TWI356097B - Ccn1 compositions and methods - Google Patents

Description

1356097 IX. Description of the invention: [Technical field to which the invention pertains] i The present invention relates to a substance involved in an intercellular matrix message molecule and a form of a peptide which is related to a cell reaction of a growth factor. In particular, the present invention relates to CCN1-related peptides, compositions thereof, and methods of using such polypeptides. The invention also relates to anti-CCN1 antibodies. [Prior Art] The role of angiogenesis or the formation of new blood vessels from existing blood vessels is a complex process that requires the cooperation of a variety of cellular events. See

Risau (1997) //aiMre 386, 671-674. The newly emerging blood vessels need to degrade the basement membrane around the original blood vessel, vascular endothelial cells to angiogenic stimulation displacement, vascular endothelial cell proliferation and arrangement into a tubular structure, and merge the new * blood vessels into a ring shape to provide the target tissue. Blood supply. See Risau. Angiogenesis is required for embryonic development, and in adults, it is important for female reproductive cycles and for the recovery of injuries. The management of freshmen is based on many pathological conditions including diabetic retinopathy, arthritis, arterial _ sclerosis, dryness, and cancer. See Folkman (1995) iVaiwre Medzczwe 1, 27-3 1. It is now understood that angiogenesis is regulated by a network of multiple inducers and inhibitors. See Bouck et al. (1996). Cwcer Λα. 69, 135-174 and Davis et al. (1999) Cwrr. Γορ.

Microbiol. Immunol. 231, 込S. The CCN family of matricellular proteins is a semi-leucine-rich, secreted protein that is associated with the intercellular matrix (ECM) but acts as a regulatory rather than a structural function. Member of the CCN family, including CCN1 5 1356097 (CYR61), CCN2 (CTGF), CCN3 (NOV), CCN4 (WISP-l), CCN5 (WISP-2), and CCN6 (WISP-3) (see Brigstock (1999) "Cr.i?ev. 20, 189-206; Lau et al. (1999) 5xp.Ce// 248, 44-5 7), which secretes the message peptide from the N-terminus followed by four insulin-like growth. Factor-binding protein homologous retention domain, Wyberburst factor C-type repeat, blood-reactive hormone (thrombospondin type 1 repeat), and c_terminal domain (CT), the C-terminal domain (CT) has heparin-binding The motif is similar to the sequence of Wenwei's factor and mucin. See Bork (1 993) 327, 125-130. While retaining its homology to ECM proteins and targeting to ECM, several CCN proteins have been shown to support cell adhesion, induce aggregate adhesion complexes, and stimulate adhesion messages. See

Kireeva et al. (1996) Mo/. Ce//. Βίο/· 16,1326-1334 ; Chen et al. (2001) «/· 5/〇/· 276,10443-10452; Chen (2001) J. Biol. Chem. 276, 47329-41/337 In the CCN family, CCN1 and CCN2 have been studied in depth. Both of these proteins stimulate cell migration, promote cell survival, and augment growth factor-induced mitosis. See Kireeva et al. (1997) Five Seals. Ce// and e.? 233, 63-77; Jedsadayanmata et al. (1999) «/· 274, 24321-24327; Babic et al. (1999) Mo/. Ce/ /. 5/〇/· 19, 2958-2966; Schober et al. (2002) B/ood 99, 4457-4465; Leu et al. (2002) «/. Bio/. C/zem. 277, 46248-46255. Both proteins are known to induce angiogenesis and chondrogenesis; see Babic et al; Wong et al. (1997) Z) ev. valence / 192, 492_508; Babic et al. (1998) Proc. atl. Acad. Sci. USA 95, 6355-6360; Shimo et al. 1356097 (1999) «/· Bioc/iew. 126, 137-145; Ivkovic et al. (2003) 130, 2779-2791. Although the CCN protein does not contain the RGD sequence motif, both CCN1 and CCN2 are direct ligands for multiple integrin receptors, and many of their activities are modulated. See Jedsadayanmata et al. (1999) /· 5ζ·ο/· C/zew. 274, 24321-24327; Schober et al. (2 002) Blood 99, 445 7-4465; Leu # Λ (2002) J. Biol. Chem Ill, 46248-46255; Kireeva % A (1998) J. Biol. Chem. 273, 3090-3096; Chen # A (2000) J. Biol. Chem. 275, 24953-24961; Grzeszkiewicz ^ A (2001) J Biol. Chem. 276, 21943-21950; Grzeszkiewicz # A (2002) Endocrinology 143, 1441-1450. Destruction of the CCA/7 gene in mice causes the embryo to die due to loss of blood vessels, while mice with an ineffective CCN2 gene die in the delivery phase due to respiratory failure due to bone deformity. See (Mo et al. (200 2) Mol. Cell Biol. 22, 8709-8720); Ivkovic ^ Λ (2003) 130, 2779-2791. These findings point to members of the CCN family as essential and non-excessive functions in development. CCN1 (rich semi-glycine 61, CYR61), an angiogenesis inducer, is a novel integrin ligand, which is a growth factor-inducible immediate-early gene. Its performance is required for proper embryonic development. Recent studies on the destruction of CC#/gene in mice have shown that ineffective embryos die during the embryonic period, mainly due to the absence of jk tubes in both the placenta and the embryo. See Mo et al. (2002) Mo/. Ce//. Price 〇/. 22, 8709-8720 » In addition to embryonic angiogenesis, CCN1 also promotes pathological angiogenesis, such as Ί356097 in tumor growth and wound repair. under. Stable transfection with CCWi in tumor cell lines can enhance tumor growth and increase ^ (^\/7- manifestation of tumor angiogenesis). See et al. (1998) Proc. iVa". dead· 5W t/.ϋ 95, 6355-6360; Xie et al. (2001) «/· 5zo/. C/iew. 276,14187-14194; Tsai et al. (2002) 0«coge«e 21,817 8-8 185. Furthermore, estrogen-induced cCNi expression was found to be associated with ongoing human breast cancer (see Xie et al. (2001) Cancer _Res. 61, 8917-8923; Sampath et al. (2001) V.docr/wo/og; 142, 2540-2548; Tsai et al. (2000) 60, 5603-5 607. Excessive manifestations of vascular and atherosclerotic lesions in restenosis have been identified, emphasizing its importance in the pathology of vascular disease. See Hilfiker et al. (2002) CBu cw/aib« 106, 254-260; Wu et al. (2000) / «ί· «/· M<?/. Mec?. 6,433-440; Grzeszkiewicz et al. 2002) 5 143, 1441-1450; Schober et al. (2002) 99, 4457-4465. In addition, the expression of CC#/ in skin-recovered wounds is related to its ability to activate the genetic program of wound healing in human skin fibroblasts, and CCN1 is proposed to play an important role in injury repair. See Latinkic et al. (1991) iVwc/eic jcz A. 19, 3261-3267; Chen et al. (2001) ·/. 5ζ·ο/· C/iem. 276, 47329-47337 ° When synthesized, CCN1 is secreted It binds to the cell surface or ECM. See Yang et al. (1991) Ce// Growi/z ά 2, 351-3 57. Previous studies have shown that CCN1 supports cell adhesion, causes cell migration, enhances growth factor-induced mitosis, and promotes cell survival in the presence of apoptotic 1356097. See Kireeva et al. (1996) Mo/. Ce". βίο,· 16, 1326-1334; Leu et al. (2002) 乂 5b/· C/ze/w. 277, 46248-46255. These cellular activities of CCN1 are partly due to their ability to interact with integrin adhesion receptors. To date, five integrins have been identified, α6β!, ανβ3, ανβ5, otIIbp3, and αΜβ2, which are CCN1 receptors in various cell types. See Schober et al. (2002)

Biooc/99, 4457-4465; Kireeva et al. (1998) ·/· B/o/· c/zew. 273,3090-3096; Jedsadayanmata et al. (1999) «/.price〇/. C/zew. 274 , 243 2 1-243 27; Grzeszkiewicz et al. (200 1) 乂_β,·〇/· C/zem. 276, 21943-21950 ; Chen et al. (2000) J·仏〇/. C/iew. 275 , 24953-2496 In an earlier study, we have demonstrated that CCN1 can cause neovascularization in the rat corneal micropocket assay. See Babic et al. (1998) iVoc. iVai/. Jcad. Scz.. t/U. 95, 6355-6360. These results are consistent with findings in vivo. In the collagen gel assay, CCN1 promotes the formation of human umbilical vein endothelial cells (HUVECs), and this process is dependent on integrin α6βι& ανβ3. See Leu et al. (2002) J•仏〇/. C/2em. 277, 46248-46255 整合 Also shown in several cell forms, integrin regulates the activity of many CCN1. CCN1 supports the adhesion of fibroblasts via interaction with integrin α6β丨 and heparin sulfate proteoglycans (HSPGs), resulting in fil〇p〇dia and layer foot (1 already 111611丨卩) 〇€^) is an extension of the mixing zone of the 1356097 region of the 〇6601 region located in the front of the pseudopod (?3611(1〇?〇<13). See Chen et al. (2001) •/.Bio/.C/zem. 276, 10443-10452. Furthermore, messages that rely on integrins are induced to cause focal adhesion kinase, paxillin, Rac, and mitotic promoter-activated protein kinases (mitogen). Activation of -activated protein kinases and upregulation of several vascular proliferation regulators. See Chen et al. (2001) 乂5沁/. C/zew. 276, 47329-47337; Chen et al. (2001) •/• Bio/.C/zew, 276, 10443-10452. In addition to fibroblasts, CCN1 also acts as an integrin on vascular smooth muscle cells and vascular endothelial cells.

