CN1835763A

CN1835763A - CCN1 compositions and methods

Info

Publication number: CN1835763A
Application number: CNA2004800232747A
Authority: CN
Inventors: L·F·劳
Original assignee: Munin Corp
Current assignee: Munin Corp
Priority date: 2003-06-20
Filing date: 2004-06-21
Publication date: 2006-09-20
Anticipated expiration: 2024-06-21
Also published as: WO2005040191A3; EP1636251A2; TW200513531A; CN1835763B; HK1095743A1; EP1636251A4; TWI356097B; WO2005040191A2

Abstract

The angiogenic inducer CCN1 (cysteine-rich 61, CYR61), a secreted matricellular protein of the CCN family, is a ligand of multiple integrins including alpha6beta1. Previous studies have shown that CCN1 interaction with integrin alpha6beta1 mediates adhesion of fibroblasts, endothelial cells, and smooth muscle cells, as well as migration of smooth muscle cells. Recently, we have reported that CCN1-induced tubule formation of unactivated endothelial cells is also mediated through integrin alpha6beta1. In this study, we demonstrate that human skin fibroblasts adhere specifically to the T1 sequence (GQKCIVQTTSWSQCSKS) within domain III of CCN1, and this process is blocked by anti-a6 and anti-b1 monoclonal antibodies. Alanine substitution mutagenesis of the T1 sequence further defines the sequence TTSWSQCSKS as the critical determinant for mediating alpha6beta1-dependent adhesion. Soluble T1 peptide specifically inhibits fibroblast adhesion to CCN1 in a dose-dependent manner. Furthermore, T1 also inhibits cell adhesion to other alpha6beta1 ligands including CCN2 (CTGF), CCN3 (NOV), and laminin, but not to ligands of other integrins. In addition, T1 specifically inhibits alpha6beta1-dependent tubule formation of unactivated endothelial cells in a CCN1-containing collagen gel matrix. To confirm that T1 binds integrin alpha6beta1 directly, we perform affinity chromatography and show that integrin alpha6beta1 is isolated from an octylglucoside extract of fibroblasts on T1-coupled Affi-gel. Taken together, these findings define the T1 sequence in CCN1 as a novel binding motif for integrin alpha6beta1, and form the basis for the development of peptide mimetics to examine the functional role of alpha6beta1 in angiogenesis.

Description

CCN1 compositions and method

Invention field

[0001] the present invention relates to material and method, it relates to the extracellular matrix signalling transduction molecule that participates in the somatomedin cell response with the form of polypeptide.More particularly, the present invention relates to CCN1 related peptides, its compositions and use the method for these polypeptide.The present invention also relates to anti-CCN1 antibody.

Background technology

[0002] angiogenesis, or from existing vascularization neovascularity, be the complex process that requirement various kinds of cell incident is finished jointly.Referring to Risau (1997) Nature 386,671-674.Vascular sprout to need basement membrane, vascular endothelial cell around the degraded mother lode pipe to angiogenesis stimulation place migration, endothelial cell proliferation be arranged in tubular structure and new vascular merges and becomes cyclic loop to provide blood supply to target tissue.Referring to Risau.Angiogenesis is essential to the embryo, and is in the adult, extremely important in female reproduction circulation and wound healing.Angiogenesis can be the basis of many pathologic conditions, comprises diabetic renal papillary necrosis, arthritis, arteriosclerosis, psoriasis and cancer.Referring to Folkman (1995) Nature Medicine 1,27-31.Very clear now, angiogenesis is regulated and control by the network system of multiple inducer and mortifier.Referring to Bouck et al. (1996) Adv.Cancer Res.69,135-174 and Davis et al. (1999) Curr.Top.Microbiol.Immunol.237,173-185.

[0003] stroma cell protein CCN family is the secreted protein that is rich in cysteine, and it is relevant with extracellular matrix (ECM), as adjusting function but not structure function.The CCN family member comprises that CCN1 (CYR61), CCN2 (CTGF), CCN3 (NOV), CCN4 (WISP-1), CCN5 (WISP-2) and CCN6 (WISP-3) are (referring to Brigstock (1999) Endocr.Rev.20,189-206; Lau et al. (1999) Exp.Cell Res.248,44-57), is made up of following institute: the terminal secreting signal peptide of N-, follow four to have the conservative territory of homology thereafter with insulin-like growth factor binding protein; C type vWF ELISA repeats; 1 type thrombospondin repeats; Have heparin binding motif, the sequence C-terminal territory (CT) similar with the mucin C-terminal to vWF ELISA.Referring to Bork (1993) FEBS Lett.327,125-130.With itself and ECM albumen homology, and what be positioned the ECM unanimity is that some CCN albumen demonstrate sustenticular cell and adhere to, and induce and stick together the speckle complex, and stimulate and adhere to the signal conduction.Referring to Kireeva et al. (1996) Mol.Cell.Biol.16,1326-1334; Chen et al. (2001) J.Biol.Chem.276,10443-10452; Chen (2001) J.Biol.Chem.276,47329-47337.

[0004] in the CCN family member, the most thorough CCN1 and the CCN2 of having identified.These two kinds of albumen can irritation cell migration, improve cell survival rate, strengthen the mitosis of growth factor-induced.Referring to Kireeva et al. (1997) Exp.Cell Bes.233,63-77; Jedsadayanmata et al. (1999) J.Biol.Chem.274,24321-24327; Babic etal. (1999) Mol.Cell.Biol.19,2958-2966; Schober et al. (2002) Blood 99,4457-4465; Leu et al. (2002) J.Biol.Chem.277,46248-46255.Known these two kinds of albumen can generate and cartilage formation by induction of vascular; Referring to Babic et al.; Wong etal. (1997) Dev.Biol.192,492-508; Babic et al. (1998) Proc.atl.Acad.Sci.U.S.A.95,6355-6360; Shimo et al. (1999) J.Biochem. (Tokyo.) 126,137-145; Ivkovic et al. (2003) Development 130,2779-2791.Although CCN albumen does not comprise the RGD sequence motifs, CCN1 and CCN2 are the direct aglucons of multiple integrin receptor, and integrin receptor has mediated their many activity.Referring to Jedsadayanmata et al. (1999) J. Biol.Chem.274,24321-24327; Schober etal. (2002) Blood 99,4457-4465; Leu et al. (2002) J.Biol.Chem.277,46248-46255; Kireeva et al. (1998) J.Biol.Chem.273,3090-3096; Chenet al. (2000) J.Biol.Chem.275,24953-24961; Grzeszkiewicz et al. (2001) J.Biol.Chem.276,21943-21950; Grzeszkiewicz et al. (2002) Endocrinology 143,1441-1450.The targeting of CCN1 gene destroys and to cause because the embryonic death that blood vessel lacks in the mice, however do not have the mice of CCN2 because skeleton deformity causes respiratory failure at perinatal death.Referring to (Mo et al. (2002) Mol.Cell Biol.22,8709-8720); Ivkovic et al. (2003) Development 130,2779-2791.These discoveries show that the CCN family member has necessary and is not unnecessary function between the period of development.

[0005] (rich cysteine 61 CYR61), by the angiogenesis inducer of the derivable instant early gene coding of somatomedin, is a kind of novel integrin aglucon to CCN1, and its expression is very important for suitable fetal development.The destructive current research of CCN1 gene target shows in the mice, and mainly due to lack blood vessel in Placenta Hominis and embryo, the embryo of no CCN1 suffers embryonic death.Referring to Mo et al. (2002) Mol.Cell Biol.22,8709-8720.Except embryonic blood vessel generates, CCN1 also can promote pathologic vessels to generate under as tumor growth and wound healing condition.Do not express in the tumor cell line of CCN1 at this, the stable transfection of CCN1 has strengthened the tumor generation, follows the vascularization of the tumor of expressing CCN1 to increase.Referring to Babic et cul. (1998) Proc.Natl.Acad.Sci.U.S.A.95,6355-6360; Xie et al. (2001) J.Biol.Chem.276,14187-14194; Tsai et al. (2002) Oncogene 21,8178-8185.In addition, the expression of the CCN1 of estrogen-induced with late period human breast carcinoma relevant.Referring to Xie et al. (2001) Cancer Res.61,8917-8923; Sampath et al. (2001) Endocrinology 142,2540-2548; Tsai et al. (2000) Cancer Res.60,5603-5607.Also at the restenosis blood vessel with observe cross expressing of CCN1 late period in the atherosis focus, the pathology importance in angiopathy of having pointed out it.Referring to Hilfiker et al. (2002) Circulation 106,254-260; Wu et al. (2000) Int.J.Mol.Med.6,433-440; Grzeszkiewicz et al. (2002) Endocrinology 143,1441-1450; Schober et al. (2002) Blood 99,4457-4465.In addition, the expression of CCN1 shows that with the ability of the genetic program of its activation wound healing in human skin fibroblast CCN1 plays an important role in injury repairing in the healing wounds of epidermis.Referring to Latinkic et al. (1991) Nucleic Acids.Res.19,3261-3267; Chen et al. (2001) J.Biol.Chem.276,47329-47337.

[0006] after synthetic, CCN1 is secreted and is connected to cell surface or ECM.Referring to Yang et al. (1991) Cell Growth ﹠amp; Differentiation 2,351-357.The previous CCN1 sustenticular cell that studies show that adheres to, and the inducing cell migration strengthens the mitosis effect of growth factor-induced, improves the survival rate of cell under the apoptosis condition.Referring to Kireeva et al. (1996) Mol.Cell Biol.16,1326-1334; Leu et al. (2002) J.Biol.Chem.277,46248-46255.The cytoactive of these CCN1 part is owing to itself and the interactional ability of integrin adhesion receptor.So far for this reason, 5 kinds of beta 2 integrin alphas ₆β ₁, α _vβ ₃, α _vβ ₅, α _IIbβ ₅, and α _Mβ ₂, in various cell types, be accredited as the CNN1 receptor.Referring to Schober et al. (2002) Blood 99,4457-4465; Kireeva et al. (1998) J.Biol.Chem.273,3090-3096; Jedsadayanmata et al. (1999) J Biol.Chem.274,24321-24327; Grzeszkiewicz et al. (2001) J.Biol.Chem.276,21943-21950; Chen et al. (2000) J.Biol.Chem.275,24953-24961.In a research more early, we have proved that CCN1 has induced neovascularization in rat cornea crystallite lattice (micropocket) are measured.Referring to Babic et al. (1998) Proc.Natl.Acad.Sci.U.S.A.95,6355-6360.With find in these bodies consistent be, CCN1 has promoted the tubule formation of human umbilical vein's endotheliocyte (HUVEC) in the collagen gel is measured, this process depends on beta 2 integrin alpha ₆β ₁And α _vβ ₃Referring to Leu et al. (2002) J.Biol.Chem.277,46248-46255.

[0007] beta 2 integrin alpha ₆β ₁Demonstrate and in some cell types, mediated many CCN1 activity.By with beta 2 integrin alpha ₆β ₁With the interaction of cell surface heparan sulfate proteoglycan (HSPG), CCN1 has supported fibroblastic adhesive attraction, causes extensively forming having the α of containing ₆β ₁The filopodium and the lamellipodium of local complex, described complex is positioned at the pseudopodium forward position.Referring to Chen et al. (2001) J.Biol.Chem.276,10443-10452.And the outside-internal signal conduction of inducing integrin to rely on causes having activated focal adhesion kinase, paxillin, Rac, mitogen-activated protein kinase, and induces some angiogenesis regulon to raise.Referring to Chen et al. (2001) J.Biol.Chem.276,47329-47337; Chen et al. (2001) J.Biol.Chem.276,10443-10452.Except fibroblast, CCN1 also can be on vascular smooth muscle cell and vascular endothelial cell and beta 2 integrin alpha ₆β ₁Interact.Referring to Grzeszkiewicz et al. (2002) Endocrinology 143,1441-1450; Leu etal. (2002) J.Biol.Chem.277,46248-46255.Recently, we have shown that in un-activation and activated HUVEC, the preceding angiogenic activity of CCN1 passes through beta 2 integrin alpha separately ₆β ₁And α _vβ ₃Mediation respectively.Leu?et?al.(2002)J.Biol.Chem.277，46248-46255。

[0008] except CCN1, other CCN family member comprises CCN2 (Connective Tissue Growth Factor, CTGF), CCN3 (nephroblastoma is crossed expression, NOV), the derivable secreted protein CCN4 of Wnt (WISP-1), CCN5 (WISP-2) and CCN6 (WISP-3) (22-24).CCN albumen can be divided into four kinds of different module territories: I) insulin-like growth factor binding protein homeodomain; II) C type vWF ELISA (vWF) duplicate domain; III) 1 type thrombospondin repeats (TSP1) territory; With IV) have heparin binding motif, a sequence C-terminal (referring to Figure 1A) similar with the mucin C-terminal to vWF.Some CCN albumen demonstrate and can interact with multiple integrin, and therefore, the location of integrin binding site provides the structure activity relationship of the stroma cell protein family that the new visual field observes this new establishment in the CCN albumen.We found in the past that the truncate CCN1 that lacks the C-terminal territory can pass through beta 2 integrin alpha ₆β ₁Induce smooth muscle cell migration.Referring to Grzeszkiewicz et al. (2001) JBiol.Chem.276,21943-21950.These discoveries show beta 2 integrin alpha ₆β ₁Binding site is arranged in three initial territories of CCN1.We are in this article at mediation α ₆β ₁17 new residue sequence have been identified, called after T1 in the adherent I type of the dependent cell CCN1 thrombospondin duplicate domain.By affinity chromatograph, we have proved α ₆β ₁Directly interact with the T1 sequence.In addition, we have proved and have passed through α ₆β ₁And the coreceptor complex of heparan sulfate proteoglycan (HSPG), the activity of CCN1 is subjected to the influence of heparin binding site H1 and H2 and T1.Heparin binding site H1 and H2 are that prolongation MAPK activation, rise Vegf and MMP-1 expression are necessary, and they are relevant with angiogenesis and matrix metabolism.We have proved that also the synthetic peptide specific that is derived from the T1 sequence blocked α ₆β ₁Dependent cell adheres to, the α of our new evaluation among the CCN1 ₆β ₁Binding site can be used as the development beta 2 integrin alpha ₆β ₁The basis of antagonist.The new α that identifies among this CCN1 ₆β ₁Binding site can be used as the development beta 2 integrin alpha ₆β ₁The basis of antagonist.

