.Matrigel thaws at 4 ℃ and spends the night.Then with the heparin (Pierce Chemical company, Illinois, USA Rockford) of itself and 20 U/ml, the bFGF (R﹠amp of 150 ng/ml; D System company, state, Minn. Minneapolis) and the Arresten of 1 μ g/ml or the endostatin mixing of 10 μ g/ml.With the pin subcutaneous injection of mixture with 21g.Control group is same mixture, does not just add the blood vessel formation inhibitor.After 14 days, kill mouse, take out the matrigel stopper, the matrigel stopper is fixed 4 hours with poly formalin PBS room temperature, forwards to then among the PBS and places 24 hours.Matrigel stopper paraffin embedding, H﹠amp is used in section; E dyeing.The section light microscopy, the blood vessel number that counting forms, calculating mean value.

When Matrigel is having in the presence of the bFGF, when being with or without the Arresten that increases concentration, under 1 μ g/ml Arresten and 10 μ g/ml endostatin, observing the blood vessel number and reduced 50%.These results show that Arresten influences new vascularization by the different step that suppresses the vascularization process.The result shows that also it is the same suppressing the effect that neovascularization is done to use in the body at Arresten under the 1 μ g/ml concentration with the endostatin of 10 μ g/ml.

Embodiment 7:Arresten suppresses in-vivo tumour to be shifted

To 1,000,000 MC38/MUC1 cell of C57/BL6 mouse mainline (GongJ. etc., 1997, Nat.Med.3:558-61).In 26 days, every other day, five control mice are injected the PBS of aseptic 10 mM, to the Arresten of six experiment mices injection 4mg/ml.After 26 days, count the lung cancer knot number of every mouse, every group is averaged.Two death have been observed for every group.Find that Arresten has significantly reduced the formation of primary tumo(u)r knot, reduce to 200 from 300 of control group.

Embodiment 8:Arresten suppresses the growth of in-vivo tumour

With 2,000,000 786-0 cell is subcutaneously injected in the male nude mouse body in 7 to 9 ages in week.First group of six mouse allows tumour grow to 700mm ³Size.Six of second group of mouse, tumour allows it grow to 100mm ³Size.The aseptic ArrestenPBS solution of injection every day was injected 700mm continuously ten days ³Injected in mice dosage be 20mg/kg.100 mm ³Injected in mice dosage be 10 mg/kg.Control mice injection BSA or PBS solution.Result such as Fig. 9 A are shown in the 9B.The curve representation of Fig. 9 A handle the back gross tumor volume from 700mm ³Increase, 10mg/kg Arresten handles (), BSA handles (+), the contrast (●).The tumour size of handling with Arresten is from 700mm ³Narrow down to 500mm ³, increase to 12000mm in 10 days of tumour size that BSA handles and control group ³Fig. 9 B represents that the tumour original size is 100mm ³Mouse, the result who handles () with Arresten causes tumour to be contracted to 80mm ³, and BSA handles in (+) 10 days the tumour size increases to 500mm ³

Collect about 5,000,000 PC-3 (adenocarcinoma of prostate cell), intravenous injection to 7 is to the male nude mouse body in 9 ages in week.Allow tumor growth 10 days.Measure the tumour size with the Vernier calipers then, with normalized form (wide 2X long * 0.52) calculate the gross tumor volume size (O ' Reilly M.S. etc., 1997, Cell, 88:277-85; O ' Reilly M.S. etc., 1994, Cell 79:315-28).Animal only divides into groups by every group of 5-6.Experimental mouse is injected Arresten (10mg/kg/day) or endostatin (10m/kg/day) every day.The contrast mouse is injected PBS solution every day.The result is shown in Fig. 9 C.Show that Arresten () is the same with endostatin (▲) good even better a little to the tumor growth retarding effect.Repeated experiments, but Arresen dosage is only used 4mg/kg/day.The result is shown in Fig. 9 D.Stop after 8 days handling (arrow), but when not applying Arresten in addition, in 12 days the obvious suppression effect is arranged still.After 12 days, the retarding effect of Arresten begins to disappear.

Embodiment 9: the transformation period of Arresten in the recycle system

Extract natural Arresten albumen from people's placenta, in the rat of body weight 200g, the dosage of every rat is 5mg by intravenous injection.Directly by of the existence of ELISA method with Arresten in the anti-Arresten antibody analysis different time serum.In contrast, also analyze the serum protein of each time point, must guarantee to use the serum of same amount.Find that the round-robin Arresten transformation period is approximately 36 hours in the serum.

Another group by and/or the subcutaneous injection mode inject people Arresten I.P., observe lung, kidney, liver, pancreas, spleen, brain, testis, the pathology sign of organs such as ovary.Carry out elisa assay, in these rat blood serums, detect Arresten antibody and exist, on glomerular basement membrane, find to have endogenic IgG settling to have this former also once observed (Kalluri, R. etc., 1994, PNAS USA 91:6201-5).Antibody settling in the kidney is not accompanied by any signs of inflammation or renal function damages.This hint Arresten be do not have pathogenic.

Embodiment 10:Arresten is to combination and the inhibition of enzyme MMP-2

MMP-2, the antibody of MMP-9 and these enzymes buy from Oncogene company and obtain.As described in the forefathers (Kalluri R. etc., 1994, PNAS USA 91:6201-5) does elisa assay with directly separating the natural A rresten that obtains from people's placenta, MMP-2 and MMP-9 have the specificity combination to Arresten.Their debond 7S structural domains.This combination does not rely on the combination of TIMP-2 and TIMP-1.

In order to assess the effect of Arresten, Matrigel is incubated 6 hours for 37 ℃ with MMP-2 and MMP-9, simultaneously slightly concussion to the degraded basilar membrane.With SDS-PAGE electrophoretic analysis supernatant, make immunoblot experiment with the antibody of type protein alpha 2 chains.Divide the beginning of testing in degraded, increase concentration gradually and add Arresten, observe the MMP2 activity inhibited.NC1 structural domain in the SDS-PAGE glue exists with monomer, dimeric forms, can do the experimental observation of the western marking with the proteic antibody of type and arrive.The Arresten that increases concentration gradually suppresses the Degradation of MMP-2 to basilar membrane.Show that Arresten can stop it to the effect of basilar membrane collagen proteolytic degradation in conjunction with MMP-2.Also obtained similar result for MMP-9.

Embodiment 11: production recombinant C anstatin in intestinal bacteria

Figure 10 represents the nucleotide sequence (SEQ IDNO:5) and the aminoacid sequence (SEQ ID NO:6) of type albumen NC1 structural domain α 2 chains.The sequence pcr amplification of coding Canstatin, with α 2 NC1 (IV)/pDS carrier is template (Neilson E.G. etc., 1993, J.Biol.Chem.268:8402-5), forward primer is 5 '-CGG GAT CCT GTC AGC ATCGGC TAC CTC-3 ' (SEQ ID NO:7), and reverse primer is 5 ' CCC AAG CTTCAG GTT CTT CAT GCA CAC-3 ' (SEQ ID NO:8).The cDNA segment that obtains is cut with Bamh I and Hind III enzyme, is connected to (Novagen company, Wisconsin, USA Madison) on pET22b (+) carrier that cuts in advance.The plasmid construction process as shown in figure 11.Be same reading frame with the downstream of Canstatin gene insertion pelB leading peptide and with it like this, albumen is secreted in the pericentral siphon chamber with the formal representation of soluble proteins.Other has one section extra carrier sequence MDIGINSD (SEQ ID NO:13) to be added on the albumen.Sequence 3 ' end links to each other with one section poly His-Tag sequence.One section carrier sequence encoding amino acid KLAAALE (SEQ ID NO:14) is arranged between cDNA and His-Tag sequence.Positive colony is confirmed through the two strands order-checking.