Grzeszkiewicz et al. (2002) V. i/ocr/wo/og;; 143, 1441-1450; Leu et al. (2002) ·/·仏0/. CAew. 277, 46248-46255. Recently, CCN1 pre-angiogenic activity has been revealed.

Pro-angiogenic activities are regulated by integrin αόβ丨 and ανβ3 in unactivated and activated HUVEC, respectively. Leu et al. (2002) «/. ΒίοΖ. C/iew. 277, 46248-46255.

In addition to CCN1, members of other CCN families include CCN2 (connective tissue growth factor (CTGF), CCN3 (nephroblastoma-overexpressed, NOV), and Wnt-induced secretion protein CCN4 ( WISP-l), CCN5 (WISP-2), and CCN6 (WISP-3) (22-24). The CCN protein line consists of four different modular domains: 1) the membrane-like growth factor binding protein homology domain, II) the Wenweibur factor (VWF) C-type repeat domain, III) thrombospondin I The type of repeat (TSP1) domain, and IV) the carboxy-terminal (CT) domain consists of 'the retinomeric domain with a heparin-binding motif and a sequence similar to vWF 1356097 c-terminal and mucin (see 1) Eight figures). Several CCN proteins have been shown to interact with a variety of integrins, and therefore, the location of the integrin binding site on the protein will provide a new perspective on the structure-function relationship of this newly established stromal cell protein family. It was previously discovered that after truncating the C-terminal domain of CCN1, it can cause smooth muscle cell migration via integrin~heart. See Grzeszkiewicz et al. (2001 W5i〇/ (10) 276, 21943 21950. This finding suggests that the integrin-intimate binding position is located in the first three domains of ccm. In this paper I am equal to (iv) thrombospondin j-type repeat domain to identify novelty The 17_residue sequence, called τι, regulates αβγ-dependent cell adhesion. By affinity chromatography, we confirmed that it can directly interact with the Τ1 sequence. In addition, via heparin sulfate proteoglycans (HSPGs) The co-receptor complex, we confirmed that the activity of CCN1 is affected by the heparin binding sites m and H2 and T1. The heparin binding sites m and H2 are required to prolong the activity of MAPKs and to regulate the upregulation of ΜΜρι. It is related to the blood neonatal and stromal metabolism. We have also confirmed that the synthetic peptide from the T1 sequence can specifically block cell adhesion, and I am equal to (10) the new 4 binding site = as an antagonist of the development of integrin αόΡι The basis of the agent. The newly identified ouPi binding position in CCN1 can be used as the beauty base for the development of antagonists of integrin αόΡι. [Invention] The present invention provides an intercellular matrix (E). CM) Message Molecules_Related Substances and Methods "In particular, the present invention relates to CCN1_ related peptides, compositions thereof, and methods of using such polypeptides. The present invention also relates to anti-CCN1 anti-1356097 aspects of the present invention. Concerning the CCN1 fragment, which comprises an antibody selected from the group consisting of: amino acid 224-240 from murine CCN1, amino acid '231-240 from murine CCN1, amino acid 226-242 from human CCN1, and amine from human CCN1 A sequence consisting of a base acid 233-242. The CCN1 fragment may comprise from 8 to 50 amino acids. The invention also relates to variants, analogs, homologs, or derivatives of the c CN1 fragment. Another aspect of the invention relates to a method for screening for angiogenesis regulators, comprising contacting a test biological sample capable of angiogenesis with an ECM message molecule and possibly a modulator. As for the control group, the second biological sample and the ECM message are also used. Molecular contact. The definition of an angiogenic regulator is based on the ability of the test sample to alter the degree of angiogenesis. The ECM message molecule can be a CCN1 fragment or a fragment, variant, analog, homolog, or derivative thereof. Another aspect of the invention relates to the use of the method to define a modulator. Another aspect of the invention relates to a method of screening for an angiogenesis regulator, the cold comprising transplanting the test implant into a test animal, wherein the test implant includes Regulators and ECM message molecules. As for the control group, the second implant including the ECM message molecule is transplanted into the test animal: it can be the same animal or different test animals. The definition of angiogenesis regulator is based on Compared to the control sample, it changed the ability of the blood vessel to the degree of fertility in the test implant. The ECM message molecule can be a CCN1 fragment or a fragment thereof, a variant, an analog, a homolog, or a derivative. The invention further relates to a modulator defined by the method. 12 1356097 Another aspect of the invention relates to a method for screening for a modulator of tumor formation comprising contacting a tumor with a potential regulator and an ECM message molecule. As for the control group, the second tumor was also contacted with the ECM message molecule. The definition of a regulator of tumor formation is based on the ability to change the extent of tumor formation in a test tumor compared to a control tumor. The ECM message molecule can be a CCN1 fragment or a fragment thereof, a variant analog, a homolog, or a derivative. Another aspect of the invention pertains to an adjustment defined by the method. Another aspect of the invention relates to a method for screening for a modulator of cell adhesion comprising contacting a test biological sample with a potential modulator and an ECM message molecule on a surface compatible with cell adhesion. As for the control group, the second test biological sample was also contacted with the ECM message molecule on the surface compatible with cell adhesion. The definition of a cell adhesion regulator is based on the ability of the test sample to change the degree of cell adhesion of the test sample. The ECM message molecule can be a CCN1 fragment or a fragment, variant, analog, homolog, or derivative thereof. Another aspect of the invention pertains to a modulator defined by the method. Another aspect of the invention relates to a method for screening for modulators of cell migration' comprising seeding cells capable of cell migration in a test gel matrix' comprising a possible modulator and an ECM message molecule in the matrix. As for the control group, cells in which cell migration can be carried out are also seeded on the second biological sample gel matrix. The matrix includes ECM message molecules. The regulation of cell migration is based on the ability of the test matrix to alter the degree of cell migration compared to the control matrix. The ECM message molecule can be a CCN1 1356097 fragment or a fragment thereof, a variant 'analog, a homolog or a derivative. Another aspect of the invention pertains to a modulator defined by the method. Another aspect of the invention pertains to antibodies which bind specifically to a CCN1 fragment or a variant, analog, homolog or derivative of the CCN1 fragment. Another aspect of the invention relates to a composition comprising an antibody that binds specifically to a ccm fragment or a variant, analog, homolog or derivative of the CCN1 fragment. [Embodiment] CCN1 is an angiogenesis inducer that plays a necessary role in normal vascular development during embryogenesis. See M〇 et al. (2〇〇2) from 〇/ Ce// 5ζ·〇/. 22, 8709-8720. See also U.S. Patent No. 6,413,735, and U.S. Patent Application Serial No. Serial No. No. No. No. No. No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No Recently, we have proposed that angiogenesis activity before CCN1 is regulated by integrin α6βι and ανβ3 in unactivated and activated HUVE, respectively. See Leu et al. (2002) «/.&〇/. CAem. 277, 46248-46255. In addition to the T1 sequence interaction in the TSP1 domain of integrin α6β# CCN1, the adhesion of fibroblasts and the unactivated endothelial cells to CCN1 also require heparin sulfate proteoglycans as co-receptors, which can be combined with Heparin binding motif in the C-terminal domain of CCN1. See Chen et al. (2000) 275, 24953-24961. In this study, we used a functional and biochemical analysis to define the 17-residue T1 sequence (GQKCIVQTTSWSQCSKS) located in domain III of CCN1 as a novel integrin. We also determined that the heparin binding sites H1 and H2 have a significant influence on the functional activity of the ad^HSPG co-receptor complex 14 1356097, such as one of the important parts of cell adhesion angiogenesis. These findings provide the basis for the development of Μ antagonists and for the purpose of: canine analysis to examine the role of the interaction of integrin a6Pi_ccNi in angiogenesis.