Summary of the invention

[0009] the invention provides extracellular matrix (ECM) signal transduction molecule relevant material and method.Particularly, the present invention relates to CCN1 related peptides, its compositions, use the method for these polypeptide.The present invention also relates to anti-CCN1 antibody.

[0010] one aspect of the present invention relates to the CCN1 fragment, and it comprises the sequence of the aminoacid 233-242 of the aminoacid 226-242 of aminoacid 231-240, people CCN1 of the aminoacid 224-240, the muroid CCN1 that are selected from muroid CCN1 and people CCN1.This CCN1 fragment can comprise 8-50 aminoacid.The present invention also relates to the segmental variant of CCN1, analog, homologue or derivant.

[0011] this aspect relate to the method for screening angiogenesis modulators on the other hand, comprise that the test organisms sample that angiogenesis can take place contacts with suspicious regulator with the ECM signal transduction molecule.In contrast, second kind of biological sample also contacts with the ECM signal transduction molecule.Identify angiogenesis modulators by the ability that in specimen, changes the angiogenesis level.The ECM signal transduction molecule can be the CCN1 fragment, or its fragment, variant, analog, homologue or derivant.Another aspect of the present invention relates to the regulator of identifying by the inventive method.

[0012] another aspect of the present invention relates to the method for screening angiogenesis modulators, comprises test implant Implantation Test animal, wherein tests implant and comprises suspicious regulator and ECM signal transduction molecule.In contrast, second kind of implant Implantation Test animal that comprises the ECM signal transduction molecule, described animal can be identical or different experimental animal.When comparing, identify angiogenesis modulators by the ability that in the test implant, changes the vascular development level with control sample.The ECM signal transduction molecule can be the CCN1 fragment, or its fragment, variant, analog, homologue or derivant.Another aspect of the present invention relates to the regulator of identifying by the inventive method.

[0013] another aspect of the present invention relates to the method that the screening tumor generates regulator, comprises tumor is contacted with the ECM signal transduction molecule with regulator.In contrast, second kind of tumor also contacts with the ECM signal transduction molecule.When comparing with control tumor, the ability evaluation tumor that can generate level by the tumor that changes the test tumor generates regulator.The ECM signal transduction molecule can be the CCN1 fragment, or its fragment, variant, analog, homologue or derivant.Another aspect of the present invention relates to the regulator of identifying by the inventive method.

[0014] another aspect of the present invention relates to the method for screening cell adhesion regulator, comprises the test organisms sample is contacted with the ECM signal transduction molecule with suspicious regulator on the surface compatible with cell adhesion.In contrast, second kind of biological sample also contacts with the ECM signal transduction molecule on the surface compatible with cell adhesion.When comparing, can pass through the ability of the cell adhesion level of change specimen, the adherent regulator of identification of cell with control sample.The ECM signal transduction molecule can be the CCN1 fragment, or its fragment, variant, analog, homologue or derivant.Another aspect of the present invention relates to the regulator of identifying by the inventive method.

[0015] another aspect of the present invention relates to the method for screening cell migration regulator, comprises that the cell inoculation that can carry out cell migration is on the test gel-type vehicle that comprises suspicious regulator and ECM signal transduction molecule.In contrast, the cell that can carry out cell migration also is inoculated on second kind of biological sample gel-type vehicle that comprises the ECM signal transduction molecule.When comparing with contrast substrate, can be by in test substrate, changing the ability identification of cell cell adhesion modulating agent of cell migration level.The ECM signal transduction molecule can be the CCN1 fragment, or its fragment, variant, analog, homologue or derivant.Another aspect of the present invention relates to the regulator of identifying by the inventive method.

[0016] another aspect of the present invention relates to specificity in conjunction with the CCN1 fragment, or in conjunction with the antibody of the segmental variant of described CCN1, analog, homologue or derivant.Another aspect of the present invention relates to the compositions that comprises antibody, and described antibody specificity is in conjunction with the CCN1 fragment or in conjunction with the segmental variant of described CCN1, analog, homologue or derivant.

The accompanying drawing summary

[0017] Fig. 1 proves segmental protein purification in recombinant C NN1 territory and the adherent ability of their sustenticular cell.Recombinant C CN1 territory fragment is expressed as the fusion rotein of six histidine marks by baculovirus expression system, and on cobalt-agarose chromatography purification.A, the segmental diagram of total length CCN1 domain and isolating territory.T1 sequence among the shaded area representative domain III (TSP1 territory).B, electrophoresis recombinant C CN1 territory fragment and total length CCN1 on the 15%SDS-polyacrylamide gel (2 μ g), and detect by Coomassie blue stain.C uses the anti-CCN1 antibody of polyclone that soluble protein is carried out immunoblotting.Referring to Kireeva et al. (1996) Mol.Cell.Biol.16,1326-1334.The molecular weight marker on the left side marks with kDa.D with the recombinant C CN1 territory fragment of purification or bovine serum albumin (BSA) (50 μ g/ml, 50 μ g/ holes) coating maleic anhydride Reacti-Bind microtiter well, spends the night under 4 ℃, uses the 1%BSA sealing.Use anti-polyhistidine monoclonal antibody (mAb), detect proteic coating efficiency by enzyme-linked immunosorbent assay.E, washed 1064SK fibroblast is suspended in the serum-free medium again, flat board be incubated at use CCN1 (20 μ g/ml) or shown on the hole that is coated with in advance of territory fragment (50 μ g/ml) (3 * 10 ⁴The every hole of individual cell).37 ℃ allow cell adhesion 20 minutes down.The secure attachment cell uses methylene blue staining, and is quantitative by absorbance at the 620nm place.Data are three average ± standard deviations of measuring.D figure and E figure have represented three experiments.

[0018] Fig. 2 proves that the territory III (TSP1 territory) of CCN1 passes through beta 2 integrin alpha ₆β ₁Supported fibroblastic adhesion.Described in Fig. 1 explanation, fibroblast adheres to the microtiter well that scribbles total length CCN1 (20 μ g/ml) or territory III fragment (50 μ g/ml), it is pointed out that wherein that cell was suspended in to contain EDTA (2.5mM), Mg before flat board is cultivated ⁺⁺(5mM), Ca ⁺⁺(5mM) or Mn ⁺⁺Serum-free medium (0.5mM).B used carrier buffer (not adding), normal mouse IgG (100 μ g/ml), anti-α before flat board is cultivated _vβ ₃Monoclonal antibody LM609 (50 μ g/ml) (Chemicon-Temecula, CA), anti-a ₆Monoclonal antibody GoH3 (50 μ g/ml) (Immunotech-Marseille, France) or anti-b ₁Monoclonal antibody P4C10 (1: 50 ascites) (Life Technologies/Gibco-BRL) cultivated 60 minutes in advance.Data are the average ± standard deviation of three experiments, have represented three experiments.

[0019] Fig. 3 proves reorganization GST-T1 fusion rotein support α ₆β ₁The fibroblast that relies on adheres to.A, the reorganization GST-peptide fusion protein that is presented in the Table I with 200 μ g/ml, sequence is coated with microtiter well.Be coated with albumen down at 4 ℃ and spend the night, use the 1%BSA sealing subsequently.The fibroblastic adhesive attraction of assessment described in Fig. 1 explanation.B, cell suspension is in containing EDTA (2.5mM), Mg ⁺⁺(5mM), Ca ⁺⁺(5mM) or Mn ⁺⁺Serum-free medium (0.5mM), and flat board is incubated on the microtiter well that is coated with glutathione S-transferase (GST) (50 μ g/ml), GST-T1 (50 μ g/ml) or CCN1 (1 μ g/ml).C used carrier buffer (not adding), normal mouse IgG (100 μ g/ml), anti-α before flat board is cultivated _vβ ₃Monoclonal antibody LM609 (50 μ g/ml), anti-a ₆Monoclonal antibody GoH3 (50 μ g/ml) or anti-b ₁Monoclonal antibody P4C10 clone (1: 50 ascites) pre-cultured cell 60 minutes.Data are the average ± standard deviation of three experiments, have represented three experiments.

[0020] Fig. 4 proves synthetic T1 peptide support α ₆β ₁The fibroblast that relies on adheres to.A uses synthetic T1, T2, T3 or T4 peptide (0.2mM) coating microtiter well to spend the night down at 4 ℃, and uses the 1%BSA sealing.Allow fibroblast under 37 ℃, to adhere to the hole of coating peptide, kept 20 minutes.B, be incubated on the hole of coating T1 at flat board before, used carrier buffer (not adding) or monoclonal antibody designated pre-cultured cell 60 minutes.Data are the average ± standard deviation of three experiments, have represented three experiments.

[0021] Fig. 5 proves that soluble T 1 peptide has suppressed α ₆β ₁The cell adhesion that relies on.A uses CCN1 (1 μ g/ml), CCN2 (2 μ g/ml) or CCN3 (5 μ g/ml) coating microtiter well, and uses the 1%BSA sealing.Use carrier buffer (not adding) or use capacitive T1, T2, T3 or the washed fibroblast of T4 peptide (0.2mM) pretreatment 30 minutes, and flat board is incubated to be coated with and specifies on the proteic hole of CCN.B and C, be incubated at use fibronectin (FN, 2 μ g/ml), vitronectin (VN, 0.4 μ g/ml), type i collagen (0.5 μ g/ml), laminin (LN at flat board, 5 μ g/ml) or before on the hole of CCN1 (1 μ g/ml) coating, in cell suspending liquid, add the T1 peptide of various concentration.Use described baculovirus expression system, purification of Recombinant muroid CCN1 albumen from serum-free insect cell conditioned medium.Referring to Kireeva et al. (1996) Mol.Cell.Biol.16,1326-1334.Rat type i collagen, vitronectin, laminin and fibronectin can (Bedford MA) buys from Collaborative Biomedical.Described in Fig. 1 explanation, measure cell adhesion.Data are the average ± standard deviation of three experiments, have represented three experiments.

[0022] Fig. 6 proves that the TTSWSQCSKS sequence has comprised α among the T1 ₆β ₁The adherent crucial determiner of dependent cell.In the T1-GST fusion rotein, replace according to the alanine fixed point of having carried out the T1 sequence described in material and the method.At protein concentration is under the 200 μ g/ml, with wild type T1 fusion rotein (GST-T1-WT), its out of order variant (scrambled variant) (GST-T1-Scram) or the metathetical mutant of alanine coat microtiter well.After using the 1%BSA sealing, begin to carry out described fibroblast and adhere to.The result is the average ± standard deviation of three experiments, has represented three experiments.

[0023] Fig. 7 proves on the link coupled Affi-gel of GST-T1, protein affinity purification beta 2 integrin alpha from the fibroblast lysate ₆β ₁Cell surface protein on the fibroblast carries out radioiodination by the lactoperoxidase-method of cracking described in material and the method.The cytolysis of labelling is in containing 200mM octyl glucoside and 0.5mM Mn ⁺⁺Initial buffer in.The lysate of this cell (swimming lane 1) can be applied to the Affi-gel agarose affinity chromatographic column that coupling has GST-T1 among out of order T1 of GST-among the A or the B.After using initial buffer (electrophoresis road 2-4) washing, use 0.35M NaCl (electrophoresis road 5-8) elution chromatography post.Under the non-reduced condition, the albumen in the elutriated fraction is resolved on the 7%SDS-polyacrylamide gel, detect by autoradiography.In C, will derive from the high eluting salt thing of GST-T1 post merge, and with anti-a ₆(GoH3) or anti-av (P3G8) monoclonal antibody carry out immunoprecipitation.Under non-reduced condition, analyze immunoprecipitation albumen.Molecular weight marker marks on the left side with kDa.The result has represented 2 experiments.

[0024] Fig. 8 proves that the T1 peptide blocked the inductive endothelium tubule of CCN1 and formed in collagen gel substrate.Unprovoked HUVEC is incubated to use not to be had (not adding) or exists on 24 orifice plates of type i collagen gel (2mg/ml) pre-coating of 50 μ g/ml CCN1, is covered on the attached cell described in second layer gel such as material and method.It is to be noted that wherein use test peptide pre-cultured cell suspension is 30 minutes before cultivating.The assessment tubule forms after 16-20 hour.The result represents three experiments that separate (amplification * 100).

[0025] Fig. 9 describes the structure and the expression of CCN1 and mutant.A, the construction sketch map of wild type CCN1 (WT) and mutant, mutant has the K239E point mutation in T1 (SM), have destruction in H1 and H2 (DM), has combinatorial mutagenesis in T1, H1 and H2 (TM).Each construction has been endowed N-terminal secretion signal and C-terminal FLAG epi-position labelling equally.By baculovirus vector at the expressed in insect cells recombiant protein.Shown that aminoacid specific in wild type T1, HI and H2 sequence and the mutant changes.B is incubated at the fibroblast flat board on the microtiter well that is coated with GST, GST-T1 peptide fusion protein or GST-T1 (K239E) peptide fusion protein (each 50 μ g/ml).Allow cell to adhere to 20 minutes down at 37 ℃.After the washing, the secure attachment cell uses methylene blue staining, and is quantitative by 620nm place absorbance.C, coomassie brilliant blue staining 10%SDS-PAGE, the wherein recombiant protein of electrophoretic separation FLAG protein affinity purification (each 2 μ g).Molecular weight marker (kDa) is presented at the left side.Use the anti-CCN1 antibody of polyclone that gel is carried out immunoblotting, be presented among the lower figure.