The plasmid that has a Canstatin is transformed into escherichia coli cell HMS174 (Novagen company, Wisconsin, USA Madison) at first.Be transformed into then in the BL21 cell and express (Novagen company, Wisconsin, USA Madison).The bacterium liquid of incubated overnight is inoculated in the 500ml LB substratum.Cultivated then about 4 hours, the time OD ₆₀₀Reach 0.6.The IPTG inducible protein that adds final concentration 5mM is expressed.After inducing two hours, the 5000g centrifugal collecting cell is with containing 6 M guanidines, 0.1M NaH ₂PO ₄, 0.01MTris.HCl, the damping fluid suspension cell of pH 8.0.The cell ultrasonic disruption that suspends, 12, centrifugal 30 minutes of 000g, supernatant cross the Ni-NTA agarose column (Qiagen, German Hilden) 4-6 time of a 5ml, flow velocity 2ml/min.The albumen of non-specific binding is respectively with the 10mM that contains of 15ml, 25mM, the 8M urea of 50mM imidazoles, 0.1MNaH ₂PO ₄, 0.01MTris-HCl, the damping fluid flush away of pH 8.0.Canstatin albumen is with respectively with containing 125mM and 250mM) the 8M urea of imidazoles, 0.1M NaH ₂PO ₄, 0.01M Tris-HCl, pH 8.0 buffer solution elution.The albumen of wash-out is dialysed twice at 4 ℃ to PBS.Wherein a part of albumen can precipitate during dialysis, collects the albumen of dialysing, and 3,500g is centrifugal, will precipitate with supernatant to separate.It is definite that the protein content of each part is analyzed (PierceChemical company, Illinois, USA Rockford) with BCA, quantitative with the SDS-PAGE electrophoretic analysis.The SDS-PAGE analysis revealed has a monomer band at 29 kDa places, and 3 extra kDa are from poly Linker and His-Tag sequence.Collection contains the proteic elution fraction of Canstatin, to the PBS dialysis, uses in order to following analysis.The Canstatin that SDS-PAGE and wester engram analysis are crossed detects with anti--His-Tag antibody.Also detect the recombinant C anstatin albumen of bacterial expression with IV collagen protein NC1 antibody.

The albumen of escherichia coli expression is mainly with the isolated in form of soluble proteins.The precipitation part accounts for 40% in the total protein, and remaining solution partly accounts for 60%.Proteinic ultimate production is approximately 15m/l.

The expression of embodiment 12:Canstatin in embryonic kidney cells 293.

PDS plasmid (the Neilson that contains α 2 (IV) NC1, E.G. etc., 1993, J.Biol.Chem.268:8402-5) be used to pcr amplification Canstatin, in this process, a segment signal peptide sequence can insert carrier for expression of eukaryon pcDNA3.1 by design and go up (In IV itrogen company, San Diego, California, USA).With the leader peptide sequences clone of 5 ' end of total length α 2 (IV) chain lead the NC1 zone 5 ' end so that protein secreting in substratum.Use the side primer that the recombinant vectors that contains Canstatin is checked order.Be further purified error-free cDNA clone, and be used for external Translation Study with the checking protein expression.Transform 293 cell (Kingstaon.R.E. with Calcium Chloride Method with the plasmid and the control plasmid that contain Canstatin, 1996, " calcium phosphate transfection " pp.9.1.4-9.1.7 sees: " up-to-date molecular biology method ", Ausubel, F.M., etc. volume, wiley and Sons company, New York, NY, USA).The clone who transforms uses gentamicin (Life Technologies/Gibco BRL company, Gaithersberg, Maryland, USA) antibiotic-screening.Three weeks of culturing cell under the situation that microbiotic exists are until there not being necrocytosis.The enlarged culturing growth is shaken in the bottle until converging at T-225 by population of cells.Collect supernatant and centrifugal concentrated then with amicon whizzer (Amicon company).Spissated supernatant SDS-PAGE, the expression of immuning hybridization and ELISA methods analyst Canstatin.Can detect the strong combination of supernatant by the ELISA method.The supernatant that contains Canstatin with the narrow spectrum antibody of Canstatin carry out affinity chromatography (Gunwar, S. etc., 1991, J.Biol.Chem.266:15318-24).Be separated to the monomer that a main peak contains the purifying of about 24 kDa, this monomer has for the immunocompetence of Canstatin antibody (anti--α 2 NC1 antibody, dilution in 1: 200).

Embodiment 13:Canstatin suppresses the propagation of endotheliocyte.

In containing the DMEM substratum of 10% foetal calf serum (FCS), cultivate calf Aorta endothelium (CAPE) cell and keep in touch and suppressed 48 hours.37 ℃ with trypsin Life Technologies/Gibco BRL company, Gaithersberg, Maryland, USA) digestion came collecting cell in 5 minutes.Be added in each hole with 24 orifice plates of 10ug/mlfibronectin bag quilt being suspended in 12,500 cells in the DMEM substratum that contains 0.5% foetal calf serum (FCS).At 37 ℃, culturing cell is 24 hours under 5%CO2 and 95% the humidity.Remove substratum and with the DMEM substratum replacing of the FCS that contains 0.5% FCS (unactivated) or 10% (cell that activates and handled).780-0, PC-3 and HEK 293 cells in contrast and grow to and converge, tryptic digestion and using the same method is layered on the flat board.Cell is handled with the Canstatin or the endostatin of triploid long-pending 0.025 to 40mg/ml concentration.Thymus pyrimidine in conjunction with test in, institute is porose the time all to add 1 millicurie in processing ³The H-thymus pyrimidine.After 24 hours, remove substratum and use PBS hole flushing three times.With the NaOH extracting cell of 1N and join and contain 4ml ScintiVerse II solution (Pennsylvania is USA) in the flicker bottle for Fisher Scientific company, Pittsburgh.Measure the combination of thymus pyrimidine with scintillometer.

The result shows as Figure 12 A and 12B.Figure 12 A is a histogram, has shown the influence of the Canstatin of different amounts for CAPE cell proliferation.Per minute thymus pyrimidine bonded counting is presented on the y-axle.0.5% is 0.5% FCS (unactivated) contrast on the x-axle, and 10% is that 10% FCS (activations) contrasts.Canstatin concentration processing with increasing amount can stably reduce the combination of thymus pyrimidine ground.Figure 12 A is a histogram, has shown at non-endotheliocyte 786-0, and the Canstatin of increasing amount influences in combination for thymus pyrimidine in PC-3 and HEK 293 cells.Per minute thymus pyrimidine bonded counting is presented on the y-axle, and what the x-axle showed is three clones handling with the Canstatin concentration of increasing amount, and 0.5% FCS (unactivated) and 10% FCS (activation) contrast.Each group is represented three times sample, and on behalf of the mean number of per minute, post add the average variance.

Also carried out the bromjophenol blue stain test.3,100 cells are added in each hole and handle as above, use then Oliver etc. method (Oliver etc., 1989, J.Cell.Science92:513-80) pair cell is counted.Each hole is cleaned once with 1 * PBS of 100ml, then each hole be added in 10% formaldehyde in the neutral buffered solution (Sigma chemical reagents corporation, St.Louis, Missouri, USA) room temperature is fixed 30 minutes.Remove formaldehyde, cell be used in 1% bromjophenol blue in the 0.01M borate buffer solution (pH 8.5) (Sigma chemical reagents corporation, St.Louis, Missouri, USA) room temperature dyeing is 30 minutes.With HCl/ ethanol (1: 1 mixture) the room temperature extracting bromjophenol blue of the 0.1N of 100ml one hour.(Califomia USA) upward measures with the photoabsorption of 655nm wavelength the painted amount of bromjophenol blue for BioRad company, Hercules at the small plate reader.