^JL As used herein, "administering" refers to a single dose or multiple doses of a composition of the invention. As used herein, "when treatment" or "treatment" refers to the protection of a mammal from the condition, which means preventing, inhibiting, suppressing or eliminating the condition. The method of P to stop this condition involves administering a composition of the invention to a mammal prior to the occurrence of the condition. A method of inhibiting this condition involves administering a composition of the invention to a mammal after causing the condition but prior to its clinical presentation. The method of suppressing this condition involves administering to the mammal a composition of the invention after the clinical manifestation of the condition, reducing or maintaining the condition. The method of eliminating this condition involves administering the composition of the present invention to the mammal after the clinical manifestation of the condition so that the mammal does not suffer from the condition again. The sputum sputum attachment is consistent with our earlier findings by the OU-HSPG, which is astringent and has been identified earlier. This truncated mutant (including CCN1 domain I-III) can cause aji-dependent smooth muscle. Cell migration (see Grzeszkiewicz et al. (2002) 143, ^41-1450), we found that the recombinant fragment corresponding to the isolated domain m (Tspi domain) of murine CCN1 is sufficient to support ο^β!-dependent fibrils Adhesion of cells. Together with ouPi, heparin sulfate proteoglycans (HSPGs) serve as co-receptors for the interaction of this 15 1356097 interaction. See Chen, N., Chen, C. C., and Lau, L. F. (2000) «/. Bz〇/. C/ι.275, 24953-24961 and Leu, S.-J.,

Lam, S. C. T., ^ Lau, L. F. (2002) J. Biol. Chem. Ill, 46248-46255. The specificity of the interaction between 〇^丨 and the mouse CCN1 domain III is due to the inability of the mouse CCN1 domain I and domain II fragments to support cell adhesion' and the specificity of anti-〇〇6 and anti-βι mAb It was confirmed by blocking the observation of cell adhesion in the CCN1 domain of the mouse (Figs. 1 and 2). In Domain III, we further accurately indicated that the integrin (xji binding position: 丨) T1_GST fusion protein and the synthetic T1 peptide in the T1 sequence CCN1 specifically support dependent cell adhesion (3rd) And 4))) 2) Integrin ^^β 丨 is purified from the T1-GST affinity matrix by the fibroblast detergent lysate, which confirms the direct interaction between the integrin and the Τ1 sequence. Effect (Fig. 7); 3) Soluble Τ1 triumphant can inhibit cell adhesion of ^丨 ligand (including CCN1, ccn2, CCN3, and laminin), but does not inhibit other integrin ligands. For example, fibrinnectin 'vitr〇nectin and collagen (Fig. 5); and 4) T1 peptide can also block unactivated HUVEC in a collagen matrix containing CCN1. Dependent tube formation (Fig. 8). It is worth noting that the soluble τι peptide is an effective inhibitor of Χδβ i-dependent cellular activity. The half-maximal inhibitory value of cell adhesion occurs between the peptide concentration of 25_ 5 〇ηιΜ (Fig. 5 C) Therefore, the inhibitory capacity of T1 can be compared with the linear RGD peptide, which can inhibit the function of other integrins, such as ανρ3, also within the range of micromoles. See Ruoslahti (1996) Ce // Dev 仏〇 / 12, 16 1356097 697-715 » Since the T1 position has been shown to affect the binding of CCN1, we have further shown that the heparin binding sites H1 and H2 cause the remaining CCN1-dependent activity. By making a complete CCN1-GST fusion protein, the protein has a mutation at the T1 position and the H1 and H2 heparin binding sites, while mutants with mutations at all three positions lose support for α6β丨-regulated fibroblast adhesion. As described in Examples 11-13, the mutants with mutations at the positions of Η1 and Η2 can only adhere to the adhesion of fibroblasts at high concentrations, and only have mutations at the Τ1 position. The concentration of wild type can still support the adhesion of fibroblasts. It also shows that the binding of heparin to CCN1 Η1 and Η2 can affect the sustained ρ42/ρ44 ΜΑΡΚ activation. By alanine substitution mutation of T1-GST fusion protein, We show that the C-terminal portion of this T1 (TTSWSQCSKS) contains a key determinant of -dependent cell adhesion. In particular, the substitution of double T231A/T232A and _W234A/K23 9A completely abolishes the ability to support cell adhesion. The fragment of this 10-residue is highly retained in other CCN family members and has only two non-reservative substitutions in CCN1, CCN2 and CCN3. Therefore, it can be inferred that oupi can also correspond to T1 in other CCN proteins. Sequence binding. Consistent with this concept, soluble T1 peptide can also inhibit CCN2 and CCN3-induced fibroblast adhesion. These results led us to infer the retention of TTZWSXCSKS sequence in CCN protein (X stands for non- Retaining residues) define a novel integrin recognition motif. An important feature of this sequence is any single alanine substitution in the residue of the residue 1356097 (also T232A, W234A, ψS235A, S238A, and K239A) caused a significant decrease in the binding activity of 〇160丨, indicating that it required multi-coordination interaction with the ligand binding pocket of integrin~01. Integrin has a limited range of ligands including laminin, CCN protein, invasin, fertilin, and collagen fragments known as tumstatin. See Sonnenberg et al. (1990) /. Ce// 仏〇/· 110, 2145-2155; Maeshima et al. (2001) _ J 5z.o/. C/zew. 276, 15240-15248; Isberg et al. (1990) Ce// 60, 86 871; Almeida et al. (1995) Ce// 81, 1095-1 104. These diverse ligands involved in various biological processes are not structurally related. Several types of ... binding sequences can be defined by screening for synthetic traits derived from certain aepi ligands. These include the NPWHSIYITRFG and TWYKIAFQRNRK sequences from the laminin chain. See Sonnenberg et al. (1990) «/. Ce// Βζ·〇/· 110, 2145-2155;

Nomizu et al. (1995) don't worry about C/iem. 270, 20583-20590;

Nakahara et al. (1996) ·/.price 〇/. C/zem_ 271,27221-27224. In addition, TDE-derived peptides derived from the disintegrin domain of the fertilin b-subunit can disrupt sperm-egg fusion, presumably by blocking the interaction of integrin αόβ丨-fertilin. See Myles et al. (1994) /Voir·ΛΓαί/· jcad· 5W. 丄 91, 4195-4198. Several other αόβ! binding peptides have also been isolated by screening for the display of spores and synthetic peptide combinations; however, these sequences are not present in any known αόβ丨 ligand. See Murayama et al. (1996) J. 18 1356097 (ΤσΛ: Less ο) 120, 445-451; Pennington et al. (1996) Mo/. 2, 19-28; DeRoock et al. (2001) Cancer 61, 3308-3313 . A comparison of the aji binding sequences in the reports to date indicates that there is no common sequence that can be used as an α^β binding motif. Furthermore, I am equal to the newly defined sequence of Τ1 in CCN1 which does not exhibit any sequence similarity to these peptides. Thus, integrins, similar to αΜβ2, recognize a wide range of binding sequences. It is still necessary to confirm whether such widely different peptide sequences can bind to the same or different positions in αόβι. Nonetheless, it is known that integrin ο^βι involves a large number of biological programs, and different α6β^-synthesis sequences can interact with different coordination positions in the binding pocket of αόβi ligands to cause different types of biological activities that can be regulated. Information path. To date, three CCN proteins have been shown to cause neovascularization in vivo. See Babic et al. (1998) Proc.

Atl. Acad. Sci. U.S.A. 95, 6355-6360 I Lin ^ A. (2003) J C7^/«. In particular; Babic et al. (1999) Mo/· Ce//. 19, 2958-2966; Shimo et al. (1999) «/. 126, 137-145; Fataccioli et al. (2002) /fwm. Gewe 77ier. 146 1 -1470. Endothelial migration, proliferation, and differentiation into tubular structures are required for the formation of new blood vessels. The activation-dependent ligand of the CCN1 line integrin in non-stimulated endothelial cells, through which the integrin receptor regulates cell adhesion and tube formation. See Le.u et al. (2002) ·/. CT^m. 277, 46248-46255. However, the intact CCN1 line is an angiogenesis elicitor's T1 peptide that acts as a sputum antagonist, blocking the formation of unactivated endothelial cells _ CCN 1-induced tube formation. Interestingly, thrombospondin 1356097 1 The T1 sequence residue in the type of repeat is similar to the domain of CCN1, and the thromboplastin is an inhibitor of angiogenesis, and its anti-angiogenic activity is located in the procollagen-like region and the class of degenerin. Type 1 repeat. See Tolsma et al. (1993) Λ Ce// 122/. 122, 497-5U. Many anti-angiogenic peptides are derived from thrombospondin, including the peptide containing the CSCTCG, which is linked to CD36 effect on endothelial cells. See also