[0026] Figure 10 proves that it is the heparin sensitivity that fibroblast adheres to mutant SM.A is incubated at 1064SK human fibroblasts flat board on the microtiter well that is coated with specified amount reorganization WT CCN1 or SM mutant, assesses cell adhesion as described in Figure 1.B before adhering to the microtiter well that is coated with WT CCN1 (2 μ g/ml), SM (2 μ g/ml) or VN (0.5 μ g/ml), does not handle or ((2 units/ml) are processed into fibrocyte for 2 units/ml) or chondroitinase abc with Heparinase I.Wherein it is to be noted, in culture medium, have the solubility heparin of 1 μ g/ml.Data are three average ± standard deviations of measuring, have represented three experiments.

[0027] Figure 11 proves that DM supports α ₆β ₁The cell adhesion of mediation.A is incubated at the fibroblast flat board on the microtiter well that is coated with specified amount DM or TM, the assessment cell adhesion.B before flat board is cultivated, uses 40 μ g/ml anti-alpha 2 integrin α _vβ ₃(LM609), beta 2 integrin alpha ₆(GoH3) or integrin β ₁The function interdiction monoclonal antibody of subunit (P4C10,1: 50 ascites) is pre-cultured cell 1 hour at room temperature.Assess cell adhesion as mentioned above.Data are three average ± standard deviations of measuring, have represented three experiments.

[0028] Figure 12 proves the effect of CCN1 mutant to MAPK activation and gene expression.A, the MAPK activation.Make 1064SK fibroblast serum starvation, and be suspended in the serum-free medium, concentration is 6 * 10 ⁵Cell/ml.The cell flat board is incubated at 1ml/ plate (35-mm plate), and as specified cultivation 1-5 hour, plate used WT CCN1 (10 μ g/ml), SM (10 μ g/ml), DM (250 μ g/ml) or laminin (LN, 10 μ g/ml) coating.The lysate of separating clarifying on 10%SDS-PAGE, and use the antibody of anti-MAPK antibody of polyclone or anti-diphosphate activation p42/p44 MAPK to carry out immunoblotting.B, the regulation and control of gene expression.Handle primary human skin flbroblast 24 hours with the WT CCN1 (WT) of each 10 μ g/ml, mutant protein (SM, DM and TM) or BSA contrast (B) before, carried out serum starvation 24 hours, separate total cell RNA.Estimate the expression of Vegf and MMP-1 after electrophoresis by RNA trace (every electrophoresis road in the total RNA of 20 μ g), the expression of monitoring glyceraldehyde 3-phosphate dehydro-genase (GAPDH) in contrast.

[0029] Figure 13 proof is passed through beta 2 integrin alpha _vβ ₃HUVEC adhere to and migration to the CCN1 mutant.A, the HUVEC that dissociates in 2.5mM EDTA also is suspended in serum-free medium again, adheres to the hole that use 15 μ g/ml CCN1 wild type CCN1 (WT) or SM or 50 μ g/ml DM or TM are coated with in advance.Wherein it is to be noted, before flat board is cultivated, use EDTA (5mM), GRGDSP peptide (RGDS, 0.2mM), anti-α _vβ ₃Monoclonal antibody LM609 (40 μ g/ml), anti-α ₆Monoclonal antibody GoH3 (40 μ g/ml) handled cell 60 minutes.Measure cell adhesion as described.B, HUVEC uses the Transwell cell to assess to the migration of CCN1 or mutant.On the lower surface of Transwell film, fix 15 μ g/mlCCN1 WT or mutant SM, 50 μ g/ml mutant DM or TM, described Transwell film has separated 2 cells.(Sigma, St.Louis MO) handle HUVEC 30 minutes with the activation integrin receptor to use 100nM PMA.Wherein it is pointed out that before flat board is incubated at the upper strata cell, use carrier buffer (not adding), normal mouse IgG (100 μ g/ml), GoH3 (50 μ g/ml), EM609 (50 μ g/ml) pre-cultured cell 30 minutes again.Allow cell migration 8 hours, in the high power field those cells to the migration of low layer cell are counted at random at 10 times.Data are three average ± standard deviations of measuring, have represented three experiments.

[0030] Figure 14 proves that the CCN1 mutant can pass through beta 2 integrin alpha _vβ ₃It is synthetic to strengthen the inductive DNA of VEGF.Use carrier buffer (not adding), LM609 (25 μ g/ml), GoH3 (25 μ g/ml) or normal mouse IgG (25 μ g/ml) to cultivate HUVEC in advance one hour.Then exist [ ³H] use during dT VEGF (5ng/ml) and/or CCN1 or mutant protein (each 5 μ g/ml) to handle cell, they mix of post-evaluation in 48 hours.Data show is three average ± standard deviations of measuring, has represented three experiments.

[0031] Figure 15 proves that the CCN1 mutant can improve the survival of HUVEC.Allow the HUVEC of serum starvation on the coverslip that uses 20 μ g/ml laminins (LN) to be coated with in advance, to adhere to 4 hours, add serum, CCN1 or mutant protein (each 5 μ g/ml) subsequently, adhered to again 16 hours.Fixed cell is measured the monitoring apoptosis by using TUNEL.Use test reagent was cultivated the anti-CCN1 antibody of polyclone 30 minutes in advance before wherein it is pointed out that in being added to culture medium.Data show is three average ± standard deviations of measuring, has represented three experiments.

[0032] Figure 16 proves that the CCN1 mutant can induce α _vβ ₃The endothelium tubule that relies on forms.Flat board be incubated at use do not have (-) have CCN1 (WT) or 24 orifice plates of type i collagen gel (2mg/ml) pre-coating of TM (each 20 μ g/ml) before, in serum-free medium, use carrier buffer (not adding) to handle HUVEC or using 5nM PMA to stimulate HUVEC.To cover on the attached cell with the second layer gel of prescription, the assessment tubule forms after 16 hours.Wherein it is pointed out that before flat board is cultivated and in cell suspending liquid, add LM609 (40 μ g/ml) or GoH3 (40 μ g/ml).The result represents 3 experiments (* 200 amplification).

Detailed Description Of The Invention

[0033] CCN1 is the Angiogenesis inducer, its embryo between the emergence period in normal blood vessels Play an important role in the growth. Referring to Mo et al. (2002) Mol.Cell Biol.22,8709-8720. Also referring to United States Patent (USP) the 6th, 413, No. 735 and U.S. Patent application series the 09/495, No. 448 (approved), all incorporated herein by reference. We disclose logical recently Cross beta 2 integrin alpha₆β ₁And α_vβ ₃Mediate CCN1 among the HUVEC of each comfortable un-activation and activation Front angiogenic activity. Referring to Leu et al. (2002) J.Biol.Chem.277,46248-46255. Except beta 2 integrin alpha₆β ₁Outside the T1 sequence interacts in the TSP1 territory of CCN1, Fibroblast and un-activation endothelial cell also need acetyl sulfate to the adhesive attraction of CCN1 The heparin proteoglycans is as the Heparin-binding motif phase in the terminal territory of coreceptor and CCN1 C Mutual effect. Referring to Chen et al. (2000) J.Biol.Chem.275,24953-24961. In this research, we have used function and biochemical analysis, to determine 17-among the III of CCN1 territory Residue T1 sequence (GQKCIVQTTSWSQCSKS) is new beta 2 integrin alpha₆β ₁In conjunction with the position The point. We also determine, heparin-binding site H1 and H2 are such as being the important section of Angiogenesis In the functions such as cell adherence of dividing, to α₆β ₁The activity of-HSPG coreceptor compound has shadow Ring. These discoveries provide development α₆β ₁The basis of antagonist, and the target that mutation analysis is provided is to detect beta 2 integrin alpha₆β ₁The effect of-CCN1 interaction in Angiogenesis.

Definition

[0034] used herein to term " administration " refer to single dose or multiple dose the present invention Composition.

[0035] used herein to term " treatment " when referring to that the protection mammal avoids disease The time, refer to prevent, suppress, suppress or eliminate a disease. Prevent disease is included in disease incidence Give the mammal present composition before. Suppress disease be included in induce after the disease but Before clinical manifestation, give mammal composition of the present invention. The compacting disease has been included in Give mammal composition of the present invention after the disease clinical manifestation, so that disease alleviates or ties up Hold. Eliminate a disease and give mammal combination of the present invention after being included in the disease clinical manifestation Thing is so that mammal is no longer ill.

Cell adherence is by α₆β ₁-HSPG coreceptor compound is regulated

[0036] finds early that with us consistent is that the truncated mutant that comprises CCN1 territory I-III can be induced α₆β ₁The smooth muscle cell migration that relies on is (referring to Grzeszkiewicz et al. (2002) Endocrinology 143,1441-1450), we find that recombinant fragment corresponding to territory III (TSP 1 territory) that separates with muroid CCN1 is enough to support α₆β ₁What rely on is fibroblastic sticking Connect. In company with α₆β ₁Together, heparan sulfate proteoglycan (HSPG) can be used as mutual work With coreceptor. Referring to Chen, N., Chen, C.C., and Lau, L.F. (2000) J.Biol. Chem.275,24953-24961 and Leu, S.-J., Lam, S.C.T., and Lau, L.F. (2002) J Biol.Chem.277,46248-46255. α₆β ₁With the interactional specificity of muroid CCN1 territory III by muroid CCN1 territory I and territory II fragment not supportint cell adhere to confirmed, and by viewed anti-α₆Resist and anti-β₁Monoclonal antibody specific inhibition cell adherence is to muroid CCN1 Territory III confirms (Fig. 1 and 2).

[0037] in the III of territory, based on following observation, we have further found out and have been beta 2 integrin alpha among the CCN1₆β ₁The T1 sequence of binding site: 1) T1-GST fusion and synthetic T1 peptide specific are supported α₆β ₁The cell adherence (Fig. 3 and 4) that relies on; 2) affine at T1-GST On the matrix from fibroblast detergent lysate purifying obtain beta 2 integrin alpha₆β ₁, prove at beta 2 integrin alpha₆β ₁And directly have an effect between the T1 sequence (Fig. 7); 3) soluble T 1 peptide Suppressed cell adhesion to the α that comprises CCN1, CCN2, CCN3 and laminin₆β ₁Aglucon, but do not suppress other integrin aglucon such as fibronectin, vitronectin and glue Former (Fig. 5); 4) the T1 peptide has also been blocked un-activation in containing the collagen stroma of CCN1 The α of HUVEC₆β ₁The dependence tubule forms (Fig. 8). It should be noted that the T1 peptide is effective α₆β ₁The dependent cell activity inhibitor. Half maximum inhibition of cell adherence occurs in peptide concentration Be 25-50mM (Fig. 5 C). Therefore, tiring that T1 suppresses has comparable with linear RGD peptide The property, the latter has also suppressed other integrin such as α in micro-molar range_vβ ₃Adhesion function. Referring to Ruoslahti (1996) Ann.Rev.Cell Dev.Biol.12,697-715.

[0038] because the T1 site can demonstrate and affect CCN1 and α₆β ₁Combination, we also disclose, heparin-binding site H1 and H2 have illustrated CCN1 remnants' α₆β ₁Dependence is lived The property. Have the complete of sudden change according to preparation at T1 site and H1 and H2 heparin-binding site Long CCN1-GST fusion has lost at the mutant of all three site failures All support α₆β ₁The ability of the fibroblast adhesive attraction of mediation. Do such as embodiment 11-13 Describe, can support during only in high concentration at the destroyed mutant of H1 and H2 site Fibroblast adheres to, and only destroyed mutant still can be near wild in the T1 site The level of type supports fibroblast to adhere to.

[0039] heparin also can demonstrate the lasting activation that influences p42/p44MAPK in H1 and the H2 combination of CCN1.

[0040] by the alanine displacement mutagenesis of T1-GST fusion rotein, the C-terminal that we disclose T1 (TTSWSQCSKS) partly contains α ₆β ₁The determiner of the cell adhesion key that relies on.It should be noted that T231A/T232A and W234A/K239A double replacement have caused losing fully of sustenticular cell adhesive capacity.This 10-residue segment is a high conservative in the CCN family member, has only 2 non-conservative substitutions in CCN1, CCN2 and CCN3.Therefore, can imagine α ₆β ₁Also can be in conjunction with corresponding T1 sequence in other CCN albumen.What meet this idea is that soluble T 1 peptide has also suppressed α ₆β ₁The fibroblast that relies on adheres to CCN2 and CCN3.These results make us reach a conclusion: TTXWSXCSKS sequence (X represents non-conserved residues) conservative in the CCN albumen has defined beta 2 integrin alpha ₆β ₁New identification motif.A key character of this sequence is that any single alanine displacement (being T232A, W234A, S235A, S238A and K239A) of conserved residues all causes losing in a large number α ₆β ₁In conjunction with active, show at α ₆β ₁In need multiple coordination with the interaction of aglucon binding pocket.