The result shows as Figure 12 C. and 12D.Figure 12 C is a histogram, has shown the influence of the Canstatin of different amounts for the absorption of CAPE stain for cell.OD ₆₅₅Absorption value is presented on the v-axle." 0.1% " representative be the contrast (unactivated) that 0.1% FCS handles, " 10% " is the contrast (activation) that 10%FCS handles.Little expression be that Canstatin with increasing amount concentration handles.In the CAPE cell, see that dyestuff absorbs minimizing in the unactivated cell that can handle at Canstatin with about 0.625-1.25 μ g/ml level.Figure 12 D is a histogram, has shown the exercising result of the Canstatin of different concns for non-endotheliocyte HEK 293 (white post) and PC-3 (column with slant lines).OD ₆₅₅Absorption value is presented on the y-axle." 0.1% " representative be the contrast (unactivated) that 0.1% FCS handles, " 10% " is the contrast (activation) that 10%FCS handles.On behalf of the relative absorbance units mean value of 655nm, post add the average variance in following 8 holes of each concentration of treatment.

Detect the ED that Canstatin suppresses for 10% serum activated endotheliocyte dose dependent ₅₀Value is about 0.5 μ g/ml (as Figure 12 A and 12C).When Canstatin dosage rises to 0.5mg/ml, do not observe for kidney tumor cell (786-0), prostate tumor cells (PC-3), perhaps human embryonic kidney cell (HEK293) has tangible action effect (as Figure 12 B and 12D).These endothelial cell specifics have shown that Canstatin may be a kind of very effective angiogenesis inhibitor reagent.

Embodiment 14:Canstatin suppresses endothelial cell migration.

In angiogenesis, endotheliocyte is not only bred but also is moved.Therefore, can estimate that Canstatin can act on the migration of endotheliocyte.Use the test of Boyden cell (Nero-Pro company, Cabin John, Maryland, USA) having detected Canstatin and endostatin has restraining effect for the FBS inductive chemotaxis of people's umbilical cord endotheliocyte (HUVECs).The HUVECs cell is grown in and contains 10%PBS and 5ng/mlDiIC18 (3) (Molecular Probes company, Eugene, Oregon, USA) M199 substratum (Life Technologies/Gibco BRL company, Gaithersberg, Maryland carries out active fluoro dyeing in USA) and spends the night.Behind the tryptic digestion, clean and diluting cells, 60,000 cells are joined add or do not add Canstatin (0.01 or 1.0mg/ml) in the hole of upper strata cell simultaneously with the M199 substratum that contains 0.5%PBS.The M199 substratum that contains 2%PBS is put into lower floor as chemoattractant.Contain the cell of cell and chemoattractant with the polycarbonate membrane in 8 μ m apertures (Poretics company, Livermore, California, USA) separately.Cell is at 37 ℃, 5%CO ₂With incubation under 95% the humidity 4.5 hours.Discard not migrating cell, go up confluent monolayer cells, scrape filter membrane, fix and be placed on the sheet glass with the PBS solution that contains 4% formaldehyde with plastic spatula with cleaning.Use high fluorescence visual field SenSys ^TMDigital camera is noted some independently same source images, and utilization PMIS (RoperScientific/Photometrics company, Tucson, Arizona, USA) image processing software is operated.Use the OPTIMIZE6.0 software program (Media Cybernetics, Rochester, NY) for cell count (Klemke, R.L. etc., 1994, J.Cell.Biol.127:859-66).

The result shows as Figure 13, this histogram has shown the number (y-axle) of the endotheliocyte of the migration in each visual field, these cells are not used VEGF (do not have VEGF or do not have serum) respectively, use to handle VEGF (containing 1%FCS and 10ng/ml VEGF), and the handling of Canstatin (Canstatin that contains 1%FCS and 10ng/mlVEGF and 1 μ g/ml) of using the Canstatin (Canstatin that contains 1%FCS and 10ng/ml VEGF and 0.01 μ g/ml) of 0.01 μ g/ml and 1 μ g/ml.

Observe when 10ng/ml Canstatin and have the obvious suppression effect for the propagation of HUVECs cell.Its not only working in a step of ability hint of Canstatin inhibition of endothelial cell proliferation and migration at vasculogenesis.Alternatively, thus Canstatin may can act on cell ground simultaneously and breeds and move as being excited ground endotheliocyte ground apoptotic signal.It is reported that apoptosis can be by agiostatin, the molecule of another angiogenesis inhibitor induce (O ' Reilly, M.S. etc., 1994, Cell 79:315-28; Lucas, R. etc., 1998, Blood 92:4730-41).

The generation of leather hose in embodiment 15:Canstatin suppresses

As the test of first Canstatin inhibition vasculogenesis ability, infer that he can stop endotheliocyte at matrigel, form pipe in promptly a kind of solid gums of mouse matrix membrane.When the mouse aortic endothelial cells was cultivated in matrigel, their very fast arrangements also formed empty tubular structure.

With matrigel (C ollaborative Biomedical Products company, Bedford, Massachusetts, USA) (320ml) be added in each holes of 24 orifice plates and make it polymerization (Grant, D.S. etc., 1994, Pathol.Res.Pract.190:854-63).Will do not contain antibiotic EGM-2 (Clonetics company, San Diego, California, USA) 25,000 mouse aortic endothelial cells (MAE) suspended substance in the substratum is added in each hole that is covered with matrigel.These cells Canstatin, BSA, aseptic PBS or α 5-NC1 district handle with the concentration that increases.All tests are all done three times.Cell was at 37 ℃ of incubation 24-28 hours and use CK2 Olympus microscopic examination (3.3 * eyepiece, 10 * object lens).Use 400 scribbles the TMAX films photograph (Kodak company) of DK.(Missouri USA) after about 4 weeks, is divided into one group of 4 mouse with these mouse to cell for Sigma chemical reagents corporation, St.Louis with the diff-quik fixing agent then.For test group (4 mouse) intradermal injection every day cumulative volume is the Canstatin of the 3mg/kg dosage of 0.2ml.Control group (4 mouse) gives the PBS of equal volume every day.Use the length of Vernier kind of calliper tumour and wide, and with the formula of standard: length x width 2 * 0.52 is calculated tumor size.Tumor size is from 26mm ³To 73mm ³, obtain differential gross tumor volume (V/V) divided by one day the volume of handling of the mat woven of fine bamboo strips with this for the gross tumor volume that calculates of fixing the date, as mentioned above.Comprise 5 mouse in each group.The result is presented among Figure 15 B, and what this figure showed is gross tumor volume (y the axle) ± standard variance of differential, to fate (x axle) mapping of handling.With respect to contrast (), the gross tumor volume that Canstatin (■) handles has only small increase, and this result and the result who handles acquisition with endostatin (zero) have tangible difference.

For the kidney cell knurl, will be in 200 ten thousand 786-0 cells be subcutaneously injected into 7 to 9 all big nude mouse bodies.Make tumor growth arrive about 100mm ³To about 700mm ³In each group 6 mouse are arranged.The Canstatin of intradermal injection every day 10mg/kg dosage injected 10 days.Control group gives the PBS of equal volume every day.The result is presented at Figure 15 C (100mm ³Tumour) and 31D (700mm ³Tumour) in.In two groups, the tumour of processing (■) contrast () of comparing has tangible atrophy.