Jimenez et al. (2000) Med. 6, 41-48; Dawson et al. (1997) «/•'Ce// 138, 707-717. The interaction of CD36 with the TSP1 domain of CCN proteins has not been confirmed; however, CD36 has been shown to bind to integrin on human platelets and melanoma cells. Mia〇 et al. (2001) 5/oocf 97, 1689-1696; Thorne et al. (2000) Price/·C/zem· 275, 35264-35275. If the CD36-~ sputum complex is also present in endothelial cells, there is an interesting possibility that the two cell surface receptors may act together to regulate angiogenesis, and the interaction between the two is via the stromal cell protein thrombus. The proximal recognition sequence in the quinone-type repeat. • 1·CCN1 fragment The present invention relates to a peptide fragment of CCN1 which modulates the activity of ccni. The peptide can be used in a therapeutic strategy designed to inhibit or cause the activity of CCN1. The peptide can be natural, synthetic or recombinant. One method is to produce a peptide having a sequence selected from the group consisting of the amino acid 224-240 of murine CCN1, the amino acid 231-240 of murine CCN1, the amino acid 226-242 of human CCN1, and human Amino acid 233_242 of CCN1. For example, a peptide with a retaining amino acid can compete with native CCN1 for its binding site on integrins and other proteins. This competitive effect thus 20 1356097 inhibits the action of natural CCN1. The invention also relates to fragments in the ccNl fragment. The peptide can be from 8 to 50 amino acids in length. The length of the inhibitory peptide 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 amino acids. The peptide may also be a homolog of the above CCN1 peptide. The homolog of the CCN1 peptide has a peptide of co-evolution with CCN1. The peptide may also be a variant of the above CCN1 peptide and homologue. A peptide variant is a peptide that has a different amino acid sequence from the original CCN1 peptide by insertion, deletion, or retention of the amino acid, but at least retains the original C CN1 peptide. active. For the purposes of the present invention, "the biological activity of a CCN1 peptide" includes, but is not limited to, the above-described activities of intact ccni, the ability to modulate CCN1 activity, and the ability to bind to a specific antibody of ccN1. The reductive substitution of amino acids, i.e., the substitution of an amino acid with a different amino acid having similar properties (e.g., hydrophilicity, extent and distribution of charged regions) typically involves minor changes. . As is known in the art, these slight changes can be identified to some extent by considering the hydropathic index of the amino acid. The heart waits for others, 乂 〇 / 〇 〇 / · 75 7..105-132 (1982). A list of amino acid fluid indices can be found in U.S. Patent No. 6,639,54, which is incorporated herein by reference. This technique is known to be substituted with an amino acid having a similar fluid index and still retains the function of a protein. In one aspect, a fluid finger 21 1356097 can be substituted with an amino acid of ±2. The hydrophilicity of the amino acid can also be used to show the substitution of the biological function of the retained protein. In order to consider the hydrophilicity of the amino acid in the peptide, it is allowed to calculate the maximum local average hydrophilicity (greatest 1〇cal average hydr〇 Philicity). It has been reported that a useful measurement method is related to antigenicity and immunity. Originally related. A list of the hydrophilicity of amino acids can be found in U.S. Patent No. 4,554,1, which is incorporated herein by reference. It is known in the art that amino acid having a similar hydrophilicity value can be substituted and still retain the biological activity of the peptide, such as immunogenicity. At a point of view, each amino acid having a hydrophilic value in the soil 2 can be substituted. Both the fluid index and the hydrophilicity value of the amine group & C are affected by the specific branching of the amino acid. Consistent with the observations, understanding the amino acid substitutions associated with biological functions is based on the similarity of amino acids and the specific branching of such amino acids, such as hydrophobicity, hydrophilicity, charge, size, and Depends on other features. In addition, computer calculus can be utilized to aid in the prediction of amino acid sequence domains that may be compatible with aqueous solvents. It is known in the art that such domains are often located proximate to the peptide, thus potentially contributing to the binding of the peptide to the determinant (including the antigenic determining factor). The winner may also be an analog of the above CCN1 peptides, homologs and variants (including non-standard amino acids or other amino acids derived from structural variations of conventional amino acids). The peptide may also be a derivative of the above CCN1 peptide, homologue and variant, which differs in the primary structure (amino acid and aminoguanidine analog). For example, the derivative differs from the original CCN 丨 peptide, the 1356097 source and the variant in that it can be glycosylated into a post-translational modification by glycosylated. For example, a polypeptide can exhibit a pattern of saccharification when expressed in a heterologous system. If the peptide retains at least one activity of the original CCN1, then such a peptide is a CCN1 derivative according to the invention. Other derivatives include, but are not limited to, covalently modified Ν- or C-terminal fusion peptides, PEGylated peptides, peptides linked to lipid moieties, alkylated peptides, A peptide that is linked to other peptides or chemicals via a functional group of an amino acid branch, as well as other modifications known in the art. Furthermore, the present invention comprises a CCN1·related peptide which, as described below, binds to the CCNi receptor. As described above, the various peptides of the present invention may be provided in the form of individual peptides or linked (e.g., covalent bonds) to other compounds. For example, an immunological vector, such as Keyhole Limpet Hemocyanin, can be incorporated into the CCN1 peptide of the present invention. The similarity of the above various fragments, variants, analogs, homologs or derivatives to the peptide is 50%, 55%, 6%, 65%, 70%, 75%, 80%, 85%, 90 %, 95%, 97%, 98% '^ or 99%» The invention also relates to isolated nucleic acids which encode the peptide. The invention also relates to a pharmaceutical composition comprising the peptide of the invention. • 2· Antibodies The present invention also relates to a pharmaceutical composition comprising an antibody that binds specifically to the CCN1 peptide of the present invention, and a pharmaceutically acceptable adjuvant, diluent, or carrier. The antibody can be made as follows, or as disclosed in WO 01/552 10, the entire contents of which are incorporated herein by reference. 23 1356097 The antibody of the present invention comprises antibodies of the IgG, IgM, IgA, IgD, and IgE classes, and fragments and derivatives thereof comprising Fab and F(ab,)2. The antibody may also be a recombinant antibody product, including, but not limited to, a single bond antibody, a chimeric antibody product, a "humanized" antibody product, and a CDR-grafted antibody product. The antibody of the present invention comprises a monoclonal antibody, a plurality of antibodies, an affinity-purified antibody, or a mixture thereof which exhibits sufficient binding specificity for the CCN1 fragment. The invention also includes antibody fragments. The antibody product comprises a Ru antibody product of the type described above as an isolated antibody or a labeled antibody. The label can be a message-generating enzyme, an antigen, other antibodies, lectins, carbohydrates, biotin, avidin, radioisotopes, toxins, heavy metals, and other combinations known in the art. Attachment techniques are also known in the art. The anti-CCN1 anti-system is used to detect the risk of tumor formation. Furthermore, anti-CCN1 antibodies can be used in the treatment of the delivery of specific cytotoxins to cells expressing CCN1 (e.g., cells involved in the neovascularization of solid tumors in spring). Such antibodies are delivered by a variety of routes of administration in the form of a pharmaceutical composition comprising a carrier or diluent which is known to those skilled in the art. • Screening of CCN1 Modulators The present invention relates to modulators of Xuexuan and CCN1-related activities. Regulators are defined as those that bind to the integrin of CCN1, thus preventing the integrin π of 楳 茚 from defining 茚 as a direct binding to CCN1 and the integration of other proteins 24 丄 097 And other proteins can directly affect the protein produced by CCN1 and the target, thus preventing CCN1 from interacting with the target. Regulators can also be defined as a combination of qualities. For the purposes of the present invention, "the job message molecule" refers to the above-mentioned eve of the film. The "ECM Message Molecule" also includes one or more other ccn peptides. The one or more additional CCN polypeptides include, but are not limited to, ccm, 吣, 咖 4, coffee 5 and CCN6, and fragments, analogs, homologs or derivatives of the one or more other (10) oxime peptides. a. Angiogenesis The method of the invention relates to the screening of modulators of angiogenesis. In one embodiment of the invention, an angiogenic biological sample is contacted with a possible modulator and an ECM message molecule in a test tube. As for the control, the second biological sample is also in contact with the ECM message molecule. The definition of angiogenesis regulators is based on their ability to alter the degree of angiogenesis in a test sample compared to a control sample. In another embodiment of the invention, the implant is transplanted into a test animal which includes the implant as a possible regulator and a message knife. As for the control group, a second implant including an ECM message molecule is transplanted into the test animal, which may be the same animal or a different test animal. The definition of a blood e-new regulator is based on its ability to alter the degree of vascular development in a test implant compared to a control sample. • b. Tumor Formation The methods of the invention are also directed to screening for modulators of tumor formation. The tumor is brought into contact with possible regulators and ECM message molecules. As for the control group, 1356097 also contacted the second tumor with the ECM message molecule. The definition of tumor formation is based on the ability to change the tumor size of a tumor compared to a control tumor. • c. Cell Adhesion The method of the invention also relates to screening for modulators of cell adhesion. The biological sample is contacted with possible regulators and ECM message molecules on a surface that is compatible with cell adhesion. As for the control group, the second biological sample was also contacted with the ECM message molecule on the surface compatible with cell adhesion. The definition of cell adhesion regulators is based on the ability of the test sample to alter the degree of cell adhesion of the test sample compared to the control sample. • d. Cell migration The methods of the invention are also directed to screening for modulators of cell migration. Cells that can undergo cell migration are seeded in a gel matrix that includes possible modulators and ECM message molecules. As for the control group, cell-transplantable cells were also seeded onto a second biological sample gel matrix, which included an ECM message molecule. The regulation of cell migration is based on the ability to alter the degree of cell migration in the test matrix as compared to the control matrix. • e. Methods of Treatment The present invention also relates to modulators of CCN1 activity, which are defined by the screening methods described above. The defined cCN1 activity modulators can be formulated into pharmaceutical compositions including pharmaceutically acceptable adjuvants, diluents, or carriers. The pharmaceutical composition comprising a modulator of CCN 1 activity can be administered to a patient to treat a disease associated with angiogenesis, tumor formation or chondrogenesis. The pharmaceutical composition can be administered alone or in combination with other compositions, such as a chemotherapeutic. The composition of the present invention can be administered by standard means, but is not limited to 'oral administration, parenteral administration, sublingual, transdermal, rectal, transmucosal, topical, via inhalation, Or administered via the buccal cavity. Parenteral administration includes, but is not limited to, intravenous, intraarterial, intraperitoneal, subcutaneous, intramuscular, intramembranous, and intraarticular administration. The present invention will now be described in detail, and other aspects and advantages of the present invention will be understood by the following illustrative embodiments. The examples are intended to be illustrative only and not to limit the scope of the invention as set forth in the appended claims. Example 1 reveals that domain III of CCN1 can support dependent cell adhesion. Example 2 reveals that the Τ1 sequence in domain III of CCN contains an integrin-mouth binding site. Example 3 illustrates the effect of soluble Τ1 peptide on αββ,-dependent cell adhesion. Example 4 illustrates the effect of alanine substitution on cell adhesion in the Τ1 sequence. Example 5 illustrates the affinity purification of integrin aji using a T1-coupled affinity matrix. Example 6 illustrates the effect of soluble T1 peptide on CCN1-induced endothelial tube formation. Example ® Example 7 illustrates the construction of a mutant peptide that will be used to inactivate the T1 binding site associated with integrin αββ!. Example 8 illustrates the adhesion assessment of a mutated CCN1 peptide (inactivation of the Τ1 binding site for binding to integrin). Example 9 illustrates a mutated CCN1 construct which is used to inactivate the Τ1 binding site for binding to integrin α6βi. Examples 10 to 14 illustrate the cell adhesion properties of CCN1 mutants. Example 1 illustrates the SM analysis using the cleavage T1 binding position and the uninterrupted heparin binding position. Example 11 illustrates the use of a broken T1 binding site and an SM analysis that inhibits the heparin binding position of 27 1356097. Example 12 illustrates the DM analysis using uninterrupted τι binding sites and cleavage heparin binding sites. EXAMPLES 13 illustrates the use of the cleavage of the butyl binding site and the cleavage of the heparin binding site. Example 14 illustrates the evaluation of cell adhesion on the side using anti-integrin. Examples 15 to 16 illustrate the effect of cleavage of H1 and H2 on the regulation of dependent MAPK activation and gene expression. Example 15 illustrates MAPK activation. Example 16 illustrates the regulation of gene expression. Examples I7 to 21 illustrate the regulation of CCN1 activity separation via ανβ3 and a6Pi_HSpGs. Example 17 illustrates that the CCN1 mutant can support the adhesion and migration of HUVEC via integrin ανβ3. Example q 8 illustrates that CCN1 or a mutant can support the migration of HUVEC. Example 197 illustrates enhanced VEGF-induced DNA synthesis via integrin ανβ3. Example 20 illustrates the effect of CCN1 mutations on HUVEC survival. Example 21 illustrates the tube forming action. These examples are intended to illustrate the invention and are not intended to limit the scope of the invention. EXAMPLES Example 1 Domain III of CCN1 (TSP1_Homologous Domain) Supports-Dependent Cell Adhesion Previous studies have established that primary human dermal fibroblasts can adhere to CCN1 via integrin and heparan sulfate proteoglycans, It causes the formation of complexes containing adi regions and activation of regional adhesion kinases, paxillin, and Rac. See Chen et al. (2000) ·/. _β/ο/· C/zew. 275, 24953-24961; chen et al. (2001) 丄./. to read .276, 28 1356097 10443-10452. The deletion assay showed that the C-terminal truncated CCN1 mutant (ie containing only the first three domains) still has chemotactic effects via integrin 〇1601 in smooth muscle cells, therefore, the binding site of integrin a6pi' The settings are in the first three domains. See Grzeszkiewicz et al. (2002) 5,143, 1441-1450 ° In order to define the CCN1 domain with integrin (Χίβ!), these three domains are expressed in insect cells by baculovirus vectors, respectively (Fig. 1) In order to enhance protein secretion, the pMelBac Β vector spring (Invitrogen Incorp.) was used to provide a N-terminal beetle melittin secretion message peptide. To generate domain I (IGFBP), domain II (VWC), domain III The genetic code sequence of (TSP1) is a PCR of CCN1 cDNA using the following primer pairs: 5,-CGCGGATCCGGCGCTCTCCACCTGC-3' and 5,-GGAATTCCCTCTGCAGATCCCTTTCAGAGCGG-3, 5,-CGCGGATCCGGCTCAGTCAGAAGGCAGAC-3' and 5'-GGAATTCCCAGGAAGCCTCTTCAGTGAGCTGCC-3' 5'-CGCGGATCCGGCTCAGTCAGAAGGCAGAC-3, and 5'-GGAATTCCCAGGAAGCCTCTTCAGTGAGCTGCC-3, the PCR product is cleaved with five coRl, and ligated to pMelBac B ^ each expressed recombinant polypeptide contains the V5 epitope and contains a polyhistamine at the C-terminus Acid-labeled, and the expressed recombinant polypeptide is purified from Sf9 cells using a serum-free baculovirus expression system as described above. See 29 135609 7