[0041] beta 2 integrin alpha ₆β ₁Have limited aglucon spectrum, it comprises laminin, CCN albumen, hyaluronidase, causes the collagen fragment of educating albumen and being called as tumor chalone.Referring to Sonnenberg et al. (1990) J.Cell Biol.110,2145-2155; Maeshima etal. (2001) J Biol.Chem.276,15240-15248; Isberg et al. (1990) Cell 60,861-871; Almeida et al. (1995) Cell 81,1095-1104.The different α that these are relevant with various biological processes ₆β ₁Aglucon does not have structural dependence.Be derived from some these α by screening ₆β ₁The synthetic peptide of aglucon has been identified some α ₆β ₁Binding sequence.These sequences comprise NPWHSIYITRFG and the TWYKIAFQRNRK sequence that derives from laminin a1 chain.Referring to Sonnenberg et al. (1990) J.Cell Biol.110,2145-2155; Nomizu et al. (1995) J.Biol.Chem.270,20583-20590; Nakahara et al. (1996) J.Biol.Chem.271,27221-27224.In addition, infer by the blocking-up beta 2 integrin alpha ₆β ₁-cause and educate proteic interaction, derive to cause and educate the TDE peptide that comprises that albumen b subunit de-connects the albumen territory and destroyed smart ovum combination.Referring to Myles et al. (1994) Proc.Natl.Acad.Sci.U.S.A.91,4195-4198.By screening phage display and synthetic peptide combinatorial libraries, some other α have also been separated ₆β ₁Binding peptide; Yet these sequences are not present in any known α ₆β ₁In the aglucon.Referring to Murayama et al. (1996) J.Biochem. (Tokyo) 120,445-451; Pennington et al. (1996) Mol.Divers.2,19-28; DeRoock et al. (2001) Cancer Res.61,3308-3313.Bao Dao α up to now ₆β ₁The relatively announcement of binding sequence can be used as α without any consensus sequence ₆β ₁Binding motif.And the T1 sequence among the CCN1 that we identify does not recently show any and these α ₆β ₁The sequence that binding peptide is similar.Therefore, beta 2 integrin alpha ₆β ₁, as α _Mβ ₂, can discern large-scale binding sequence.At present, no matter whether these many different peptide sequences are at α ₆β ₁In still have to be determined in conjunction with identical or different site.Even so, because beta 2 integrin alpha ₆β ₁Many biological processes have been involved, different α ₆β ₁Binding sequence can be at α ₆β ₁With different coordination site interactions, mediate the active unlike signal pathway of different biological in the aglucon binding pocket to induce.

[0042] up to now, three kinds of CCN albumen demonstrate and induce neovascularization in vivo.Referring to Babic et al. (1998) Proc.Natl.Acad.Sci.U.S.A.95,6355-6360; During Lin et al (2003) J.Biol.Chem. delivers; Babic et al. (1999) Mol.Cell.Biol.19,2958-2966; Shimo et al. (1999) J.Biochem. (Tokyo.) 126,137-145; Fataccioli et al. (2002) Hum.Gene Ther.13,1461-1470.Endothelial cell migration, to breed and be divided into little tubular construction be essential for neovascularization.Non-be excited in the endotheliocyte CCN1 for and beta 2 integrin alpha ₆β ₁The irrelevant aglucon of activation, adhere to and tubule formation by this integrin receptor mediated cell.Referring to Leu et al. (2002) J.Biol.Chem.277,46248-46255.In view of complete CCN1 is the angiogenesis inducer, the T1 peptide is as α ₆β ₁The tubule that antagonist has been blocked the inductive un-activation chrotoplast of CCN1 forms.What is interesting is that the 1 type thrombospondin that the T1 sequence is present in CCN1 repeats in the homeodomain, thrombospondin is an angiogenesis inhibitor, and its anti-angiogenesis activity is positioned at the procollagen homology region and properdin sample 1 type repeats.Referring to Tolsma et al. (1993) J.Cell Biol.122,497-511.Many anti-angiogenic peptides are derived from thrombospondin, comprise the peptide that contains CSVTCG, its on endotheliocyte with the CD36 effect.Referring to Jimenez et al. (2000) Nat.Med.6,41-48; Dawson et al. (1997) J.Cell Biol.138,707-717.Do not prove the interaction in CD36 and CCN albumen TSP1 territory yet, yet CD36 demonstrates on human blood platelets and melanoma cells and beta 2 integrin alpha ₆β ₁Associating.Miao?et?al.(2001)Blood?97，1689-1696；Thome?et?al.(2000)J.Biol.Chem.275，35264-35275。If CD36-α ₆β ₁Complex also is present on the endotheliocyte, so a kind of interesting may be that the recognition sequence that closes on during these two kinds of cell surface receptors repeat by 1 type thrombospondin with stroma cell protein interacts, regulate angiogenesis jointly.

1.CCN1 fragment

[0043] the present invention relates to the fragments of peptides of CCN1, it has regulated the activity of CCN1.This peptide can be used to be designed to suppress or induce the active therapeutic strategy of CCN1.This peptide can be natural, synthetic or reorganization.A kind of method can be used for producing the aminoacid 226-242 of aminoacid 231-240, people CCN1 of the aminoacid 224-240, the muroid CCN1 that comprise the peptide that is selected from following sequence: muroid CCN1 and the aminoacid 233-242 of people CCN1.For example, comprise conserved amino acid peptide can with the binding site on natural CCN1 competition integrin and other albumen.Therefore this competition can suppress the effect of natural CCN1.The present invention also relates to the segmental fragment of CCN1.The length of this peptide can be 8-50 aminoacid.Inhibitory peptide length can be 8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49 or 50 aminoacid.

[0044] peptide of the present invention can be the homologue of above-mentioned CCN1 peptide.The homologue of CCN1 peptide is to share common peptide of evolving with CCN1.This peptide also can be the variant of above-mentioned CCN1 peptide and homologue.The peptide variant is by amino acid whose insertion, disappearance or conservative substitution, and with the different peptide of natural CCN1 peptide, but it still keeps the biological activity of at least a natural CCN1 peptide on aminoacid sequence.For the object of the invention, " biological activity of CCN1 peptide " include but not limited to above-mentioned total length CCN1 activity, regulate the active ability of CCN1 and by the bonded ability of CCN1 specific antibody.

[0045] amino acid whose conservative substitution is promptly used the different aminoacids substituted amino acid of similar quality (for example degree of hydrophilic, charged region and distribution), by being understood this area, typically comprises slight variations.These slight variations can part be identified by the hydrophilic index of considered amino acid, by being understood this area.Kyte?et?al.，J.Mol.Biol.157：105-132(1982)。The tabulation of amino acid pro aqua index can be at United States Patent (USP) the 6th, 639, finds in No. 054, and it is attached to herein by reference.What be known in the art is, the aminoacid of similar hydrophilic index can be substituted and still keep protein function.Have on the one hand, ± aminoacid of 2 hydrophilic indexes replaced.

[0046] amino acid whose hydrophilic also can be used to disclose the proteic displacement that finally causes keeping biological function.The hydrophilic of considered amino acid allows to calculate the maximum local average hydrophilic of this peptide in the scope of peptide, and this useful yardstick has been in the news relevant with antigenicity and immunogenicity well.The tabulation of amino acid whose hydrophilic index can be at United States Patent (USP) the 4th, 554, finds in No. 101, and it is attached to herein by reference.Amino acid replacement with similar hydrophilicity value can cause the peptide that forms to keep biological activity, and immunogenicity for example is by being understood this area.On the one hand, use each other the aminoacid of hydrophilicity value in ± 2 to replace.Amino acid whose hydrophilic index and hydrophilicity value all are subjected to the influence of the concrete side chain of aminoacid.With observing consistent be, should understand with the amino acid replacement of biological function compatibility and depend on amino acid whose relative similarity, specifically depend on amino acid whose side chain, as by hydrophobicity, hydrophilic, electric charge, size and the announcement of other character.

[0047] in addition, computerized algorithm can aid forecasting may arrive the aminoacid sequence territory of aqueous solvent.These territories are known in the art, and often to the outside arrangement of peptide, therefore might help to comprise antigenic determinant in conjunction with determiner.

[0048] peptide of the present invention also can be the analog of above-mentioned CCN1 peptide, homologue and variant, comprises that off-gauge aminoacid or other derive from conventional amino acid whose structural change thing.Peptide of the present invention also can be the derivant of above-mentioned CCN1 peptide, homologue, variant and analog, and different modes is outside primary structure (aminoacid and amino acid analogue).By the explanation mode, by the post translational modification of glycosylation or a kind of form, derivant can be different from natural CCN1 peptide, homologue and variant.For example, owing to express in the allos system, polypeptide can show glycosylation pattern.If these peptides keep at least a biological activity of natural CCN1, these peptides are exactly CCN1 derivant of the present invention.Other derivant include but not limited to have the N-of covalent modification or C-end fusogenic peptide, Pegylation peptide, peptide, alkylation peptide in conjunction with lipid components, be attached to the peptide of other peptide or chemicals and other trim of understanding for this area by amino acid side chain functional group.In addition, the present invention has also considered the CCN1 related peptides in conjunction with the CCN1 receptor, as described below.

[0049] various peptide of the present invention as mentioned above, can be provided as isolating peptide, is what connect perhaps, as connect other chemical compound by covalent bond.For example, immunogenic carrier such as keyhole limpet hemocyanin can be incorporated into CCN1 peptide of the present invention.Above-mentioned various fragment, variant, analog, homologue or derivant can have 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% concordance with peptide of the present invention.The present invention also relates to the nucleotide of this peptide of separated coding.The present invention also relates to comprise the pharmaceutical composition of peptide of the present invention.

2. antibody

[0050] the present invention also relates to comprise the pharmaceutical composition that specificity can be accepted accessory drugs, diluent or carrier in conjunction with the antibody and the medicine of CCN1 peptide of the present invention.Can production antibody as described below, perhaps described in WO 01/55210, their content integral body by reference is attached to herein.

[0051] antibody of the present invention comprises that IgG, IgM, IgA, IgD and IgE antibody-like and it comprise Fab and F (ab ') ₂Fragment and derivant.Antibody also can be the antibody product of reorganization, includes but not limited to the antibody product of single-chain antibody, chimeric antibody product, " humanization " antibody product and CDR-grafting.Antibody of the present invention comprises antibody or its mixture of monoclonal antibody, polyclonal antibody, affinity purification, and it shows enough CCN1 fragment binding specificities.

[0052] the present invention has also considered antibody fragment.The antibody product comprises the antibody product of the above-mentioned type, as the antibody of isolated antibody or linkage flag.Labelling can be enzyme, antigen, other antibody, agglutinin, carbohydrate, biotin, avidin, radiosiotope, toxin, heavy metal and other compositions known in the art that produces signal; Interconnection technique also is known in the art.

[0053] anti-CCN1 antibody can be used for the risk that diagnosing tumour takes place.In addition, anti-CCN1 antibody can be used for the treatment of, and is designed to selectively targeted cytotoxin is transported to the CCN1 cell of expression, for example participates in the cell of solid tumor neovascularization.Can carry these antibody by the multiple approach that gives in the pharmaceutical composition that comprises carrier or diluent, this is understood by those skilled in the art.

3. screen the regulator of CCN1

[0054] the present invention relates to screen the regulator of CCN1 related activity.Can identify and the regulator of the integrin binding site of CCN1 reaction, therefore stop CCN1 and target integrin and other albumino reaction.Also can identify direct target integrin and other proteic regulator, therefore stop CCN1 and above-mentioned target integrin and other proteic property as a result (productively) to interact in conjunction with CCN1.Also can identify the bonded regulator of remote-effects CCN1 and target protein.

[0055] for the object of the invention, " ECM signal transduction molecule " refers to above-mentioned CCN1 fragment.The purposes of " ECM signal transduction molecule " has also been considered one or more additional C CN polypeptide.This one or more additional C CN polypeptide includes but not limited to fragment, variant, analog, homologue or the derivant of CCN1, CCN2, CCN4, CCN5 and CCN6 and described one or more other CCN polypeptide.

A. angiogenesis

[0056] method of the present invention relates to the screening angiogenesis modulators.In one embodiment of the invention, the biological sample that can carry out angiogenesis contacts with the ECM signal transduction molecule at external and suspicious regulator.In contrast, second kind of biological sample also contacted with the ECM signal transduction molecule.When comparing, can identify angiogenesis modulators by the ability that changes specimen angiogenesis level with control sample.

[0057] in another embodiment of the invention, the implant that comprises suspicious regulator and ECM signal transduction molecule is implanted in the experimental animal.In contrast, second kind of implant that comprises the ECM signal transduction molecule also is implanted in the experimental animal, and described animal can be same animals or different experimental animals.When comparing, can identify angiogenesis modulators by in the test implant, changing the ability of vascular development level with control sample.

B. tumor takes place

[0058] method of the present invention also relates to the tumorigenic regulator of screening.Tumor is contacted with the ECM signal transduction molecule with suspicious regulator.In contrast, second kind of tumor also contacts with the ECM signal transduction molecule.When comparing, can identify tumorigenic regulator by changing the ability of test tumor tumor occurred level with control tumor.

C. cell adhesion

[0059] method of the present invention also relates to screening cell adhesion regulator.Biological sample is contacted with the ECM signal transduction molecule with suspicious regulator on the surface compatible with cell adhesion.In contrast, second kind of biological sample also contacts with the ECM signal transduction molecule on the surface compatible with cell adhesion.When comparing with control sample, can be by changing the ability of specimen cell adhesion level, the adherent regulator of identification of cell.

D. cell migration

[0060] method of the present invention also relates to the regulator that screens cell migration.The cell inoculation that can carry out cell migration is on the gel-type vehicle that comprises suspicious regulator and ECM signal transduction molecule.In contrast, the cell that can carry out cell migration also is inoculated on second kind of biological sample gel-type vehicle that comprises the ECM signal transduction molecule.When comparing with contrast substrate, can be by in test substrate, changing the ability of cell migration level, the adherent regulator of identification of cell.