The Canstatin that produces in the intestinal bacteria suppresses little (100mm ³Tumour, Figure 15 C) and big (700mm ³Tumour, Figure 15 D) growth of kidney cell knurl.For the human prostate tumour (PC-3) in the serious comprehensive immunodeficient mouse (SCID), the Canstatin of 10mg/kg dosage makes the relative volume of tumour be 55% of the mouse of only injecting carrier.In nude mouse, used the Canstatin and the endostatin of relative low dosage, the Canstatin of 3mg/kg dosage has the effect the same with the endostatin of 8mg/kg dosage.In the research, mouse shows as health in all bodies, old and feeble sign do not occur, does not have mouse death in the test.

Embodiment 17:Canstatin handles the CD31 immunohistochemistry of mouse

The minimizing of gross tumor volume has shown the restraining effect that these tumour medium vesselses are formed in the in vivo test.For detecting the tumour pipe, carried out the CD31 antibody alkaline phosphatase immunohistochemistry of paraffin embedding tumor biopsy.The tumor tissues that downcuts is cut into several thick fritters of about 3-4mm with razor used 4% formaldehyde fixed 24 hours then.Before dehydration and paraffin embedding, organize and change among the PBS.Behind paraffin embedding, tissue is cut into the 3mm slab and is fixed up.Section is taken off cured, aquation again, and with the proteolytic enzyme XX IV of 300mg/ml (Missouri is USA) 37 ℃ of pre-treatment 5 minutes for Sigma chemical reagents corporation, St.Louis.Ethanol termination reaction with 100%.It is air-dry to cut into slices, and seals in aquation and with 10% rabbit anteserum.Then slide with 1: 50 the dilution mouse-anti mouse CD31 monoclonal antibody (PharMingen company, San Diego, California, USA) be incubated overnight at 4 ℃, then at 37 ℃, incubation twice in 30 minutes, uses the anti-rat immune globulin of rabbit (DAKO) and the mouse APAAP (DAKO) of dilution in 1: 50 respectively.Carry out color reaction with new magenta.The section haematoxylin redyeing.

Can observe the reduced number of the tumour comparison of Canstatin processing according to the blood vessel of tumour.Embodiment 18: the recombinant production of Tumstatin and Tumstatin mutant in intestinal bacteria

Nucleotide sequence in the NC1 district of α 3 chains of type (SEQ ID N0:9) and aminoacid sequence (SEQ ID NO:10) be shown in Figure 16.The Tumstatin encoding sequence is from α 3 NC1 (IV)/pDS carrier (Neilson, E, G. etc., 1993, J.Boil.Chem.268:8402-5) come through pcr amplification, the front end primer that uses is: 5 '-CGG GATCCG GGT TTG AAA GGA AAA CGT-3 ' (SEQ ID NO:11), the rear end primer is: 5 '-CCC AAG CTT TCA GTG TCT TTT CTT CAT-3 ' (SEQ IDNO:12).The fragment that obtains is with BamH I and the enzymic digestion of Hind III, be connected to predigested pET22b (+) (Novagen company, Madison, Wisconsin, USA) on.Make up as shown in figure 17.Tumstatin connects and is fused to the downstream of pelB leading peptide, thereby makes it can navigate to periplasmic space and expression soluble protein.Add the additional sequence encoding amino acid sequence MDIGINSD (SEQ ID NO:13) on the protein to.3 ' end of sequence is connecting the polyhistidine binding sequence.At the 3 ' end of cDNA and the carrier sequence encoding amino acid sequence KLAAALE (SEQ IDNO:12) of the interpolation between the Histidine binding sequence.Positive colony checks order on two chains.The plasmid structure of coding Tumstatin be transformed into before this intestinal bacteria HMS174 (Novagen company, Madison, Wisconsin, USA) in, (Wisconsin expresses in USA) for Novagen company, Madison to be transformed into BL21 then.At 500ml LB substratum (Fisher Scientific company, Pittsburgh, Pennsylvania, USA) middle incubated overnight bacterial cultures.Culture is grown 4 hours up to reaching OD ₆₀₀Value be 0.6 add then IPTG to final concentration be the expression of 1mM with induced protein.After inducing 2 hours, also use 6M guanidine, 0.1M NaH with the centrifugal collection thalline of 5,000 * g ₂PO ₄, 0.01M Tris-HCl, pH 8.0 resuspended thalline.Resuspended thalline ultrasonic disruption, with 12,000 * g centrifugal 30 minutes.The supernatant part is passed through Ni-HTA agarose column (Qiagen company, Hilden, Germany) 4-6 time of 5ml with the speed of per minute 2ml.With the imidazoles that contains 10mM and 25mM, 8M urea, 0.1M NaH ₂PO ₄, 0.01M Tris-HCl, the solution of pH 8.0 cleans pillar and removes the protein of non-specific binding.At 8M urea, 0.1M NaH ₂PO ₄, 0.01MTris-HCl, the concentration (50mM, 125mM, and 250mM) that increases imidazoles in the damping fluid of pH 8.0 washes down protein from pillar.The protein of wash-out is dialysed twice to PBS at 4 ℃.Part in dialysis procedure in the total protein precipitates.Collect the protein of dialysis, it is divided into insoluble part (precipitation) and soluble part (supernatant) in that about 3,500 * g is centrifugal.

The Tumstatin of escherichia coli expression mainly separates with soluble protein, and SDS-PAGE analyzes the band that is shown as one 31 kDa.3 kDa that increase are from joint and Histidine binding sequence.Contain the test that this big part is used to next step.(Illinois USA) detects proteinic concentration in each part for Pierce chemical company, Rockforrd, uses the scanning concentration measuring apparatus to carry out quantitative SDS-PAGE and analyzes by the BCA test.Can see the band that has to represent dimeric 60 kDa of Tumstatin under non-reduced condition, this band is reduced to the monomer of 31 kDa under reductive condition.Proteinic total harvest yield is every liter of 5mg.

The truncated Tumstatin (Tumstatin-N53) of reorganization lacks the amino acid of 53 N end, expression in escherichia coli and purifying such as another mutant (Kalluri, R. etc., 1996, J.Biol.Chem.271:9062-8).This mutant as shown in figure 18, this composition diagram has shown truncated amino acid whose position in α 3 (IV) NC1 monomer.53 amino-acid residues that filled circles has been represented to leave out N-end from Tumstatin with produce " Tumstatin-N53 " (Kalluri, R. etc., 1996, J.Biol.Chem.271:9062-8).Disulfide linkage indicates with short-term, and their location is that different (Siebold waits 1988, J.Biochem.176:617-24) in α 1 (IV) NC1 and α 2 (IV) NC1.For clarity sake, among the figure only indicate two disulfide linkage structures that may exist.

The preparation method of the rabbit antibody of anti-people α 3 (IV) NC1 as previously mentioned (Kalluri, R. etc., 1997, J.Clin.Invest.99:2470-8).Mono-clonal mouse-anti mouse CD31 monoclonal antibody available from PharMingen company (PharMingen company, San Diego, California, USA).FITC bonded sheep anti-mouse igg antibody, FITC bonded goat anti-rabbit igg antibody, and in conjunction with the goat anti-rabbit igg antibody of horseradish peroxidase available from Sigma chemical reagents corporation (Sigma chemical reagents corporation, St.Louis, Missouri, USA).