Grzeszkiewicz et al. (2001) J. Biol Chem 276, 21943-21950. .. Briefly, 'cell lines were cultured in EX-CELL '400 medium (JRH Biosciences, Lenexa, KS) under serum-free conditions, with a viral infection dose of 1 multi (multiplicity of infecti〇n 〇f 1〇) Infection, and collection was performed 42 to 46 hours after infection. The collected medium was cleared by centrifugation and then concentrated 10 to 15 times with a Bi〇max-5 centrifugal filter (Millip〇re, Billerica, ΜΑ) and The original buffer (5 〇 Lu mM TiOO and 10 mM Hepes 'pH 7.4, 0.5 M NaCi) was dialyzed overnight at 4 ° C and then applied to the TALON cobalt-Sepharose column (cl〇 Ntech,

Palo Alto, CA). The column was washed with the original binding buffer at pH 7.0, and the product was analyzed by SDS-PAGE with 50 mM phosphate pH 7.0, 0·3 M NaC and 150 mM sodium sulphate. Coomassie Brilliant Blue staining and immunoturbation. The fractions of the aggregated domain fragments were dialyzed against 20 mM Hepes, pH 7.4, 150 mM NaCl overnight at 4 eC to remove the imidazole. Each domain® fragment has the expected molecular mass (domain I, domain II, and domain 〜, respectively, ~u kDa, 18 kDa, and 9 kDa), and is immunoreactive with anti-CCNi multi-strain antibodies (Figures 1B and C) ). Human 1 064 SK fibroblasts are used to express the ability of each domain to support cell adhesion. Primary human foreskin fibroblasts 1064SK (ATCC CRL-2076, passage 2) were stored at 37 ° C and 5% C 〇 2 in Iscove's with 10% fetal bovine serum (Intergen, Purchase, NY) adjusted In Dulbecco's medium (Invitrogen, Carlsbad, CA). All experiments 30 1356097 used cell lines between the 5th to 20th generations. The test protein was applied to a 96-well microtiter plate (Becton 'Dickinson, NJ) in PBS (50 » ml per well) and the well was treated with 1% BSA (Sigma, St. Louis, MO) at room temperature. Block for 1 hour. To enhance coating efficiency, the CCN1 domain polypeptide was covalently linked to a maleic anhydride Reacti-Bind microtiter plate (Pierce, Rockford, IL) at 4 °C overnight and then blocked with BSA for 2 hours at 37 °C. . The cell-adhesion was performed in an amount of 5 X 10 5 cells/ml using the washed unconfluent cells as described, and the unconfluent cell line was resuspended in the basal medium without blood. See Chen et al. (2001) /. Ao/. Ckw. 276, 10443-10452. Named here, the cell line is pre-incubated with EDTA, peptide, or functional-blocking mAb for 3 min before seeding. All three domains were coated to the microtiter wells with similar efficiency. The microtiter wells were coated with six histidine-tagged proteins or BSA (50 ml per well) overnight at 4 ° C, followed by blocking with 1% BSA for 2 hours at room temperature. Protein coating efficiency was achieved by incubation with anti-polyhistidine mAb (Invitrogen) (at 37 (2 hours) followed by horseradish peroxidase-conjugated secondary antibody (Amersham)