E. Therapeutic Method

[0061] the CCN1 active regulator that the present invention also relates to use above-mentioned screening technique to identify.The CCN1 active regulator of identifying can be made and comprise the pharmaceutical composition that medicine can be accepted accessory drugs, diluent or carrier.Can give the patient with the pharmaceutical composition that comprises the CCN1 active regulator, be used for the treatment of with angiogenesis, tumor and take place or cartilage formation diseases associated.Can be separately or with other compositions for example chemotherapeutics unite and give pharmaceutical composition.

[0062] present composition can give with standard mode, includes but not limited to oral, parenteral, Sublingual, transdermal, rectum, saturating mucosa, part, through sucking or buccal administration.Parenteral includes but not limited in intravenous, intra-arterial (intraareterial), intraperitoneal, subcutaneous, intramuscular, the sheath and intra-articular administration.

[0063] now generally described the present invention, other aspects and advantages of the present invention will be understood on the basis of considering following illustrative embodiment.These embodiment are just in order to illustrate the present invention, and not in order to limit the scope of the invention that proposes in the appended claim.The territory III that embodiment 1 discloses CCN1 supports α ₆β ₁The cell adhesion that relies on.The T1 sequence that embodiment 2 discloses among the III of CCN territory contains beta 2 integrin alpha ₆β ₁Binding site.Embodiment 3 has described soluble T 1 peptide to α ₆β ₁The influence of the cell adhesion that relies on.Embodiment 4 has described the adherent influence of alanine displacement pair cell in the T1 sequence.Embodiment 5 has described the affinity substrate protein affinity purification beta 2 integrin alpha that uses coupling T1 ₆β ₁Embodiment 6 has described the influence that soluble T 1 peptide forms the inductive endothelium tubule of CCN1.Embodiment 7 has described the beta 2 integrin alpha of T1 ₆β ₁The structure of the mutant peptide of binding site place deactivation.Embodiment 8 has described the beta 2 integrin alpha at T1 ₆β ₁The adhesion assessment of the sudden change CCN1 peptide of binding site place deactivation.Embodiment 9 has described the beta 2 integrin alpha at T1 ₆β ₁The structure of the sudden change CCN1 of binding site place deactivation.Embodiment 10-14 has described the cell adhesion character of CCN1 mutant.Embodiment 10 has described and has used destructive T1 binding site and do not destroy the SM mensuration that the heparin binding site carries out.Embodiment 11 has described and has used destructive T1 binding site and suppress the SM mensuration that the heparin binding site carries out.Embodiment 12 has described the DM that uses unbroken T1 binding site and destructive heparin binding site to carry out and has measured.Embodiment 13 has described the TM that uses destructive T1 binding site and destructive heparin binding site to carry out and has measured.Embodiment 14 has described and has used anti-alpha 2 integrin to be used to assess the cell adhesion of DM.Embodiment 15-16 has described H1 and H2 and has destroyed influence to CCN1 dependency MAPK activation and gene expression regulation.Embodiment 15 has described the activation of MAPK.Embodiment 16 has described gene expression regulation.Embodiment 17-21 has described and has passed through α _vβ ₃And α ₆β ₁The active decomposition of CCN1 of-HSPG mediation.Embodiment 17 has described and has passed through beta 2 integrin alpha _vβ ₃HUVEC adhere to and move to the CCN1 mutant.Embodiment 18 has described the migration of HUVECS to CCN1 or mutant.Embodiment 19 has described and has passed through beta 2 integrin alpha _vβ ₃It is synthetic to strengthen the inductive DNA of VEGF-.Embodiment 20 has described the influence of CCN1 mutant to the HUVEC survival.Embodiment 21 has described tubule formation.These embodiment are for the present invention being described, should not being interpreted as limiting the scope of the invention.

Embodiment 1

The territory III of CCN1 (TSPI-homology territory) supports α ₆β ₁The cell adhesion that relies on

[0064] the previous primary human skin flbroblast of having determined is passed through beta 2 integrin alpha ₆β ₁Adhere to CCN1 with heparan sulfate proteoglycan, induce to form and contain α ₆β ₁Focus complex and activation focal adhesion kinase, paxillin and Rac.Referring to Chen et al. (2000) J.Biol.Chem.275,24953-24961; Chen et al. (2001) J.Biol.Chem.276,10443-10452.Deletion analysis shows that the C-terminal truncate CCN1 mutant only contain junior three territory is retained in and passes through beta 2 integrin alpha in the smooth muscle ₆β ₁Induce chemotactic ability, therefore the integrin binding site is positioned in three initial territories.Referring to Grzeszkiewicz et al (2002) Endocrinology 143,1441-1450.

[0065] in order to define and beta 2 integrin alpha ₆β ₁Interactional CCN1 domain, we have expressed each (Figure 1A) in these three territories via baculovirus vector in insect cell.In order to strengthen protein excretion, we have used pMelBac B carrier (InvitrogenIncorp.) so that N-terminal bee variety phallotoxins secreting signal peptide to be provided.In order to produce the sequence of encoding domain I (IGFBP), territory II (VWC) and territory III (TSP1), we use following primer sets that CCN1cDNA is carried out PCR respectively;

5 '-CGCGGATCCGGCGCTCTCCACCTGC-3 ' and

5’-GGAATTCCCTCTGCAGATCCCTTTCAGAGCGG-3’

5 '-CGCGGATCCGGCTCAGTCAGAAGGCAGAC-3 ' and

5’-GGAATTCCCAGGAAGCCTCTTCAGTGAGCTGCC-3’

5 '-CGCGGATCCGGTCTTTGGCACC-3 ' and

5′-GGAATTCCCTTTTAGGCTGCTGTACACTGGTTGTC-3′

[0066] uses BamH1 and EcoR1 digestion PCR product, connect into pMelBac B.The recombinant polypeptide of every kind of expression contains V5 epitope and polyhistidine labelling at C-terminal, uses described serum-free baculovirus expression system purification from the Sf9 cell.Referring to Grzeszkiewicz et al. (2001) J.Biol.Chem.276,21943-21950.

[0067] in brief, cell maintain serum-free EX-CELL 400 culture medium (JRHBiosciences, Lenexa, KS) in, and infect 42-46 hour collecting cell after infection with infection multiplicity 10.Culture medium by the centrifugal clarification collection, use Biomax-5 centrifugal type filter (Millipore subsequently, Billerica, MA) concentrate 10-15 doubly, under 4 ℃ to natural buffer (50mM sodium phosphate and 10mM Hepes, pH7.4,0.5M NaCI) dialysed overnight, be splined on then TALON cobalt-agarose column (Clontech, Palo Alto, CA).Under pH7.0 with natural binding buffer liquid at the pH7.0 column scrubber, use 50mM phosphate, 0.3MNaCl and 150mM imidazoles eluting post then.Carry out coomassie brilliant blue staining and immunoblotting, assay products behind the SDS-PAGE.The segmental merging fraction in each territory under pH7.4 to 20mMHepes, 150mM NaCl 4 ℃ of following dialysed overnight, remove imidazoles.Each territory fragment has expection molecular weight (territory I, territory II and territory III respectively do for oneself～11kDa, 18kDa and 9kDa), carries out immunoreation (Figure 1B and C) with anti-CCN1 polyclonal antibody.

[0068] our end user 1064SK fibroblast is determined the adherent ability of each territory sustenticular cell.Elementary human foreskin fibroblast 1064SK (ATCC CRL-2076 goes down to posterity 2) is at 37 ℃ and 5%CO ₂Be maintained at down Iscove improvement Dulbecco ' s culture medium (Invitrogen, Carlsbad, CA) in, its have 10% hyclone (Intergen, Purchase, NY).The cell in the 5th to the 20th generation is used in all tests.In PBS (the every hole of 50ml) will test albumen coat 96 orifice plates (Becton Dickinson, NJ) on, (MO) blind hole is 1 hour for Sigma, St.Louis at room temperature to use 1%BSA.In order to strengthen coating efficiency, (Pierce, Rockford IL), spend the night, and use BSA to seal 2 hours down at 37 ℃ subsequently under 4 ℃ the CCN1 domain polypeptide to be covalently attached to maleic anhydride Reacti-Bind microtitration plate.Use washed branch junction (Subconfluent) cell that is suspended in again in the serum-free basal medium, with 5 * 10 ⁵Cell/ml carries out cell adhesion as described.Referring to Chenet al. (2001) J.Biol.Chem.276,10443-10452.Wherein it is pointed out that before flat board is cultivated, used EDTA, peptide or function interdiction monoclonal antibody pre-cultured cell 30 minutes.

[0069] all three kinds of territories is coated on the microtiter well with similar efficient.Use albumen or BSA (50ml/ hole) coating microtiter well under 4 ℃ of six histidine marks to spend the night, sealed 2 hours with 1%BSA under the room temperature subsequently.By using anti-polyhistidine monoclonal antibody (Invitrogen) (2 hours, under 37 ℃) cultivate, (Amersham Pharmacia Biotech-Piscataway, NJ) (1 hour, 37 ℃ under) measures the albumen coating efficiency to use the second antibody of horseradish peroxidase subsequently.Chromogenic reaction is by at A ₄₂₀Measure quantitatively.

[0070] have only territory III can support fibroblast to adhere to (Fig. 1 D and E).Fibroblast attaches to territory III and is suppressed by EDTA (2.5mM), and this inhibition can be by adding Mg in test media ⁺⁺Alleviate (Fig. 2 A).Cell adhesion also is Ca ⁺⁺(5mM) suppress, be Mn ⁺⁺(0.5mM) strengthen.This bivalent cation sensitivity spectrum is similar to total length CCN1, meets and passes through beta 2 integrin alpha ₆β ₁Cell adhesion.Referring to Chen et al. (2000) J.Biol.Chem.275,24953-24961.In order to determine which kind of specificity integrin receptor mediated cell adheres to territory III, we have tested the inhibitory property of function interdiction monoclonal antibody.Use anti-a ₆(GoH3) or b ₁(P4C10) monoclonal antibody is cultivated the cell adhesion that fibroblast has been blocked territory III and total length CCN1, wherein anti-alpha 2 integrin α in advance _vβ ₃Monoclonal antibody or control mice IgG do not have effect (Fig. 2 B).These results show that together human skin fibroblast adheres to isolating CCN1 territory III, and as adhering to total length CCN1, this passes through beta 2 integrin alpha ₆β ₁Mediation.

Embodiment 2

The T1 sequence contains beta 2 integrin alpha among the III of CCN1 territory ₆β ₁Binding site

[0071] we have used other screening system strategy to find out beta 2 integrin alpha among the CCN1 ₆β ₁Binding site.By peptide being expressed as the fusion rotein that is connected with GST, prepare the overlapping peptide (table 1) in a series of covering whole C CN1 three territories.By polymerase chain reaction (PCR), be the encode coded sequence of various peptides (Fig. 3) of template amplification with CCN1 cDNA.The primer that uses corresponding with suitable coded sequence, and contain BamHI and the EcoRI restriction enzyme site that is useful on the clone.For example, use following primer to produce the T1 peptide-coding sequence:

5 '-CGGGATCCGCGGGCCAGAAATGCATCGTT-3 ' and

5 '-CCGGAATTCCGCTCTTGGAGCACTGGGACC-3 ' is purified pcr product on polyacrylamide gel, uses BamHI and EcoRI digestion, is connected in the pGEX-4T-2 carrier (Amersham Pharmacia Biotech).Prove conclusively all clone's steps by sequence analysis.Produce the GST-peptide fusion protein in escherichia coli (E.coli) bacterial strain BL21, (Amersham Pharmacia Biotech) carries out purification by the glutathion affinity chromatograph, subsequently under 4 ℃ to the thorough dialysed overnight of PBS.

[0072] these fusion rotein is purified near homogeneity, has similar coating efficiency in the air, as use anti-GST antibody by ELISA detected (data not shown) in microtitration.Assess every kind of peptide-gst fusion protein and supported the adherent ability of fibroblast.Have only a kind of peptide-gst fusion protein, promptly, can adhere to (Fig. 3 A) by sustenticular cell from the T1 of territory III.In addition, EDTA and Ca in measuring culture medium ⁺⁺Be suppressed to fibrocyte and adhere to T1-GST, and Mn ⁺⁺Strengthen its effect (Fig. 3 B).Equally, use anti-a ₆(GoH3) or b ₁(P4C10) the monoclonal antibody pre-cultured cell can be blocked cell adhesion in T1-GST, but other integrin agent interfering such as GRGDSP peptide (Life Technologies/Gibco-BRL) or anti-α _vβ ₃(LM609) do not have effect (Fig. 3 C), show that the T1-GST fusion rotein passes through beta 2 integrin alpha ₆β ₁Support fibroblastic adhesive attraction.Equally, T1-GST also supports α in other cell type ₆β ₁The cell adhesion of mediation comprises endotheliocyte, smooth muscle cell and PC3 prostatic cell (data not shown).

[0073] contains beta 2 integrin alpha in order further to establish the T1 sequence ₆β ₁Binding site, synthesized the tetrapeptide (table 1) of striding CCN1 territory III, and tested the adherent ability of its sustenticular cell.(purification on reversed-phase high-performance liquid chromatography passes through analytical reagent composition subsequently for Huntsville, AL) the synthetic peptide of preparation by ResGen Inc.Be similar to the result who uses the GST-peptide fusion protein to obtain, synthetic T1 peptide rather than other 3 kinds of peptides (T2, T3 and T4) are supported fibroblast adhesive attraction (Fig. 4 A).And cell adhesion to immobilization T1 peptide is anti-α ₆(GoH3) or anti-b ₁(P4C10) suppress, rather than anti-α _vβ ₃(LM609) or control mice IgG (Fig. 4 B).These results show that again T1 contains beta 2 integrin alpha ₆β ₁Binding site.