With the expression of Tumstatin in the spissated supernatant of SDS-PAGE and immuning hybridization analysis acquisition, method is (Kalluri, R. etc., 1996, J.Biol. Chem.271:9062-8) as previously mentioned.Carry out one to SDS-PAGE in the discontinuous buffering system of separation gel 12%.Isolating protein transduction is moved on on the nitrocellulose filter BSA room temperature sealing with 2% 30 minutes.After having sealed remaining binding site, clean film with cleaning buffer solution, containing 1%BSA, incubation among the PBS who resists of dilution in 1: 1000.Room temperature is incubated overnight on shaking table.Clean film with cleaning buffer solution then, use in conjunction with two anti-room temperature shaking table incubations of horseradish peroxidase 3 hours.And then the cleaning Hybond membrane, add substrate (in the phosphate buffered saline buffer of 0.05M, contain 0.01% cobalt chloride and nickel ammonium and diamino is connected benzene) and room temperature incubation 10 minutes.Pour out substrate solution then, add the substrate buffer solution that contains hydrogen peroxide.After demonstrating band, with distilled water termination reaction and desciccator diaphragm.Can see the band of one 31 kDa.

The expression of embodiment 19:Tumstatin in 293 embryonic kidney cells

Use eukaryotic vector pcDNA3.1, human Tum-5 statin can be used as a kind of excretory soluble protein and expresses in 293 embryonic kidney cells.This recombinant protein (not containing any purifying and detection site) can carry out purifying with affinity chromatography, and detects a kind of pure monomeric form with SDS-PAGE and immuning hybridization analysis from main peak.

Contain α 3 (IV) NC1 (Neilson, E.G. etc., 1993, J.Biol.Chem.268:8402-5) pDS plasmid is used to pcr amplification Tumstatin, in this process, can add one section leader peptide sequences and be fused to carrier for expression of eukaryon pcDNA3.1 (Invitrogen company with reading frame, San Diego, California, USA) on.The leading peptide of 5 ' end of total length α 3 (IV) chain is cloned into the 5 ' end in NC1 district so that protein can be secreted in the substratum.Use the side primer that the recombinant vectors that contains Tumstatin is checked order on two chains.Be further purified error-free cDNA clone, and be used for external Translation Study with the checking protein expression.By Calcium Chloride Method with contain the plasmid of Tumstatin and control plasmid transform 293 cells (Sambrook, J. etc., 1989, the molecular cloning experiment guide, cold spring harbor laboratory, Cold Spring Habour, New York, USA, pps.16.32-16.40).The clone who transforms uses gentamicin (Life Technologies/Gibco BRL company, Gaithersberg, Maryland, USA) antibiotic-screening.Three weeks of culturing cell under the situation that microbiotic exists are until there not being necrocytosis.The enlarged culturing growth is shaken in the bottle until converging at T-225 by population of cells.Collect supernatant and centrifugal concentrated then with amicon whizzer (Amicon company).Spissated supernatant SDS-PAGE, the expression of immuning hybridization and ELISA methods analyst Tumstatin.Can detect the strong combination of supernatant by the ELISA method.

The supernatant that contains Tumstatin with the narrow spectrum antibody of Tumstatin carry out affinity chromatography (Gunwar, S. etc., 1991, J.Biol.Chem.266:15318-24).Be separated to the monomer that a main peak contains the purifying of about 31 kDa, this monomer has for the immunocompetence of Tumstatin antibody (anti--α 2 NC1 antibody, dilution in 1: 200).

Embodiment 20:Tumstatin inhibition of endothelial cell proliferation

The protein that uses intestinal bacteria to produce passes through ⁵The H-thymus pyrimidine detects the antiproliferative activity of Tumstatin to the C-PAE cell in conjunction with test.

Clone and culture 786-0 (kidney tumor cell system), PC-3 (human prostate tumor cell line), C-PAE (ox pulmonary artery endothelial cell system), MAE (mouse aortic endothelial cells system) is available from USS culture collection center.786-0 and C-PAE clone are deposited in DMEM (Life Technologies/Gibco BRL company, Gaithersberg, Maryland, USA) in, ECV-304 clone is in M199, MAE clone is at EGM-2 (Clonetics company, San Diego, California, USA) in, be supplemented with 10% foetal calf serum (FCS), the gentamicin of 100 units/ml and the Streptomycin sulphate of 100mg/ml in the substratum.

Proliferation test .C-PAE cell is grown in the DMEM substratum that contains 10% foetal calf serum (FCS) and keeps in touch and suppressed 48 hours.The C-PAE cell is used to second and the quadravalence section.786-0 and PC-3 cell are used as the contrast of non-endotheliocyte in this experiment.With trypsin Life Technologies/Gibco BRL company, Gaithersberg, Maryland, USA) came collecting cell in 5 minutes 37 ℃ of digestion.To be added at 12,500 cells in containing the DMEM substratum of 0.5% foetal calf serum (FCS) in each hole with 24 orifice plates of 10 μ g/mlfibronectin bag quilt.At 37 ℃, 5%CO ₂With culturing cell under 95% the humidity 24 hours.Remove substratum and change with the DMEM substratum that contains 0.5% FCS.Unactivated control cells is cultivated with 0.5% FCS.Cell is handled with 0.01 Tumstatin to 10mg/ml concentration.Porose 1 millicurie that after beginning to handle 12 hours, all adds ³The H-thymus pyrimidine.After 24 hours, remove substratum and with dyeing and take a picture once more (Grant, D.S. etc., 1994, Pathol.Res.Pract.190:854-63).Observe ten visuals field, pipe is counted and averaged.

The result shows as Figure 14, the figure illustrates and is using BSA () respectively, and the endothelium tubing that Canstatin (■) and α 5NC1 (zero) handle forms the per-cent (y axle) of quantity and contrast (PBS processing) formation.The longitudinal axis is represented the square root of the variance of mean value.The result shows that greatly having reduced the endothelium pipe with respect to contrast ground Canstatin forms.

Leather hose ground formed in Canstatin dose dependent ground selectivity suppressed, can be near the formation (Figure 14) of killer tube fully under the Canstatin of 1mg protein concn.And control protein, bovine serum albumin (BSA), the NC1 district of α 5 chains of type demonstrates in this test all for not influence of the formation of interior leather hose, for restraining effect be narrow spectrum, rather than add proteinic cause.It is a kind of angiogenesis inhibitor reagent that this result demonstrates Canstatin.

Embodiment 16:Canstatin suppresses the interior growth of body of tumour.

Collector's prostate cancer cell from culture (PC-3 cell) also is subcutaneously injected into 200 ten thousand cells in aseptic PBS in 7 to 9 all big SCID male mouse bodies.Tumor growth is divided into one group of 4 mouse with these mouse after 4 weeks.For test group intradermal injection every day cumulative volume is the Canstatin of the 10mg/kg dosage of 0.1ml.Control group gives the PBS of equal volume every day.When on-test (0 day), the tumor size of control mice is 88-135mm ³, and the mouse of handling is 108-149mm ³Comprise 5 mouse in each group.Obtain differential gross tumor volume (V/V) divided by first day the volume of handling with this for the gross tumor volume that calculates of fixing the date.The result is presented at Figure 15 A, and what this figure showed is gross tumor volume (v the axle) ± standard variance of differential, to fate (x axle) mapping of handling.With respect to contrast (), the gross tumor volume that Canstatin (■) handles has only small increase.