Biotech-Piscataway, NJ) culture (1 hour at 37 ° C) for detection of β color reaction was visualized and quantified by Auo measurement. Only domain III can support the adhesion of fibroblasts (Fig. 1). Adhesion of fibroblasts supported by domain 111 can be achieved by EDTA (25 mM) (4) and this inhibition can be added to the assay medium. Released by Μ广(5mM) (2nd 8th). Cell adhesion can also be borrowed by: Μ (5' is inhibited and by round ++ (〇5, promoted. 31 31 31 1356097 The side of the divalent cation sensitivity is similar to the complete CCN1, and with the integrator via aji The cell adhesion is consistent. See Chen et al. (2000) · 275, 24953-2496. In order to determine which specific integrin receptors can regulate cell adhesion supported by domain III, we utilize functional-blocking mAbs. To test for inhibition. Pre-incubation of fibroblasts with anti-a6 (GoH3) or b! (P4C 10) mAbs eliminates cell adhesion supported by domain III and intact CCN1, whereas anti-integrin ανβ3 (LM609) Or the control group of mouse IgG mAb has no effect (Figure 2). In summary, these results show that, similar to the adhesion supported by intact CCN1, human skin supported by CCN1 alone Fibroblast adhesion is regulated by integrin. Example 2 The domain of CCN The T1 sequence in ΠΙ contains the integrin binding site. We used another systematic screening strategy to precisely determine the integrin binding site in CCN1. CCN1 One of the first three domains series of partial sequence overlap peptides (Table 1) was prepared by expressing the peptide as a fusion protein linked to GST. The CCN1 cDNA was used as a template to polymerize by chain reaction (PCR). The coding sequence of the peptide is increased. A primer relative to the appropriate coding sequence is used and the position is cleaved with the EcoRI restriction enzyme for colonization. For example, the following primers are used to generate the T1 peptide coding sequence: 5,-CGGGATCCGCGGGCCAGAAATGCATCGTT-3, With 5,-CCGGAATTCCGCTCTTGGAGCACTGGGACC-3,

The PCR product was purified by polyacrylamide gel, and the selection step was confirmed by sequence analysis using BamHI and five coRI 32 (1) 6097 cut "J and ligated to the pGEX_4T 2 vector (AmeMham pharmacia Bi〇teCh). GST-win: The fusion protein was produced in E. coli BL21 strain and by glutathione affinity chromatography (Amersham Pharmacia)

Purification by Biotech) followed by extensive dialysis of pBs overnight. Purification of these fusion proteins to near homogeneity and similar degree of coating efficiency on microtiter wells (this uses anti-antibodies) Called for detection by ELISA, no data shown). The ability of each peptide to fuse the protein to support fibroblast adhesion was assessed. Only - a win - _ melt. The protein, which is derived from the domain of τ, can support cell adhesion (Fig.) Re-human, the adhesion of fibroblasts supported by ΤΙ-GST can be inhibited by the bribes and Ca++ in the assay medium, and by Μη+ Promotion (Fig. 3) Furthermore, cell adhesion supported by T1_GST can be blocked by pre-incubation of cells with anti-a6 (GoH3) or anti-bi (P4Cl〇) secrets, but not Will be affected by other integrin-interrupting agents (such as GRGDSP Life Technologws/Gibco-BRL) or anti-ανβ3 (LM6〇9) (Fig. 3C), indicating that the T1_GST fusion protein supports fibrinogen via integrin (tetra) 1 Cell adhesion. Similarly, '71_Caf also supports cell adhesion regulated by α6βι in other cell types, including endothelial cells, smooth muscle cells, and PC3 prostate cancer cells (data not shown). In order to further determine that the T1 sequence contains integrin~...binding position 〇 into four & spanning the CCN1 domain ΙΠ peptide (see Table 〇 and testing its ability to support cell adhesion. The synthetic peptide system 纟 ^ 33 1356097 ( Huntsville, AL) preparation, followed by purification by reverse phase high performance liquid chromatography and 卩 mass spectrometer analysis. Results obtained by fusion of protein with GST_ peptide. Similarly, synthetic T1 peptide, but not other 3 peptides ( T2, Τ3 and, can support fibroblast adhesion (Fig. 4A). Furthermore, cell adhesion supported by immobilized τι-peptide can be inhibited by anti-% (g〇H3) or anti-(P4C10) 'But will not be affected by anti-heart (LM6q9) or control rats

IgG effects (Fig. 4B). These results again indicate that T1 contains the integrin α6ρι binding position. · (Example 3 Soluble Τ1 peptide can inhibit α6Ρι_dependent cell adhesion. We predict that soluble Τ1 peptide can block the cell adhesion that is known to bind to the receptor of integrin cxdi. As shown, the addition of 0.2 mM Τ1 to the cell suspension effectively blocked C (:Ni-supported fibroblast adhesion, while T2, T3, or T4 had no effect. τι adhered to CCN1-supported cell adhesion The inhibitory effect is dose-dependent and achieves maximum inhibition at 100 μΜ (Fig. 5c). Other members of the cCN protein steroid, CCN2 (CTGF) and CCN3 (NOV), also show support fibers via integrin αδβi Protocell adhesion (see

Chen et al. (2001) */.Exhibition/·C/iew. 276,10443-10452; Lin et al. (2003) ·/. _βζ·ο/· C/iew. In press)' and these CCN proteins The corresponding T1 sequences all exhibit homology to the degree of sputum. The $ a graph shows that T1 can also specifically inhibit the adhesion of cells supported by CCN2 and CCN3, and speculate that the T1 sequence in the CCN protein is a common binding site of integrin α6|3ι. 1356097 To further demonstrate the specificity of T1 inhibition, we tested its ability to block cell adhesion supported by receptors that bind to other integrins. Compared with the cell-adhesive effect supported by ccm in a dose-dependent manner (Fig. 5C), it is compatible with fibronectin (ligand of integrin (4)) and vitronectin (integrin). The cell adhesion supported by the ligand and the collagen Z integrin ligand has no significant effect (Fig. 5B). The cell adhesion supported by the layer white (known as integrin-pure ligand) can be partially inhibited by T1 peptide (~15%). This partial inhibition may be associated with incomplete inhibition by the anti-a6 mAb GoH3 stagnation. Other integrins may exist, for example, as adhesion receptors for laminin. In summary, these results show that the soluble T1 peptide specifically inhibits dependent cell adhesion, thus providing further support for the binding site of the Τ1 sequence containing integrins. Example 4 Effect of alanine substitution on cell adhesion in the sequence of Τ1 In order to determine the decisive factor for the aePi-dependent cell adhesion in the T1 sequence, we prepared a series of GST-peptide fusions with T1 backbone. The τι backbone has a single or dialanine substitution in the CCN1, CCN2, and CCN3 retention residues and is then tested for its ability to support cell adhesion. In order to generate a fixed-point alanine substitution in the T1 peptide (Fig. 6), the synthesized oligonucleotide was anneai in PCR to generate an appropriate coding sequence and the sequence was cloned to pGEX_4T_2. The coding sequence of the T1 sequence was prepared using the following primers and cloned to ρ〇ΕΧ-4Τ-2: ?5 1356097 5,-GATCCGGTCAAAAATGTATTGTTCAAACTACTTCTTGG TCTCAATGCTCTAAATCTGG-3' and

5'-AATTCCAGATTTAGAGCATTGAGACCAAGAAGTAGTA GTTTGAACAATACATTTTTGACCG-3' is to generate a mutant peptide, and the important codon is changed to GCA or GCT for alanine substitution. As shown in Figure 6, the substitution of the alanine at residue K226, 1228, or Q230 does not affect the ability of the peptide to support cell adhesion. However, a single mutation in T23 1 or T2 32 partially reduced cell adhesion, and the combination of T23 1 and T232 alanine substitution completely abolished the ability of T1 to support cell adhesion. Furthermore, a single substitution in W234, S235, S238, or K239 lost T1 activity > 90%. When the mutations of W234 and K239 were combined, cell adhesion was completely eliminated. These results indicate that the TTSWSQCSKS T1 regulates the core sequence of aji binding. These data may also explain the ineffectiveness of the T2 peptide, which overlaps with the T1 peptide but lacks the TT residue in the core sequence, so as to be unable to inhibit αβγ-dependent cell adhesion. Example 5 Integrin using the Τ1-coupled affinity matrix A6h affinity purification In order to confirm that the T1 peptide can be directly combined with integrin aji, we use the cell surface protein on the surface of fibroblasts for affinity chromatography to separate the integration on the T1-coupled affinity column. Unconjugated 1064SK fibroblasts were separated by 2 mM EDTA and 0.05% BSA in PBS, washed three times and resuspended in PBS containing 36 1356097 20 mM glucose in an amount of 2 X 107 cells/ml. For surface labeling, the cell suspension was incubated with glucose oxidase (100 mg/ml) and 200 mg/ml lactooxidase (Calbiochem-Novabiochem, La Jolla, CA) at 4 °C. ), and ~400 mCi/ml of carrier-free Na Ϊ (Amersham Pharmacia Biotech) was gently shaken for 30-60 minutes. 10 ml of cell culture medium was added to terminate the labeling. Wash the labeled cells and dissolve in 1 ml of cell lysis buffer (50 mM Hepes, pH 7.4, 200 πιΜ octyl-bD-glucopyranoside, octyl-bD-glucopyranoside, protease inhibitors) Proteinase inhibitor cocktail), and 〇.5 mM Mn++). As for affinity chromatography, the GST_T1 or GST_ hybrid T1 protein is associated with