Embodiment 3

Soluble T 1 peptide has suppressed α ₆β ₁The cell adhesion that relies on

[0074] we predict that soluble T 1 peptide can block cell adhesion to known integrin binding α ₆β ₁Substrate.As shown in Fig. 5 A, in cell suspending liquid, add 0.2mM T1 and effectively blocked fibroblast adhesion CCN1, otherwise T2, T3 and T4 do not have effect.The inhibition effect that the T1 pair cell adheres to CCN1 is dose-dependent, and the maximum inhibitory action of acquisition is at 100 μ M (Fig. 5 C).The member CCN2 (CTGF) of other CCN protein family and CCN3 (NOV) also show and pass through beta 2 integrin alpha ₆β ₁Support fibroblast to adhere to (referring to Chen et al. (2001) J.Biol.Chem.276,10443-10452; During Lin et al. (2003) J.Biol.Chem. delivers), in these CCN albumen, there is homology highly in the corresponding T1 sequence.Fig. 5 A shows T1 also specificity inhibition cell adhesion CCN2 and CCN3, shows the T1 sequence beta 2 integrin alpha in the CCN albumen ₆β ₁Common binding site.

[0075] in order further to prove the inhibiting specificity of T1, we have detected the ability of the substrate of its other integrin binding of blocking-up cell adhesion.Compare (Fig. 5 C) with the effect of T1 dose-dependent inhibition cell adhesion CCN1, it adheres to fibronectin (beta 2 integrin alpha to fibroblast ₆β ₁Aglucon), vitronectin (a _vThe aglucon of integrin) and collagen (β ₁The aglucon of integrin) there is not remarkable result (Fig. 5 B).Cell adhesion is to known beta 2 integrin alpha ₆β ₁The aglucon laminin can be suppressed (～15%) by the T1 peptide moiety.This part suppresses to be similar to by anti-a ₆(data are for showing) that monoclonal antibody GoH3 obtained.May be because there be other integrin in incomplete inhibition of T1 and GoH3, as α ₂β ₁, it is also as the adhesion receptor of laminin.These results show that together soluble T 1 peptide specific has suppressed α ₆β ₁Therefore the cell adhesion that relies on has further supported the T1 sequence to contain beta 2 integrin alpha ₆β ₁Binding site.

Embodiment 4

The adherent influence of alanine displacement pair cell in the T1 sequence

[0076] in order to determine which residue is α in the T1 sequence ₆β ₁The adherent important determiner of dependent cell, we have prepared a series of GST-peptide fusions, it has carried the T1 skeleton, and the conserved residues place between CCN1, CCN2 and CCN3 has single or two alanine displacements, and has tested the adherent ability of their sustenticular cell.In order to produce the fixed point alanine displacement of T1 peptide, the annealing synthetic oligonucleotide carries out PCR, producing suitable coded sequence, and is cloned into pGEX-4T-2.Use following primer preparation to be used for the coded sequence of T1 sequence, and be cloned into pGEX-4T-2:

5 '-GATCCGGTCAAAAATGTATTGTTCAAACTACTTCTTGGTCTCAATGCTCTAAATCT GG-3 ' and

5 '-AATTCCAGATTTAGAGCATTGAGACCAAGAAGTAGTAGTTTGAACAATACATTTTT GACCG-3 ' is in order to produce mutant peptide, and changing the relevant codon of alanine is GCA or GCT.

[0077] as shown in Figure 6, the alanine displacement at residue K226, I228 or Q230 place does not influence the adherent ability of peptide sustenticular cell.And the single mutation at T231 or T232 place will cause the part of cell adhesion to reduce, and the adherent ability of T1 sustenticular cell has been eliminated in the combination alanine displacement of T231 and T232 fully.In addition, the displacement of the list among W234, S235, S238 or the K239 causes the T1 loss of activity greater than 90%.In when, in W234 and K239 combinatorial mutagenesis taking place when, the cell adhesion complete obiteration.These results show that TTSWSQCSKS mediates α among the T1 ₆β ₁Bonded core sequence.These data have explained that also T2 peptide ability has not suppressed α ₆β ₁The cell adhesion that relies on, but itself and the overlapping TT residue that lacks core sequence of T1 peptide.

Embodiment 5

Use the affinity substrate affinity purification beta 2 integrin alpha of coupling T1 ₆β ₁

[0078] in order to confirm the direct integrin binding α of T1 peptide ₆β ₁, we use the cell surface protein on the fibroblast to carry out affinity chromatograph, to separate beta 2 integrin alpha on the affinity column of coupling T1 ₆β ₁Use the 2mM EDTA among the PBS to separate the synthetic fibers cell 1064SK of branch with 0.05%BSA, wash three times, with 2 * 10 ⁷Individual cell/ml is suspended among the PBS that contains the 20mM glucose again.For carrying out surface markers, 4 ℃ use down 100mU/ml glucoseoxidases, 200mg/ml lactoperoxidase (Calbiochem-Novabiochem, La Jolla, CA) and～400mCi/ml carrier-free Na ¹²⁵I (AmershamPharmacia Biotech) is gentle to rotate pre-cell suspension thing 30-60 minute.Be end mark, add the 10ml cell culture medium.The cell of washing labelling is at 1ml lysis buffer (50mMHepes, pH7.4,200mM octyl group-b-D-pyranglucoside, protease inhibitor cocktail and 0.5mM Mn ⁺⁺) middle cell lysis.For affinity chromatograph, with 10mg/ml Affi-Gel 10 suspensions (Bio-Rad Laboratories, Hercules, CA) with GST-T1 or the out of order T1 albumen coupling of GST-in gel.The cellular lysate of labelling is applied to (3ml/ml gel) on the affinity substrate, cultivated 2 hours down for 4 ℃.Use the lysis buffer column scrubber of 30 times of column volumes, use the 0.3M NaCl eluting in the lysis buffer subsequently.By on 7% polyacrylamide gel, carrying out electrophoresis under the non-reduced condition, and carry out autoradiography subsequently, analyze the labelled protein in the elutriated fraction.In immunoprecipitation analysis, use the anti-a of 5mg ₆(GoH3) or anti-α _v(P3G8) monoclonal antibody (Chemicon, Temecula, CA) incubation labelled protein as shown.On Protein G-Sepharose, collect immunoprecipitation albumen, and on 7% polyacrylamide gel, resolving under the non-reduced condition.

[0079] uses the out of order T1 sequence preparation contrast post that merges with GST, not eluting labelled protein band (Fig. 7 A) from the out of order T1-GST post.By comparison, under the 0.35M NaCl from the T1-GST affinity column eluting two protein bands, the corresponding integrin a of apparent molecular weight ₆(～150kDa) and b ₁The subunit of (～130kDa) (swimming lane 5-7, Fig. 7 B).In order to confirm that bonded labelled protein is beta 2 integrin alpha really ₆β ₁Complex uses GoH3 (anti-a ₆) or (the anti-α of P3G8 in contrast _v) eluent is carried out immunoprecipitation.Fig. 7 C has shown the GoH3 immunoprecipitation and has derived from the labelled protein band of eluent, and P3G8 can not the protein precipitation complex in control sample.In a word, we reach a conclusion, beta 2 integrin alpha ₆β ₁Directly be attached to the T1 sequence among the CCN1.

Embodiment 6

Soluble T 1 peptide has destroyed the inductive endothelium tubule of CCN1 and has formed

[0080] the some CCN albumen that comprise CCN1, CCN2 and CCN3 are effective angiogenesis inducer.Referring to Babic et al. (1998) Proc.Natl.Acad.Sci.U.S.A.95,6355-6360; During Lin et al. (2003) J.Biol.Chem. delivers; Babic et al. (1999) Mol.Cell Biol.19,2958-2966.In addition, when making collagen gel, CCN1 can induce the tubule of unactivated Human umbilical vein endothelial cells (HUVEC) to form, and this process is by anti-a ₆Monoclonal antibody GoH3 blocks.Referring to Leu et al. (2002) J.Biol.Chem.277,46248-46255.Because the T1 sequence has been represented beta 2 integrin alpha in CCN1 ₆β ₁Main binding site, we have checked that the tubule whether soluble T 1 peptide suppresses the inductive un-activation HUVEC of CCN1 forms.Form in order to measure the endotheliocyte tubule, when existing or not having described test proteins or peptide, detected the Human umbilical vein endothelial cells (HUVEC) in the three-dimensional collagen gel.Referring to Leu et al. (2002) J.Biol.Chem.277,46248-46255.As shown in Figure 8, when collagen gel is prepared with CCN1, induce Human umbilical vein endothelial cells to form tubule.Before flat board is cultivated, HUVEC and T1 (0.2mM) precincubation were suppressed the inductive tubule formation of CCN1 in 30 minutes fully.By comparison, contrast T2, T3 and T4 peptide do not have effect.In sum, these results show, go up beta 2 integrin alpha by blocking-up CCN1 and un-activation HUVEC ₆β ₁Interaction, T1 has suppressed the inductive tubule of CCN1 and has formed.

Embodiment 7

Beta 2 integrin alpha at T1 ₆β ₁The structure of the mutant peptide of binding site place inactivation

[0081] the some CCN albumen that comprise CCN1, CCN2 and CCN3 are effective vascularization inducer.Be used for determining novel α ₆β ₁During binding site, the metathetical GST-T1 fusogenic peptide of alanine described in the embodiment 4 is proved to be and is not suitable for measuring the function of T1 site in the CCN1 activity.The mutant of expressing is assembled, and at cell interior formation occlusion body (data not shown), shows that these mutants have taked deleterious conformation, has stoped their secretion.In order to create at α ₆β ₁The active mutant that is kept other function by specifically inactivating of-HSPG-mediation, we use two kinds of algorithms, PSIPRED and FRAGFOLD (McGuffin, L.J., Bryson, K. and Jones, D.T. (2000) Bioinformatics.16,404-405, and Jones, D.T. (2001) Proteins Suppl 5,127-132), made up fusion rotein, with may changing that prediction can cause by suddenling change in secondary structure.

[0082] according to Leu, S.J., Liu, Y., Chen, N., Chen, C.C., Lam, S.C., and Lau, L F. (2003) J.Biol.Chem.278, method described in the 33801-33808, we have prepared the gst fusion protein and the mutant peptide of CCN1 peptide.

[0083] by using primer sets, is template, made up mice CCN1 with C-terminal FLAG labelling with mice Ccnl cDNA.

F1:5 '-CGCAATTGGAAAAGGCAGCTCACTGAAGAGGC-3 ' and

F2:5 '-CCGGAATTCCTACTTGTCATCGTCATCCTTGTAGTCGTCCCTGAACTTGTGGATGT CATTG-3 ' has therefore produced and has contained the PCR product of Ccnl termination codon back immediately following FLAG marker coding sequence and termination codon.Use NcoI and EcoRI double digested PCR product, connect into the carrier of precut, in the pBlueBac4.5 carrier, to replace NcoI, EcoRI-digestion fragment (Grzeszkiewicz, T.M., the Kirschling of total length mice CcnlcDNA, D.J., Chen, N., and Lau, L.F. (2001) J.Biol.Chem.276,21943-21950).In order to be consistent, similarly made up employed WT CCN1 and all mutants in this research, they have identical N-terminal secretion signal and C-terminal FLAG epi-position labelling.

[0084] can not be that the T1 district of CCN1 presents sensitivity highly to interference by secretion is consistent with alanine replacement mutation body.Use almost any amino acid replacement to cause the acute variation of predicted protein structure in the decisive T1 residue (W234, S235, S238, K239).Yet we find a sudden change, and promptly K239E can not cause the prediction conformation change.Destroy and α in order to test this sudden change ₆β ₁Interactional effectiveness, we have made up the fusion rotein that GST is connected with the T1 peptide, and wherein T1 carries K239E sudden change (GQKCIVATTSWS QCSES).

Embodiment 8

Be evaluated at the beta 2 integrin alpha of T1 ₆β ₁The adhesion of the sudden change CCN1 peptide of binding site place inactivation

[0085] compare with the GST-T1 fusions with WT sequence, we have estimated fibroblast and have adhered to constructed T1 (K293E) mutant peptide according to embodiment 7.GST-T1 has similar coating efficiency with GST-T1 (K239E), measures (data not shown) as ELISA.

[0086] we place fibroblast on the microtiter well that is coated with GST, GST-T1 peptide fusions or GST-T1 (K239E) peptide fusion protein (each 50 μ g/ml).37 ℃ allow cell adhesion 20 minutes down.After the washing, the secure attachment cell uses methylene blue staining, and is quantitative by the absorbance at 620nm place.

[0087] as expected, fibroblast adheres to the GST-T1 fusions and does not adhere to BSA and GST contrast (Fig. 9 B).Referring to Leu, S.J., Liu, Y., Chen, N., Chen, C.C., Lam, S.C., and Lau, L.F. (2003) J.Biol.Chem.278,33801-33808.GST-T1 (K239E) can not adhere to by sustenticular cell fully, shows that T1 and beta 2 integrin alpha can be fully eliminated in the K239E sudden change of charge reversal ₆β ₁Combination.

Embodiment 9

Beta 2 integrin alpha at T1 ₆β ₁The structure of the sudden change CCN1 of binding site place inactivation

[0088] we have made up the K239E single mutation in total length CCN1, and called after SM (Fig. 9 A) uses the two-step pcr method to carry out rite-directed mutagenesis, as Koskinen, P., Lehvaslaiho, H., MacDonald Bravo, H., Alitalo, K., and Bravo, R. (1990) Oncogene.5 is described in the 615-618.Inner primer is to being:

5 '-GTCTTGGTCCCAGTGTTCCGAGAGCTGCGG-3 ' and

5 "-this construction thing liquid of CACTGGGACCAAGACGTGGTCTGAACGATGC-3 ' can produce silent mutation at the C237 place, thereby provide selection markers by removing the BSP12861 restriction enzyme site.The outside primer that uses in PCR is the foregoing description A ₁Described F1 and F2.The mice Ccnl cDNA that use has the FLAG labelling makes up SM as pcr template.