In another one PC-3 test, collecting the PC-3 cell from culture also will be in 300 ten thousand cells be subcutaneously injected into 6 to 7 all big nude mouse bodies.The big PBS hole flushing of tumor growth three times.With the NaOH extracting of 1N put cell and join contain 4mlScirtiVerse II solution (Fisher Scientific company, Pittsburgh, Pennsylvania, USA) flicker bottle in.Measure the combination of thymus pyrimidine with scintillometer.

The result shows as Figure 19 A and 19B.Figure 19 A is a histogram, shown when the Tumstatin (x axle) with different amounts handles, ³H-thymus pyrimidine (y axle) and C-PAE cell (Figure 19 A), the combination of 786-0 cell (Figure 19 B) and PC-3 cell (Figure 19 C).Each group is represented three times sample.Tumstatin obviously has dose dependent for 20%FCS activated endotheliocyte and suppresses ED ₅₀Value is about 0.01mg/ml (as Figure 19 A.).Even when dosage rises to 20mg/ml, do not observe for kidney tumor cell (786-0), perhaps prostate tumor cells (PC-3) has tangible antiproliferative effect (as Figure 19 B and 19C).These endothelial cell specifics have shown that Tumstatin may be a kind of very effective angiogenesis inhibitor reagent.In the cell that Tumstatin (0.1-1.0mg) handles and in the control cells, ³During the difference of H-thymus pyrimidine bonded mean value significantly (P＜0.5).When handling PC-3 cell or 786-0 cell, do not observe restraining effect (as Figure 19 B. and 19C) with Tumstatin.The data that have the asterisk mark are significant, use OneTailed Student ' s to detect and confirm its P＜0.05.

The generation of leather hose in embodiment 21:Tumstatin suppresses

With matrigel (Collaborative Biomedical Products company, Bedford, Massachusetts, USA) (320ml) be added in each holes of 24 orifice plates and make it polymerization (Grant, D.S. etc., 1994, Pathol.Res.Pract.190:854-63).Will do not contain antibiotic EGM-2 (Clonetics company, San Diego, California, USA) 25,000 mouse aortic endothelial cells (MAE) suspended substance in the substratum is added in each hole that is covered with matrigel.These cells Tumstatin, BSA or 7S district handle with the concentration that increases.Control cells is handled with aseptic PBS.All tests are all done three times.Cell was at 37 ℃ of incubation 24-28 hours and use CK2 Olympus microscopic examination (3.3 * eyepiece, 10 * object lens).Use 400 scribbles the TMAX films photograph (Kodak company) of DK.Then cell with the diff-quik fixing agent (Sigma chemical reagents corporation, St.Louis, Missouri, USA) dyeing and take a picture once more (Grant, D.S. etc., 1994, Pathol.Res.Pract.190:854-63).Observe ten visuals field by two for the uncomprehending viewer of this testing sequence, pipe is counted and averaged.

The result shows as Figure 20.When the mouse aortic endothelial cells is cultivated at matrigel, in the time of in a kind of solid gums of mouse matrix membrane, their very fast arrangements and form empty tubular structure (Haralabopoulos, G.C etc., 1994, Lab.Invest.71:575-82).By the Tumstatin that produces in 293 cells, compare the formation (Figure 20) of leather hose in dose dependent suppresses in the MAE cell significantly with the BSA contrast.After handling with the protein of 1mg/ml, the percentage BSA that the endothelium pipe forms is 98.0 ± 4.0, and Tumstatin is 14.0 ± 4.0.The Tumstatin that uses intestinal bacteria to produce also can obtain identical result.The 7S district of type (the terminal non-collegenous dormain of N-) is for the not effect of formation of interior leather hose.When concentration was 800-1000ng/ml, Tumstatin can obtain maximum restraining effect.That handle with Tumstatin (●, 0.1 to 10g/ml) and contrast [BSA ()], the difference of the average percentage value of 7S district (zero) is tangible (P＜0.5).Every bit is represented the SE in mean value ± three holes.This tests triplicate.The pillar that has the asterisk mark is the follow-up test that the result did to P＜0.5.In the test that the 7S district handles, can observe the pipe of good formation.Handle the formation that the MAE cell demonstrates the pipe of minimizing with 0.8mg/ml Tumstatin.

For estimating the effect that Tumstatin forms for new kapillary in vivo, carried out matrigel Implantation Test (Passaniti, A. etc., 1992, Lab Invest.67:519-29).(Marine USA) obtains the C57/BL6 mouse in 5 to 6 ages in week for Jackson laboratory, Bar Harbor from the Jackson laboratory.(Massachusetts is USA) 4 ℃ of soaked overnight for Collaborative BiomedicalProducts company, Bedford with matrigel.Before giving the injection of C57/BL6 mouse, with the 20U/ml heparin (Pierce chemical company, Rockdord, Illinois, USA), the bFGF (R﹠amp of 150ng/ml; D System company, Minneapolis, Minnesota, USA) and the Tumstatin of 1mg/ml mix.There is not angiogenesis inhibitor in the control group.Syringe needle subcutaneous injection matrigel mixture with No. 21.After 14 days, put to death mouse and take out the matrigel implant.Formaldehyde with 4% (among the PBS) room temperature is the matrigel implant fixedly, places 24 hours in changing over to then.Implant is carried out paraffin embedding, section and H﹠amp; E dyeing.Inspection is cut into slices and in 10 high power fields the blood vessel number is counted under opticmicroscope.All sections all are to be numbered and observed by a pathologist who does not know this research process.

When the matrigel of implanting contained with heparin, whether what no matter use was the Tumstatin that intestinal bacteria produce, and can both observe angiopoietic reduced number 70% with the Tumstatin processing of 1m/ml.Number of blood vessel under each high power field is, Tumstatin, and 2.25 ± 1.32, and contrast, 7.50 ± 2.17.Each pillar has been represented mean value ± SE of every group of 5-6 mouse.Tumstatin (1mg/ml) and the formation of handling with PBS of comparing the intravital neovascularity of obvious inhibition.The difference of animal that Tumstatin handles and the average percentage value of control animal is tangible (P＜0.05).The processing of Tumstatin is significantly, and its P＜0.05th detected determined by One Tailed Student ' s.Embodiment 22:Tumstatin and Tumstatin mutant suppress intravital tumor growth

Collect 500 ten thousand PC-3 cells and be subcutaneously injected into the back of male nude mouse in 7 to 9 ages in week.Use the length of Vernier kind of calliper tumour and wide, and with the formula of standard: length x width ²* 0.52 calculates tumor size.Make tumor growth to 100mm ³, these mouse are divided into one group of 5 or 6 mouse.Use Tumstatin or mouse endostatin abdominal injection every day (20mg/kg) among the aseptic PBS respectively for each test group, injected altogether 10 days.Control group gives vector injection (BSA or PBS).Tumor size of calculating in per 2 or 3 days after 10 days.The result shows as Figure 21 A, the figure illustrates gross tumor volume (mm ³) for contrasting () with PBS, 20mg/kg Tumstatin (●) and 20mg/kg endostatin (zero) handle the figure that fate (x axle) is done (y axle).The Tumstatin that produces in the intestinal bacteria can obviously suppress the growth (as Figure 21 A) of PC-3 human prostate tumour cell.The Tumstatin of 20mg/kg and the endostatin of 20mg/kg can suppress growth of tumor equally.The restraining effect that can observe significantly for tumor growth at the tenth day (contrasts 202.8 ± 50.0mm ³, Tumstatin82.9 ± 25.2mm ³, endostatin68.9 ± 16.7mm ³).With compare, inject the growth that Tumstatin or endostatin can suppress human prostate tumour cell (PC-3) every day.This test is to work as gross tumor volume less than 100mm ³In time, begin.