Affi-Gel 10 (Bi〇-Rad Laboratories, Hercules, CA) coupled in a 10 mg/ml gel suspension. The labeled cell lysate was applied to the affinity matrix (matriee) and cultured for 2 hours at 4 . The column was washed with 3 管 column volume of cell lysis buffer and then eluted with lysis buffer containing 〇·35 μ NaCl. The labeled protein in the eluate was passed through 7/. The polyacrylamide gel was subjected to electrophoretic analysis under non-reducing conditions followed by automatic radioactive development. In the immunosuppressive analysis, the labeled protein was cultured with 5 mg of anti-a6 (G〇H3) or anti-_av (P3G8) mAbs (Chemicon, Temecula, CA) as indicated. The immunoprecipitated protein was collected from the protein G_SepharQse and analyzed under non-reducing conditions with a 7% polyacrylamide gel. A control column was prepared by GST fusion to a mixed T1 sequence, and the labeled protein broad band was not eluted from the self-mixed T1-GST and 0·^·丄, s columns (Fig. 37 1356097 7A). Conversely, 'from the τΐ-GST affinity column, when eluted with 〇35 μ NaCl, two protein broad bands have molecular weights equivalent to integrin “·. (~150 kDa) and b丨 (~13〇kDa) subunits ( Column 5_7, Figure 7B). Confirmation that the bound labeled protein is indeed an integrin "... complex, using G〇H3 (anti- or P3G8 (anti-~) as a control group to make the eluate Immunoprecipitation was performed. Figure 7C shows that g〇H3 can immunoprecipitate the labeled protein broad band from the eluate, whereas in the control sample, p3G8 cannot, and the protein complex is precipitated. In summary, integration Suichi 01 can directly bind to the T1 sequence in CCN1. Example 6 Dissolved T1 peptide disrupted CCN1-induced endothelial tube formation Several CCN protein lines containing CCN1, CCN2, and CCN3 have potential angiogenesis Inducing matter. See Babic et al. (1998) Proc iVai/.Jcad. «Scj?· Π/4. 95,6355-6360 ; Lin et al. (2003) «/. 5zo/. CAew. In press; Babic et al. (1999) Mo/. Ce" 19 2958-2966. Furthermore, when formulated in collagen gel, ccNl can induce Of the human umbilical cord vascular endothelial cells (HUVEC) tube formation of action, and this program can be blocked by anti -a6 mAb GoH3 See Leu et al. (2002)

/· 5ζ·ο/· C/zem. 277,46248-46255. Since the T1 sequence represents the integrin in CONI (the major binding site of 3⁄46β1, we tested whether this soluble T1 succulent can inhibit CCN1-induced tube formation in unactivated HUVEC. To analyze endothelial cell tube formation, humans Umbilical cord vascular endothelial cells (HUVEC) are detected in a collagen gel in a three-degree space in the presence or absence of the test protein or peptide described. See Leu et al. 38 1356097 (2002) */. 5z' 〇/· C/ze/w_ 277,46248,46255. As shown in Figure 8, when CCN1 is formulated in the Shengyuan gel, the human umbilical cord endothelial cells are induced to form a tube. Before inoculation, HUVEC and T1 ( 0.2 mM) pre-incubation for 30 minutes can completely inhibit the formation of CCN1-Embroidered tubes. Conversely, the T2, T3 and T4 peptides of the control have no effect. Therefore, these results indicate that the T1 can block CCN1. Inhibition of CCN1-induced tube formation by interaction with integrin on unactivated HUVECs. Example 7 Constructs of mutations that are inactivated at the binding site of Τ1 binding to integrin ouPi include ccm, CCN2 And CCN3 several kinds of C The CN protein is a potent angiogenesis agent. The alanine-substituted GST-T1 fusion protein described in Example 4 proves unsuitable for determining the function of the T1 position in the action of CCN1 when used to determine the novel α6Ρι binding position. Sexual role. The expressed mutants will cluster together and form in the cells to form inclusion bodies (data not shown), indicating that the mutants employ an unfavorable configuration to prevent their own ploidy cells from secreting. In order to allow the mutant to specifically lose activity in the action of cuP^HSPG-regulation while retaining other functions, we utilized two algorithms, ppsiRED and FRAGFOLD (McGuffin, LJ, Bryson, K.^ JoneSj DT

(2000) 16, 404_4〇5, and J〇nes, d T (2001) palpitations such as Suppl 5, 127_132) establish fusion proteins to predict possible changes in the secondary structure that may cause possible mutations. 1356097 according to Leu, SJ, Liu, Υ., Chen, N., Chen, C. C., Lam, SC, A Lau, LF (2003) J. Biol. Chem. 278, 33801-33808 ^ A GST fusion protein of a CCN1 peptide and a mutant peptide was prepared. Using the following primer pair to establish a C-terminal mouse CCN1 with FLAG tag using mouse cDNA as a template

Fl: 5'-CGCAATTGGAAAAGGCAGCTCACTGAAGAGGC-3' and

F2: 5'-CCGGAATTCCTACTTGTCATCGTCATCCTTGTAGTC GTCCCTGAA CTTGTGGATGTCATTG-3',

Thus a PCR product is obtained which contains the FLAG tag coding sequence followed by the last codon of the stop codon. The PCR product was double-cleaved with iVcoI and five coRI, and ligated into a pre-cleaved vector to replace the fragment of the complete mouse Cc«7 cDNA cut into iVcoI, jEcoRI in the pBlueBac4.5 vector (Grzeszkiewicz, Τ · M., Kirschling, DJ, Chen, N. and Lau, LF (2001) J. Biol. Chem. 276, 21943-21950). For consistency, WT CCN1 and all mutants used in this study have an N-terminal secretion message and a C-terminal FLAG epitope tag. Consistent with the inability to secrete alanine-substituted mutants, the T1 region of CCN1 showed high sensitivity to perturbation. The substitution in the decisive T1 residue (W234, S235, S23 8, K239) almost any amino acid will cause significant changes in the predicted protein structure. However, we found that a mutation, also known as K239E, did not cause a change in the predicted configuration. To test the efficacy of this mutation in disrupting the interaction with aji, we established a fusion protein that links GST to the T1 peptide, which has a Κ239Ε mutation (GQKCIVATTSWSQCS£S). 'Example 8 * Evaluation of Adhesion of Mutant CCN1 Peptide (wherein the T1 binding site for binding to integrin is inactivated) Evaluation of fiber supported by ΤΙ (K293E) mutant peptide established according to Example 7 Protocell adhesion and comparison with GST-T1 fusions with WT sequences. GST-T1 and GST-T1 (K239E) have similar coating efficiency (data not shown by ELISA). Fibroblasts were seeded into microtiter wells coated with GST, GST-T1 peptide blends, or GST-T1 (K239E) peptide fusion proteins (50 μg/ml each). The cells were allowed to adhere at 37 ° C for 20 minutes. After washing, the adherent cells were fixed, stained with methylene blue, and quantified by the absorbance at 62 Onm. As predicted, the GST-T1 fusion supported adhesion of fibroblasts, whereas the BSA or GST control did not (Fig. 9B). See also Leu, S. J., Liu, Y., Chen, N., Chen, C. C., Lam, S. C. and Lau, L. F. (2003) /.仏〇/· C/zew. 278, 33801-33808. GST-T1 (K23 9E) is completely unable to support cell adhesion, indicating that the charge-opposite K239E mutation may be sufficient to disrupt the binding of T1 to integrin. Example 9 Mutant CCN1 construct inactivated at the T1 binding site bound to integrin Using site-directed mutagenesis using Koskinen, P., Lehvaslaiho, H., 1356097

MacDonald Bravo, Η., Alitalo, Κ· and Bravo, R. (1990) Owcogewe. 5, 615-618, a two-step PCR procedure to establish a single K239E mutation in intact CCN1, called SM (9A) Figure). The internal primer pair is 5'-GTCTTGGTCCCAGTGTTCCGAGAGCTGCGG-3' and 5.-CACTGGGACCAAGACGTGGTCTGAACGATGC-3'. This construct also creates a silent mutation at C237, thus providing a screening marker by removing the BSP12861 restriction position. The external primer used in the PCR was F1 and F2 described in Example 7. SM was established using a mouse cDNA having a FLAG tag as a PCR template. A DM coding sequence was also created in which the heparin binding sites H1 and H2 were disrupted as shown in Figure 9A. Under the same conditions as CCN1, these mutations changed H1 from KGKKCSKTKKSPEPVR to AGAACSATAKSPEPVR and H2 from FTYAGCSSVKKYRPKY to FTYAGCSSVAAYAPKY. Using the above procedure, the DM coding sequence is used to generate a DM construct having a FLAG tag. The TM sequence was created using DM as a template. The K239E mutant was generated using the above method. All constructs were confirmed by direct sequence analysis. Example 10 SM analysis - broken T1 binding site, uninterrupted heparin binding site Human fibroblasts were seeded onto microtiter wells coated with a specified amount of recombinant WT CCN1 or SM mutant, and as in Example 1. The evaluation of cell adhesion. Surprisingly, SM can support the adhesion of fibroblasts in a dose-dependent manner 1356097, which is often similar to WT CCNl # and achieves maximum cell adhesion at a coating concentration of 1 μg/ml (10A) Figure). This result confirms that SM can still support cell adhesion via the adrHSPG co-receptor, indicating that there may be other unidentified potential a^rHSPG-binding sites, or that the H1 and H2 heparin binding sites are sufficient to support via the aJrHSPG co-receptor complex. Cell adhesion. Example 11 SM analysis - T1 binding position of cleavage, inhibition of heparin binding position