[0089] we have also made up the DM coded sequence, and it has destructive heparin binding site H1 and H2, described in Fig. 9 A.These sudden changes become AGAACSATAKSPEPVR with H1 from KGKKCSKTKKSPEPVR, and H2 is become FTYAGCSSVAAYAPKY from FTYAGCSSVKKYRPKY, and background is identical with CCN1.We use the DM coded sequence, use said method to produce the DM construction with FLAG labelling.Use DM as template, use said method to produce the K239E sudden change, we have created the TM sequence.Confirm all constructions by direct sequence analysis.

Embodiment 10

SM mensuration-destructive Tl binding site, unbroken heparin binding site

[0090] we are incubated at the microtiter well that uses specified amount reorganization WT CCN1 or the coating of SM mutant with 1064SK human fibroblasts flat board, as evaluation cell adhesion as described in the embodiment 1.Surprisingly, SM can support fibroblast to adhere in dose-dependent mode, resembles very much WT CCN1, obtains maximum cell and adhere to (Figure 10 A) when coating concentration is 1 μ g/ml.This result proves that SM still can pass through α ₆β ₁-HSPG coreceptor sustenticular cell adheres to, and shows to exist other also not obtain the α that identifies ₆β ₁-HSPG binding site, or H1 and H2 heparin binding site pass through α ₆β ₁-HSPG coreceptor complex is enough to sustenticular cell and adheres to.

Embodiment 11

SM mensuration-destructive Tl binding site suppresses the heparin binding site

[0091] because SM is keeping complete heparin binding site, we have tested cell adhesion SM and whether have depended on HSPG.Before adhering to the microtiter well that uses WT CCN1 (2 μ g/ml), SM (2 μ g/ml) or VN (0.5 μ g/ml) coating, we use Heparinase I (2 units/ml) or chondroitinase abc (2 units/ml) (from Sigma, St.Louis MO) is processed into fibrocyte.Control wells does not process.It is pointed out that wherein the solubility heparin concentration that exists is 1 μ g/ml in culture medium.

[0092] as expected, no matter be the solubility heparin that has saturated CCN1 heparin binding site, also be to use Heparinase I to handle cytoclasis cell surface HSPG, can both block cell adhesion WT CCN1.Referring to Chen, N., Chen, C.C., and Lau, L.F. (2000) J.Biol.Chem.275,24953-24961.Equally, solubility heparin or heparinase are handled and have been suppressed cell adhesion SM similarly, show that interaction partners cell adhesion to this CCN1 mutant of cell surface HSPG and SM is vital.Using chondroitinase abc to handle cell does not have effect, confirms at special on HSPG rather than the chondroitin sulfate proteoglycan of relating to of CCN1.

Embodiment 12

DM mensuration-unbroken T1 binding site, destructive heparin binding site

[0093] be combined in effect in the CCN1 function in order further to estimate heparin, we use embodiment 9 described DM mutants to detect the effect of eliminating heparin binding activity.The sudden change of the alkaline residue of H1 or H2 reduces the heparin combination, the combinatorial mutagenesis among H1 shown in Fig. 9 A and the H2 blocked the heparin binding activity among the CCN1 (Chen, N., Chen, C.C., and Lau, L.F. (2000) J.Biol.Chem:275,24953-24961).Because having lost heparin binding activity, DM kept T1 α ₆β ₁Binding site, we suppose that DM may be at α ₆β ₁Impaired on the activity of-coreceptor-mediation, but α may when high coating concentration, be passed through ₆β ₁Sustenticular cell adheres to, and is similar to cell adhesion GST-T1 peptide fusion protein (Fig. 9 B).

[0094] in fact, cell adhesion has reached peak (Figure 10 A) at 1 μ g/ml to CCN1, and DM can not adhere to as adhering to the substrate sustenticular cell, unless be coated with much higher concentration, the maximum adhesion effect occurs in 50-100 μ g/ml (Figure 11 A).

Embodiment 13

TM mensuration-destructive Tl binding site, destructive heparin binding site

[0095] as described in example 9 above, we have also produced the mutant (TM) of combination K293E sudden change in the DM background, thereby have destroyed the beta 2 integrin alpha of T1 ₆β ₁Binding site, and the H1 of heparin and H2 binding site.

What [0096] cause raised eyebrows is, all can not adhere to by sustenticular cell at any concentration TM that tried, and it is invalid fully to keep, even when being coated with 250 μ g/ml.This discovery shows that DM can pass through α ₆β ₁The specificity sustenticular cell adheres to, and this activity can be by the beta 2 integrin alpha of T1 ₆β ₁K239E sudden change in the binding site is removed.

Embodiment 14

Use the cell adhesion of anti-alpha 2 integrin with assessment DM

[0097] eliminates α in order to verify by K239E T1 sudden change ₆β ₁Adhesive attraction, we have detected the cell adhesion that DM supports and whether have passed through α ₆β ₁Mediation.We used 40 μ g/ml anti-alpha 2 integrin α before flat board is cultivated _vβ ₃(LM609), beta 2 integrin alpha ₆(GoH3) or integrin β ₁The function interdiction monoclonal antibody room temperature pre-cultured cell of subunit (P4C10,1: 50 ascites) 1 hour.Estimate cell adhesion then as mentioned above.

[0098] unanimity is anti-alpha 2 integrin α ₆(GoH3) or β ₁(P4C10) monoclonal antibody has been blocked the fibroblast adhesion of DM or CCN1 support, and anti-α _vβ ₃Monoclonal antibody LM609 does not have effect (Figure 11 B).In sum, these results are shown as fibrocyte adhesion CCN1 needs α ₆β ₁-HSPG coreceptor and three kinds of binding sites interact: T1, H1 and H2.α is still passed through in sudden change among H1 and the H2 (DM) ₆β ₁When high coating concentration, allow cell adhesion, and the destruction of all three kinds of binding sites (TM) has been eliminated fully and has been passed through α ₆β ₁The cell adhesion of-HSPG (Figure 11 A).

Embodiment 15

The MAPK activation

[0099] CCN1 have rare ability induce as the p42/p44MAPK continuous activation that adheres to substrate (Chen, C.-C., Chen, N., and Lau, L.F. (2001) J.Biol.Chem.276,10443-10452).In order to test sudden change in T1, H1 and the H2 binding site to active influence, fibroblast is adhered to WT CCN1, SM, DM or laminin, continue the different time (Figure 12 A).In order to assess the activation of MAPK, the 1064SK fibroblast is carried out serum starvation, with 6 * 10 ⁵Individual cell/ml is suspended in the culture medium of serum-free again, and flat board is incubated at the plate that is coated with CCN1 (10 μ g/ml), SM (10 μ g/ml), DM (250 μ g/ml) or laminin (10 μ g/ml) in advance, cultivates as shown 1-5 hour.On 10%SDS-PAGE clarifying cellular lysate is carried out electrophoresis, (Progema, Madison WI) carry out immunoblotting in the antibody of diphosphate p42/p44MAPKS to use anti-MAPK antibody or specific action.

[0100] as shown in Figure 12 A, the cell that adheres to laminin produces the typical case and sees the quick and of short duration p42/p44MAPK activation that adheres to the ECM substrate, reaches active top level after flat board is cultivated 1 hour, drops to background level afterwards.On the contrary, the activation of MAPK is kept the longer time in the cell of adhesion WT CCN1 or SM, even reaches back 5 hours of dull and stereotyped cultivation.The MAPK activation of this prolongation loses in the cell that adheres to DM.Therefore, the sudden change in H1 and the H2 site does not influence the short-term activation of MAPK, but specificity has been eliminated CCN1 and induced the activatory ability of lasting MAPK.

Embodiment 16

Gene expression regulation

[0101] CCN1 has activated genetic program in fibroblast, the proteic encoding gene that causes relating to angiogenesis and matrix metabolism raises, described albumen comprises vascularization inducer VEGF and matrix metalloproteinase MMP-1 (Chen, C.-C., Chen, N., and Lau, L.F. (2001) J.Biol.Chem.276,10443-10452).Because activatory MAPK by dystopy to nucleus, but their phosphorylations and activating transcription factor (Hazzalin there, C.A. and Mahadevan, L.C. (2002) Nat.Rev.Mol.Cell Biol.3,30-40), continue the activatory disappearance of MAPK and can change the ability that the CCN1 regulator gene is expressed.

[0102] loses solubility CCN1 (WT) and CCN1 mutant (SM in order to test lasting MAPK activation, DM, TM) influence of regulator gene ability to express, we use 10 μ g/ml albumen to handle the primary human skin flbroblast 24 hours of serum starvation under serum-free condition.Separate total cell RNA, on the agarose formaldehyde gel, resolve, use the standard scheme trace on nylon membrane.We as described will by random primer labelling ³²P-dCTP mixes people VEGF-A, MMP-1 and GAPDH cDNA, produced radioactivity cDNA probe (Chen, C.-C., Chen, N., and Lau, L.F. (2001) J.Biol.Chem.276,10443-10452).Our (0.1 * SSC under high stringent condition then; 0.1%SDS, 65 ℃) the washing trace, and use Phosphorimager analyze they (Molecular Dynamics, Sunnyvale, CA).By RNA trace (the total RNA of 20 μ g in each swimming lane), assess the expression of Vegf and MMP-1 after electrophoresis, the expression of monitoring glyceraldehyde 3-phosphate dehydro-genase (GAPDH) in contrast.

[0103] as shown in Figure 12B, SM can raise the expression of Vegf and MMP-1, be similar to WT CCN1, and DM and TM is short of this activity fully.Therefore, the MAPK of prolongation activation and rise Vegf and MMP-1 express and need heparin binding site H1 and H2.

Embodiment 17

Pass through beta 2 integrin alpha _vβ ₃HUVEC adheres to and moves to the CCN1 mutant

[0104] we have detected activatory HUVEC and have adhered to the CCN1 mutant.HUVEC dissociates with 2.5mM EDTA and is suspended in serum-free medium again, is adhered to the hole of using 15 μ g/ml wild type CCN1 (WT) or SM or 50 μ g/ml DM or TM coating in advance.Wherein it is pointed out that before flat board is cultivated we use EDTA (5mM), GRGDSP peptide (RGDS, 0.2mM), anti-α _vβ ₃Monoclonal antibody LM609 (40 μ g/ml), anti-α ₆β ₁Monoclonal antibody GoH3 (40 μ g/ml) handled cell 60 minutes.Measure cell adhesion as described.

[0105] WT CCN1 and SM adhere to (Figure 13 A) with the similarity degree sustenticular cell.EDTA has suppressed HUVEC fully and has adhered to WT and SM, GRGDSP peptide or anti-α _vβ ₃Monoclonal antibody LM609 partly suppresses HUVEC and adheres to WT and SM, shows to relate to beta 2 integrin alpha _vβ ₃Cell adhesion is also by anti-α ₆Monoclonal antibody GoH3 partly suppresses, and meets α ₆β ₁-HSPG coreceptor role (Leu, S.-J., Lam, S.C.T., and Lau, L.F. (2002) J.Biol.Chem.277,46248-46255).On the contrary, HUVEC adhesion DM and TM are entirely EDTA, GRGDSP peptide and LM609 to be suppressed, but is not suppressed by GoH3 fully.These results show that DM and TM can pass through α _vβ ₃Support activatory HUVEC to adhere to, but lost support α ₆β ₁The ability of the cell adhesion of mediation.

Embodiment 18

HUVEC is to the migration of CCN1 or mutant

[0106] use the Boyden chamber of improvement to measure, we find WT CCN1 and α ₆β ₁Mutant in the-HSPG binding site can stimulate HUVEC migration (Figure 13 B).Use the migration of assessment HUVEC in Transwell chamber to CCN1 or mutant.Fix the CCN1WT of 15 μ g/ml or mutant DM or the TM of mutant SM, 50 μ g/ml on the lower surface of Transwell film, this Transwell film has been separated two chambers.Use 100nM PMA to handle HUVEC 30 minutes with the activation integrin receptor.Wherein it is to be noted, on flat board is incubated at before the chamber, use carrier buffer (not adding), normal mouse IgG (100 μ g/ml) (Sigma, St.Louis, MO), GoH3 (50 μ g/ml) (Chemicon, Temecula, CA), LM609 (50 μ g/ml) (Immunotech, Marseille, France) pre-cultured cell is other 30 minutes.Allow cell migration 8 hours, count the cell that move downward chamber in the high power field at random at 10.

[0107] cell is anti-α to the migration of CCN1 and all mutants _vβ ₃Monoclonal antibody LM609 institute suppresses fully, and anti-α ₆Monoclonal antibody GoH3 does not have effect.Therefore, has destructive α ₆β ₁The CCN1 mutant of-HSPG binding site can stimulate α _vβ ₃The HUVEC migration that relies on is similar to WT.

Embodiment 19

Pass through beta 2 integrin alpha _vβ ₃It is synthetic to strengthen the inductive DNA of VEGF

[0108] it is synthetic and itself is not had mitogenesis that CCN1 has strengthened the DNA of growth factor-induced.Referring to Kireeva, M.L, Mo, F.-E., Yang, G.P., and Lau, L.F. (1996) Mol.Cell.Biol.16,1326-1334.We use carrier buffer (not adding), LM609 (25 μ g/ml), GoH3 (25 μ g/ml) or normal mouse IgG (25 μ g/ml) to cultivate HUVEC in advance 1 hour.Then we use VEGF (5ng/ml) and/or CCN1 or mutant protein (each 5 μ g/ml) exist [ ³H] handle cell during dT, it mixes post-evaluation in 48 hours.Data are three meansigma methods ± standard deviations of measuring, have represented three experiments.