In mouse, studied simultaneously the effect of Tumstatin for another primary tumo(u)r.Collect 200 ten thousand 786-0 cells and be subcutaneously injected into the back of male nude mouse in 7 to 9 ages in week.Make tumor growth arrive about 600mm ³To about 700mm ³, these mouse are divided into one group of 6 mouse.Be used in Tumstatin abdominal injection every day (6mg/kg) among the aseptic PBS, injected altogether 10 days.Control group gives PBS injection.The result shows as Figure 21 B, the figure illustrates gross tumor volume (mm ³) (y axle) figure of being done for the fate (x axle) after handling with PBS contrast () and 6mg/kg Tumstatin (●).The Tumstatin that produces in the intestinal bacteria can obviously suppress the growth (as Figure 21 B) of PC-3 people's kidney tumor cell when 6mg/kg.The restraining effect that can observe significantly for tumor growth at the tenth day (contrasts 1906 ± 179.7mm ³, Tumstatin 619 ± 120.7mm ³).With compare, inject the growth that Tumstatin can suppress people's kidney tumor cell (786-O) every day.This test be when gross tumor volume be 600-700mm ³In time, begin.Mean value ± SE of 5-6 mouse in each group of every bit representative.The pillar that has the asterisk mark is the follow-up test that the result did to P＜0.5.

The part in the NC1 district of α 3 chains of type [α 3 (IV) NC1] is antigenic determinant (Butkowski, R.J. etc., 1987, the J.Biol.Chem.262:7874-7 of the cause of disease of Goodpasture syndromes; Saus, J. etc. 1988, J.Biol.Chem.263:13374-80; Kalluri, R. etc., 1991, J.Biol.Chem.266:2401 8-24).The Goodpasture syndromes is a kind of autoimmune disease, shows as that lung goes out and/or glomerulonephritis (Wilson, C.﹠amp; F.Dixon, 1986, The Kidney, W.B.Sanders company, Philadelphia, Pennsylvania, USA, pps.880-89; Hudson, B.G. etc., 1993, J.Biol.Chem.268:16033-6).These symptoms are because the combination of the autoantigen of anti-α 3 (IV) NC1 causes the basilar membrane of renal glomerulus and alveolar be interfered (Wilson, 1996, supra; Histon 1993, supra).There are several research groups to attempt to predict position [Kalluri.R. etc., 1995, the J.Am.Soc.Nephrol.6:1178-85 of Goodpasture syndromes autoantigen on α 3 (IV); Kalluri.R. etc., 1996, J.Biol.Chem.271:9062-8; Lery, J.B. etc., 1997, J.Am.Soc.Nephrol.8:1698-1705; Kefalides, N.A. etc., 1993, Kidney Int.43:94-100; Quingones, S. etc., 1992, J.Biol.Chem.267:19780-4 (correcting errors in printing) at 269:17358; Netzer, K.O etc., 1999, J.Biol.Chem.274:11267-74], at the N end, it is position at the bottom of the antigenic determinant that the residue in C end and mid-way all was in the news.Recently, the cause of disease determinant at the N end of more possible disease-related is identified out (Hellmark, T. etc., 1999, Kidney Int.55:936-44), further determines it is 40 amino acid of N end.Designed a kind of brachymemma 53 the amino acid whose tumstatin (Tumstatin-N53) that lack the N end, take place relevant with the cause of disease of the autoantigen determinant of Goodpasture.This mutain is used for following test.

200 ten thousand 786-0 cells are subcutaneously injected into the back of the male nude mouse in 7 to 9 ages in week.Making tumor growth is 100-150mm to about size ³, these mouse are divided into one group of 5 mouse.With Tumstatin abdominal injection every day (20mg/kg) that lacks 53 N terminal amino acid residues of expression in escherichia coli, injected altogether 10 days.Control mice gives PBS injection.The result shows as Figure 22, the figure illustrates gross tumor volume (mm ³) increase (y axle) for contrast mouse (), the figure that the fate (x axle) after 20mg/kg Tumstatin mutant (●) is handled is done.The Tumstatin-53 that produces in the intestinal bacteria can obviously suppress the growth (as Figure 22) of 786-0 people's kidney tumor cell when 20mg/kg.With compare, inject every day Tumstatin can obviously suppress people's kidney tumor cell (786-O) growth (the tenth day: the contrast 110.0 ± 29.0mm ³, Tumstatin345.0 ± 24.0mm ³).This test be when gross tumor volume be 600-700mm ³In time, begin.Mean value ± SE of 5-6 mouse in each group of every bit representative.The data that have the asterisk mark are significant, detect with One Tailed Student ' s and determine its P＜0.05.

Embodiment 23: the immunohistochemical staining analysis of α 3 (IV) NC1 and CD31

Get the 7-9 male C57/BL6 mouse kidney and the skin histology in age in week, be used for the immunofluorescence microscopy analysis through handling.Tissue sample is handled with liquid nitrogen freezing, and it is standby to be cut into the 4mm slab.Tissue with as the processing of the described IiT of forefathers (Kalluri, R. etc., 1996, J.Biol.Chem.271:9062-8).Refrigerated section one antibody, i.e. anti-CD31 antibody of polyclone (dilution in 1: 100) or the anti-α 3 of polyclone (IV) NC1 antibody (dilution in 1: 50) dyeing.Add two then and resist, the anti-human IgG antibody that Chinese People's Anti-Japanese Military and Political College's mouse IgG antibody that FITC connects or FITC connect.Check immunofluorescence with Olympus fluorescent microscope (Tokyo, Japan).In negative control, replace one to resist with an incoherent preceding immune serum.

In the mouse kidney, in GBM and basement membrane of blood vessel, observe the expression of α 3 (IV) NC1.In renal glomerulus endotheliocyte and vascular endothelial cell, can be observed CD31, the expression of PECAM-1.In mouse skin, in basement membrane of epithelium and basement membrane of blood vessel, there is not α 3 (IV) NC1.In dermal endothelial cell, observe the expression of CD331.In mouse kidney renal glomerulus endotheliocyte and little blood vessel, observe the expression of CD31.Outside glomerular basement membrane and renal glomerulus, observe the expression of α 3 (IV) NC1 in the basement membrane of blood vessel.In mouse skin, observe the expression of CD31 in the little blood vessel of endothelium.In basement membrane of epithelium, do not have the expression of α 3 (IV) NC1, almost do not observe the expression of α 3 (IV) NC1 in the little basement membrane of blood vessel of epithelium.These results show the embodiment that the canstatin restriction distributes.

Embodiment 24: the mutant of anti-angiogenic proteins and segment

The pseudomonas elastin enzymic digestion of proteic mutant of Arresten and canstatin and segment (Mariyama etc., 1992, J.Biol.Chem.267:1253-8) obtain.Digestion product is handled with gel-filtration HPLC, and the SDS-PAGE electrophoretic analysis of resulting segment is with above-described interior cutaneous vessel analytical method analysis and evaluation.These segments comprise the 12 kDa segments of Arresten, the segment of 8 kDa of Arresten, the segment of one 10 kDa of canstatin.In addition, obtain the segment (" 333 " and " 334 ") of two canstatin by the PCR clone.

As shown in figure 23, foregoing interior cutaneous vessel analysis, the segment [19 kDa (▲)] of two Arresten segments [12 kDa () and 8 kDa (■)] and canstatin suppresses interior cutaneous vessel and forms, its effect even also stronger than Arresten (●) or canstatin (zero).Figure 24 represents that canstastin segment 333 (●) is the same with 334 (zero) effect and likens to the canstatin (▲) of contrast is strong, with BSA (■) and α 6 chains () in contrast.