[0109] as expected, VEGF in HUVEC (5ng/ml) handles and to have induced DNA synthetic, and adds CCN1 separately or mutant does not have effect (Figure 14).Yet, use CCN1, SM, DM or TM to handle cell when having VEGF and strengthened synthetic about 2 times of the inductive DNA of VEGF.LM609 blocks this enhancing, and therefore reducing DNA is blended into the independent inductive level of VEGF.On the contrary, contrast IgG and GoH3 all without any effect.Therefore, SM, DM and TM can both pass through beta 2 integrin alpha in endotheliocyte _vβ ₃The DNA that strengthens growth factor-induced is synthetic, is similar to WT CCN1.

Embodiment 20

The CCN1 mutant has promoted the HUVEC survival

[0110] we before determined, under the condition that lacks somatomedin, CCN1 passes through beta 2 integrin alpha _vβ ₃Improved the survival rate of endotheliocyte.Referring to Leu, S.-J., Lam, S.C.T., and Lau, L.F. (2002) J.Biol.Chem.277,46248-46255.In order to determine whether the CCN1 mutant can improve the survival rate of cell, we use TUNE to measure, and have measured them and flat board have been incubated at the effect (Figure 15) of the HUVEC on the laminin.Allow the HUVEC of serum starvation to adhere to use the coverslip 4 hours that 20 μ g/ml laminins (LN) are coated with in advance.We add serum, CCN1 or mutant protein (each 5 μ g/ml) then, continue 16 hours again.Fixed cell uses TUNEL to measure the monitoring apoptosis.It is pointed out that wherein our use test reagent was cultivated the anti-CCN1 antibody of polyclone 30 minutes in advance before adding culture medium.

[0111] when cell maintains in 20% serum, detect seldom apoptotic cell, and in serum-free medium greater than 70% apoptosis.Adding CCN1, SM, DM or TM can both make the decreased number of apoptotic cell to about 40%.Exist anti-CCN1 antibody can reverse this effect, show that improving cell survival rate is the activity of CCN1 polypeptide.Also monitor the DNA synthetic (data not shown) of the HUVEC that under similarity condition, handles.Consistent with result shown in Figure 14 is that the DNA synthesis rate is not subjected to the influence of independent CCN1 or mutant, shows the not increase cell proliferation increase no thanks to of apoptotic cell.These results show that the CCN1 mutant passes through beta 2 integrin alpha under the condition that lacks somatomedin _vβ ₃The activity of mediation still can promote the survival of endotheliocyte.

Embodiment 21

Tubule forms

[0112] in the time of in being incubated at collagen gel, CCN1 also passes through beta 2 integrin alpha _vβ ₃Induce the tubule in the activation endotheliocyte to form.Referring to Leu, S.-J., Lam, S.C.T., and Lau, L.F. (2002) J.Biol.Chem.277,46248-46255.In order to detect this activity in the CCN1 mutant, flat board be incubated at use do not have (-) have CCN1 (WT) or 24 orifice plates of type i collagen gel (2mg/ml) pre-coating of TM (each 20 μ g/ml) before, use carrier buffer (not adding) to handle HUVEC, or use the 5nM PMA in the serum-free medium to stimulate HUVEC.The second layer gel of same recipe is covered on the adherent cell, and the post-evaluation tubule formed in 16 hours.Wherein it is pointed out that before flat board is cultivated and add LM609 (40 μ g/ml) or GoH3 (40 μ g/ml) to cell suspending liquid.The result represents 3 experiments (* 200 amplification).

When [0113] not having CCN1, unprovoked HUVEC does not demonstrate tubule and forms, and 20 μ g/ml CCN1 do not induce tubule to form (Figure 16).Yet the activatory HUVEC of PMA replys WT CCN1 or TM by forming tubule.Before flat board is cultivated, add LM609 (40 μ g/ml) and suppress tubule formation, do not have effect and add GoH3 (40 μ g/ml) to cell suspending liquid.Obtained identical result (data not shown) with SM and DM.These results show that the CCN1 mutant can induce α in activated endothelial cells _vβ ₃The tubule that relies on forms.

Sequence table

<110>Lau，Lester?F.

＜120〉CCN1 compositions and method

<130>PCT/US2004/019766

<150>US?Provisional?60/480,161

<151>2003-06-21

<160>24

<170>PatentIn?version?3.3

<210>1

<211>10

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>1

Thr?Thr?Ser?Trp?Ser?Gln?Cys?Ser?Lys?Ser

1 5 10

<210>2

<211>17

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>2

Gly?Gln?Lys?Cys?Ile?Val?Gln?Thr?Thr?Ser?Trp?Ser?Gln?Cys?Ser?Lys

1 5 10 15

Ser

<210>3

<211>10

<212>PRT

＜213〉artificial sequence

<220>

＜223〉the identification motif of integrin a6b1

<220>

<221>misc_feature

<222>(3)..(3)

＜223〉Xaa can be any natural amino acid

<220>

<221>misc_feature

<222>(6)..(6)

＜223〉Xaa can be any natural amino acid

<400>3

Thr?Thr?Xaa?Trp?Ser?Xaa?Cys?Ser?Lys?Ser

1 5 10

<210>4

<211>12

<212>PRT

＜213〉artificial sequence

<220>

＜223〉derive from the synthetic peptide of integrin a6b1

<400>4

Asn?Pro?Trp?His?Ser?Ile?Tyr?Ile?Thr?Arg?Phe?Gly

1 5 10

<210>5

<211>12

<212>PRT

＜213〉artificial sequence

<220>

＜223〉derive from the synthetic peptide of integrin a6b1

<400>5

Thr?Trp?Tyr?Lys?Ile?Ala?Phe?Gln?Arg?Asn?Arg?Lys

1 5 10

<210>6

<211>25

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>6

cgcggatccg?gcgctctcca?cctgc 25

<210>7

<211>32

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>7

ggaattccct?ctgcagatcc?ctttcagagc?gg 32

<210>8

<211>29

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>8

cgcggatccg?gctcagtcag?aaggcagac 29

<210>9

<211>33

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>9

ggaattccca?ggaagcctct?tcagtgagct?gcc 33

<210>10

<211>22

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>10

cgcggatccg?gtctttggca?cc 22

<210>11

<211>35

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>11

ggaattccct?tttaggctgc?tgtacactgg?ttgtc 35

<210>12

<211>29

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>12

cgggatccgc?gggccagaaa?tgcatcgtt 29

<210>13

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>13

ccggaattcc?gctcttggag?cactgggacc 30

<210>14

<211>58

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>14

gatccggtca?aaaatgtatt?gttcaaacta?cttcttggtc?tcaatgctct?aaatctgg 58

<210>15

<211>61

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>15

aattccagat?ttagagcatt?gagaccaaga?agtagtagtt?tgaacaatac?atttttgacc 60

g 61

<210>16

<211>32

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>16

cgcaattgga?aaaggcagct?cactgaagag?gc 32

<210>17

<211>61

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>17

ccggaattcc?tacttgtcat?cgtcatcctt?gtagtcgtcc?ctgaacttgt?ggatgtcatt 60

g 61

<210>18

<211>17

<212>PRT

＜213〉artificial sequence

<220>

＜223〉sudden change of the K239E in the T1 peptide

<400>18

Gly?Gln?Lys?Cys?Ile?Val?Ala?Thr?Thr?Ser?Trp?Ser?Gln?Cys?Ser?Glu

1 5 10 15

Ser

<210>19

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>19

gtcttggtcc?cagtgttccg?agagctgcgg 30

<210>20

<211>31

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>20

cactgggacc?aagacgtggt?ctgaacgatg?c 31

<210>21

<211>16

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>21

Lys?Gly?Lys?Lys?Cys?Ser?Lys?Thr?Lys?Lys?Ser?Pro?Glu?Pro?Val?Arg

1 5 10 15

<210>22

<211>16

<212>PRT

＜213〉artificial sequence

<220>

＜223〉mutant H1 site

<400>22

Ala?Gly?Ala?Ala?Cys?Ser?Ala?Thr?Ala?Lys?Ser?Pro?Glu?Pro?Val?Arg

1 5 10 15

<210>23

<211>16

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>23

Phe?Thr?Tyr?Ala?Gly?Cys?Ser?Ser?Val?Lys?Lys?Tyr?Arg?Pro?Lys?Tyr

1 5 10 15

<210>24

<211>16

<212>PRT

＜213〉artificial sequence

<220>

＜223〉mutant H2 site

<400>24

Phe?Thr?Tyr?Ala?Gly?Cys?Ser?Ser?Val?Ala?Ala?Tyr?Ala?Pro?Lys?Tyr

1 5 10 15

Claims

1. isolating CCN1 fragment, described fragment comprises x aminoacid, and wherein said fragments sequence is in being selected from following sequence:

(a) the aminoacid 224-240 of muroid CCN1;

(b) the aminoacid 231-240 of muroid CCN1;

(c) the aminoacid 226-242 of people CCN1;

(d) the aminoacid 233-242 of people CCN1, or described segmental variant, analog, homologue or derivant, condition is that x is 8-50.

2. method of screening angiogenesis modulators, described method comprises:

(a) first biological sample that enables to experience angiogenesis contacts with suspicious regulator with the ECM signal transduction molecule;

(b) second biological sample is contacted with the ECM signal transduction molecule;

(c) the angiogenesis level that obtains of comparison step (a) and step (b), thus by changing the ability discriminating angiogenesis modulators of comparing the angiogenesis level with step (b),

The CCN1 fragment that wherein said ECM signal transduction molecule is a claim 1 or its fragment, variant, analog, homologue or derivant.

3. the method for claim 2, wherein the described biological sample of step (a) and (b) also contacts with fragment, variant, analog, homologue or the derivant of one or more CCN polypeptide or described one or more CCN polypeptide, and described CCN polypeptide is selected from CCN1, CCN2, CCN3, CCN4, CCN5 and CCN6.

4. method of screening angiogenesis modulators, described method comprises:

(a) will comprise first cornea of the first implant Implantation Test animal of ECM signal transduction molecule and suspicious regulator;

(b) will comprise second cornea of the second implant Implantation Test animal of ECM signal transduction molecule;

(c) vascular development that obtains of comparison step (a) and step (b) by changing and the ability of step (b) medium vessels developmental phase than step (a) medium vessels developmental level, is differentiated angiogenesis modulators thus,

5. the method for claim 4, wherein step (a) and implant (b) also comprise fragment, variant, analog, homologue or the derivant of one or more CCN polypeptide or described one or more CCN polypeptide, and described CCN polypeptide is selected from CCN1, CCN2, CCN3, CCN4, CCN5 and CCN6.

6. method of screening tumor generation regulator, described method comprises:

(a) give the first tumor ECM signal transduction molecule and suspicious regulator;

(b) give the second tumor ECM signal transduction molecule;

(c) the tumor occurred level that obtains of comparison step (a) and step (b), thus by changing the ability discriminating tumor generation regulator of comparing the tumor occurred level with step (b),

7. the method for claim 6, wherein also give fragment, variant, analog, homologue or the derivant of one or more CCN polypeptide of step (a) and tumor (b) or described one or more CCN polypeptide, described CCN polypeptide is selected from CCN1, CCN2, CCN3, CCN4, CCN5 and CCN6.

8. method of screening the cell adhesion regulator, described method comprises:

(a) on the surface compatible, ECM signal transduction molecule and suspicious regulator are added first biological sample with cell adhesion;

(b) on the surface compatible, the ECM signal transduction molecule is added second biological sample with cell adhesion;

(c) the cell adhesion level of measuring in comparison step (a) and the step (b) is compared the ability identification of cell cell adhesion modulating agent of cell adhesion level thus with step (b) by change,

9. the method for claim 8, wherein also give fragment, variant, analog, homologue or the derivant of one or more CCN polypeptide of step (a) and biological sample (b) or described one or more CCN polypeptide, described CCN polypeptide is selected from CCN1, CCN2, CCN3, CCN4, CCN5 and CCN6.

10. method of screening the cell migration regulator said method comprising the steps of:

(a) can experience the cell inoculation of cell migration in first gel-type vehicle that comprises ECM signal transduction molecule and suspicious regulator;

(b) can experience the cell inoculation of cell migration in second gel-type vehicle that comprises the ECM signal transduction molecule;

(c) the cell migration level of measuring in comparison step (a) and the step (b) is moved regulator by changing the ability identification of cell of comparing the cell migration level with step (b) thus,

11. the method for claim 10, wherein (a) and substrate (b) also comprise fragment, variant, analog, homologue or the derivant of one or more CCN polypeptide or described one or more CCN polypeptide, and described CCN polypeptide is selected from CCN1, CCN2, CCN3, CCN4, CCN5 and CCN6.

12. a regulator, described regulator is differentiated by each method of claim 2-11.

13. comprising the regulator and the medicine of claim 12, a pharmaceutical composition, described compositions can accept accessory drugs, diluent or carrier.

14. a pharmaceutical composition, described compositions comprise CCN1 fragment or its fragment, variant, analog, homologue or the derivant of claim 1, and medicine can be accepted accessory drugs, diluent or carrier.

15. an antibody, described antibody specificity is in conjunction with CCN1 fragment or the segmental fragment of described CCN1, variant, analog, homologue or the derivant of claim 1.

16. comprising the antibody and the medicine of claim 15, a pharmaceutical composition, described compositions can accept accessory drugs, diluent or carrier.

17. a method of regulating CCN1 among the patient, described method comprise each compositions of the patient of needs claim 14-16 is arranged.