The document of all citations, patent, and patent application is all by all being incorporated in this paper in this citation.Though the present invention has done special explanation and description at this with embodiment, but to those skilled in the art, under the condition of not leaving essence of the present invention in the claim and scope, change on the various forms of routine techniques and details can also be arranged.

Claims

1. isolating protein, be selected from one of following: the NC1 district of α 1 chain of type, the NC1 district of α 2 chains of type, the perhaps NC1 district of α 3 chains of type, perhaps their fragment, analogue, derivative or their mutant, wherein, protein, fragment, analogue, derivative or their mutant all have the angiogenesis inhibitor characteristic.

2. the isolating protein of claim 1, protein wherein is a kind of monomer.

3. the isolating protein of claim 2, protein wherein is to catch peptide (Arresten).

4. the isolating protein of claim 2, protein wherein is Canstain.

5. the isolating protein of claim 2, protein wherein is Tumstatin.

6. the proteinic polymer of a claim 1, perhaps their fragment, analogue, derivative or their mutant, polymer wherein has the characteristic of angiogenesis inhibitor.

7. chimeric protein, it comprises the protein of one or more claims 1, perhaps their fragment, analogue, derivative or their mutant, chimeric protein wherein has the characteristic of angiogenesis inhibitor.

8. the chimeric protein of claim 7, further contain and be selected from one of following at least a kind of protein molecular: endostatin or its fragment, angiostatin or its fragment, restin or its fragment, apomigren or its fragment, perhaps other anti-angiogenic proteins matter or its fragment.

9. a composition as a kind of bioactive ingredients, contains the protein of one or more claims 1.

10. the composition of claim 9, and pharmaceutically-compatible filler.

11. composition, as a kind of bioactive ingredients, the protein that contains one or more claims 1, also contain and be selected from following at least a protein molecule: endostatin or its fragment, angiostatin or its fragment, restin or its fragment, apomigren or its fragment, perhaps other anti-angiogenic proteins matter or its fragment.

12. a composition as a kind of bioactive ingredients, contains the polymer of claim 6.

13. a composition as a kind of bioactive ingredients, contains the chimeric protein of claim 7.

14. an isolating polynucleotide, the protein of coding claim 1, perhaps their fragment, analogue, derivative or their mutant, chimeric protein wherein has the characteristic of angiogenesis inhibitor.

15. a kind of isolating polynucleotide of claim 14, polynucleotide wherein can be operationally connected on the expression control sequenc.

16. polynucleotide transformed host cells with claim 15.

17. the host cell of claim 16, host cell wherein is selected from bacterium, yeast, mammals, insect or vegetable cell.

18. an isolating polynucleotide, the protein of coding claim 3.

19. an isolating polynucleotide, the protein of coding claim 4.

20. an isolating polynucleotide, the protein of coding claim 5.

21. a method of producing the polynucleotide encoded protein matter of claim 14, this method comprises:

(a) the polynucleotide transformed host cells of cultivation claim 14, the host cell of nucleic acid is selected from bacterium, yeast, mammals, insect or vegetable cell;

(b) protein purification from culture;

Produce the polynucleotide encoded protein matter of claim 14 thus.

22. an isolating polynucleotide obtains according to following technology:

(a) a kind of or more polymerized nucleoside acid probe of preparation, this probe under certain condition and suitable rigorous degree can with the polymerized nucleoside acid hybridization of claim 14;

(b) with this probe and mammalian DNA hybridization;

(c) separate the DNA polynucleotide that arrives with probe in detecting; The nucleotide sequence of the isolating polynucleotide of nucleic acid is consistent with the nucleotide sequence of the polynucleotide of claim 14.

23. give the method for Mammals anti-angiogenic proteins matter, this method comprises mammalian cell is imported in the mammalian body, described mammalian cell is at the polynucleotide of extracorporeal treatment with insertion claim 14, and going out the anti-angiogenic proteins matter of dose therapeutically effective at this mammiferous expression in vivo, this dosage is enough to suppress the angiogenic activity in the Mammals.

24. the method for claim 23, the expression of antagonism angiogenin protein is a transient expression.

25. the method for claim 23, the cell of nucleic acid is selected from: blood cell, til cell, medullary cell, vascular cell, tumour cell, liver cell, muscle cell, fibroblast.

26. the method for claim 25, the polynucleotide of nucleic acid inserts in the cell by virus vector.

27. specificity is in conjunction with the antibody of isolating protein, analogue, deutero-homologue or their mutant of claim 1.

28. method that in mammalian tissues, suppresses angiogenic activity, this method comprises with a kind of composition handles tissue, said composition contains following one or several: the isolating protein of claim 1 or their fragment, analogue, derivative or their mutant, the proteinic polymer of claim 1, the segmental polymer of claim 1, a kind of proteinic chimeric protein that comprises one or several claims 1, perhaps a kind of chimeric protein that contains the protein fragments of claim 1.

29. the method for claim 28, the disease of nucleic acid comprises: angiogenesis-dependent tumors, innocent tumor, rheumatic arthritis, diabetic retinopathy, psoriasis, vision vasculogenesis disease, the Osler-Webber syndromes, cardiac muscle vasculogenesis, sheet vasculogenesis, telangiectasis, the hemophilia tight knot, hemangiofibroma, wound granulation, intestinal adhesion, arteriosclerosis, scleroderma, antihyperuricemic, the cat scratch fever disease, helicobacter pylori ulcer, angiostenosis due to dialysis is transplanted, contraception and obesity.

30. the method for claim 29, the disease of nucleic acid is a tumour.

31. use the composition of claim 28 to suppress to have the disease of the active symptom of blood vessel hyperplasia, this method comprises the patient's administration for this disease, said composition is in conjunction with radiotherapy, chemotherapy and immunotherapy.

32. a peptide species, its 2nd of comprising SEQ ID NO:10 is to the 125th amino acids, and this polypeptide has anti-angiogenesis activity.

33. the polynucleotide of the polypeptide of the claim 32 of encoding.

34. a peptide species, its 125 of mat woven of fine bamboo strips that comprise SEQ ID NO:10 is to the 245th amino acids, and this polypeptide has anti-angiogenesis activity.

35. the polynucleotide of the polypeptide of the claim 34 of encoding.

36. the NC1 district fragment of α 1 chain of the type with anti-angiogenesis activity, this fragment obtains by the PCR cloning process.

37. the NC1 district fragment of α 2 chains of the type with anti-angiogenesis activity, this fragment obtains by the PCR cloning process.

38. the NC1 district fragment of α 3 chains of the type with anti-angiogenesis activity, this fragment obtains by the PCR cloning process.

39. the NC1 district fragment of α 1 chain of the type with anti-angiogenesis activity, this fragment obtains by pseudomonas (Pseudomonas) elastoser digestion method.

40. the angiogenesis inhibitor fragment of claim 39, this segmental size is 12 KDa.

41. the angiogenesis inhibitor fragment of claim 39, this segmental size is 8 KDa.

42. the NC1 district fragment of α 2 chains of the type with anti-angiogenesis activity, this fragment obtains by pseudomonas (Pseudomonas) elastoser digestion method.

43. the angiogenesis inhibitor fragment of claim 42, this segmental size is 10 KDa.

44. the NC1 district fragment of α 3 chains of the type with anti-angiogenesis activity, this fragment obtains by pseudomonas (Pseudomonas) elastoser digestion method.