Since SM retains the intact heparin binding site, 遂 tests whether cell adhesion by δΜ depends on HSPG. Heparinase I (heparinase) before adhesion of fibroblasts to microtiter wells coated with WT CCN1 (2 μg/ml), SM (2 μg/ml), or VN (0.5 μg/ml) /ml) or chondroitinase ABC (2 units / ml) (all purchased from Sigma, St. Louis, MO). The cells in the control group were not treated. Named here, soluble heparin is present in the medium in an amount of 1 μg/ml. WT CCN1-supported cell adhesion was blocked as predicted by either saturating the CCN1 heparin binding site in the presence of soluble heparin or treating cells with heparinase 伤害 to damage cell surface HSPG. See

Chen, N., Chen, C. C. and Lau, L. F. (2000) /.扪0/· G hidden 275, 24953-24961. Similarly, SM-supported cell adhesion was similarly inhibited by treatment with soluble heparin and heparinase, suggesting that the interaction of HSPG and SM on the cell surface is important for the adhesion of the CCN1 mutant to the supporting cells. of. Chondroitinase ABC treatment has no effect on cells 43 1356097. It is determined that CCN1 lineage specificity relates to HSPG rather than chondroitin sulfate proteoglycan. Example 12 DM analysis - uninterrupted T1 binding position, cleavage heparin binding position To further assess the role of heparin binding in CCN1 function, we used DM mutants as described in Example 9 to detect heparin binding activity After the impact. As shown in Figure 9A, mutations in the basic residues of H1 or H2 reduce heparin binding, and mutations in combination H1 and H2 can eliminate heparin-binding activity in CCN1 (Chen, N., Chen, CC&amp ; Lau, LF (2000) X 5z'o/. C/jew. 275, 24953-24961). Since DM has lost heparin-binding activity but retains the T1 ouPi binding position, we hypothesized that DM may impair co-receptor-regulating activity, but may support cell adhesion via α6β1 at high coating concentrations, similar to GST -T1 peptide fusion protein supports cell adhesion (Fig. 9). Indeed, when CCN1-supported cell adhesion reached a level of 1 μg/ml (Figure 10), DM could not support cell adhesion unless coated at very high concentrations as an adherent, and maximum adhesion occurred at 50. -100 μg/ml (page 11). Example 13 TM analysis - T1 binding position of cleavage, heparin binding position of cleavage As described in Example 9, we also created a mutant (TM) which binds to the K293E mutation in the DM background, thus disrupting the T1 binding position , and the binding sites of H1 and H2 of heparin. Significantly, TM could not support cell adhesion at any of the tested concentrations. 44 Ϊ 356097 Effect' Even when the S coating concentration was 250 μg/ml, it was completely ineffective. This result indicates that DM can specifically adhere to the cell adhesion by 'supporting the K239E mutation in the T1 binding position of αόβι, and this effect disappears. Example 14 Evaluation of cell adhesion of DM using anti-integrin In order to verify the disappearance of adhesion caused by the K239E T1 mutation, it was examined whether DM-supported cell adhesion was regulated via α6|3ι. Blocking mAb with 40 μg/ml anti-integrin ανβ3 (LM609), anti-integrin~(G〇H3), or anti-integrin βι subunit (P4C10, 1:5〇 ascites) before inoculation Incubate for 1 hour at room temperature. Cell adhesion was then assessed as described above. Consistently, the anti-integrin tt6 ((3〇113) or β^ραίΟ) mAb blocked the adhesion of DM- or CCN1-supported fibroblasts, while the anti-Guada LM609 had no effect (Fig. 11B). Thus, these results indicate that ccni-supported fibroblast adhesion requires a6Pi_HSpG co-receptors that interact with three binding sites: τι, H1, and H2. Mutations in m and H2 (DM) can still support cell adhesion via αόβι at high coating concentrations, and all three sites are destroyed (TM) and then completely abolished by aJi-HSPG-supported cell adhesion (p. UA map). Example 15 MAPK Activation CCN1 has an abnormal monthly force that causes the sustained activation of ρ42/ρ44 ΜΑρκ (Chen, C.-C " M 38 τ〇τ ι., Chen, Ν. and Lau, LF (2001) • Ugly 45 1356097 C/^w_ 276, 10443-10452). In order to test the effect of mutations in the binding sites of τΐ, HI and H2 on this activity, fibroblasts were adhered to WT CCN1, SM, DM, or laminin for a specified period of time (Fig. 12). To assess MAPK activation, '1064SK fibroblasts lack serum and resuspend in serum-free medium at 6 X 105 cells/ml' and culture the cells in WT CCN1 (1 μg/ml) as indicated , SM (10 μg/ml), DM (250 μg/ml), or laminin (10 μg/ml) coated 35-mm culture dish for ι_5 hours. The clarified cell lysate was electrophoresed on 10% SDS-PAGE, and immunized with multiple anti-MAPK antibodies or antibodies specific for the phloridizing activity p42/p44 MAPKS (Progema, Madison, WI). Turning. As shown in Figure 12A, cells adherent to laminin can rapidly and transiently activate p42/p44 MAPK, just as ECM-derived cell adhesion acts to the background after maximal activation 1 hour after inoculation. value. Conversely, MAPK activation in cells adhering to WT CCN1 or SM persisted with a greater degree of activation even after 5 hours of inoculation. This prolonged MAPK activation was not seen in cells adhering to dm. Thus, mutations in the 'H1 and H2 positions have no effect on short-term MAPK activation, but specifically eliminate the ability of CCN1 to cause sustained MAPK activation. EXAMPLE 16 Modulation of Gene Expression CCN1 activates a gene program in fibroblasts that results in genes encoding proteins involved in angiogenesis and matrix metabolism (including angiogenesis-inducing VEGF 1 and membrane metal-binding protease ΜΜΡ-l) Upward adjustment (Chen, C.-C., Chen, N.> S. Lau, LF (2001) J. Biol. Chem. '276, 10443-10452). Since activated MAPK is transferred to the nucleus, it phosphorylates and activates transcription factors in the nucleus (Hazzalin, CASl Mahadevan, LC (2002) Nat. Rev. Mol. Cell Biol. 3, 30-40), If sustained MAPK activation is lost, Bay J may alter the ability of CCN1 to regulate gene expression. To test the effect of loss of sustained MAPK activity on the ability of soluble CCN1 φ (WT) and CCN1 mutants (SM, DM, TM) to regulate gene expression, primary human skin fibroblast was 10 μg. /ml of protein was treated for 24 hours in the absence of serum. Total cellular RNA was then isolated, resolved in an agar-formaldehyde gel and disrupted to a nylon membrane using standard laboratory procedures. As described, the 32P-dCTP was combined with human VEGF-A, ΜΜΡ-1, and GAPDH cDNAs by random primers to create a radioactive cDNA probe (Chen, C.-C., Chen, N., and Lau). , LF (2001) «/.

Chm·276, 10443-10452). The wash was then washed under high stringency conditions (0.1 X SSC; 0.1% SDS at 65 ° C) and analyzed using a Phosphorimage Scanning Analyzer (Phosphorimager, Molecular Dynamics, Sunnyvale, CA). The expression of Feg/ and ΜΜΡ-l was evaluated by RNA disruption (20 μg total RNA in each column) followed by electrophoresis, and monitoring of Glyceraldehyde 3-phosphate dehydrogenase , GAPDH) performance as a control group. As shown in Figure 12B, similar to WT CCN1, SM can be adjusted upwards. 47 1356097

The performance of Feg/ and MMP-1, while DM and TM completely lack this activity. Therefore, the heparin binding sites HI and Η2 are required to prolong the activation of · and to upregulate the expression of Feg/ and MMP-1. Example 17 CCN1 Mutant Supports Adhesion and Migration of HUVEC via Integrin ανβ3 We examined the adhesion of activated HUVECs supported by CCN1 mutants. HUVECs, which were separated in 2.5 mM EDTA and resuspended in serum-free medium, were adhered to wells previously coated with 15 μg/ml of wild-type CCN1 (WT) or SM, or 50 μg/ml of DM or TM. . It is indicated here that the cells are EDTA (5 mM), GRGDSP peptide (RGDS, 0.2 mM), anti-ανβ3 mAb LM609 (40 μg/ml), anti-a6 mAb GoH3 (40 μg/ml) before culture. Handle for 60 minutes. The adhesion of the cells was measured as described above. WT CCN1 and SM support cell adhesion to a similar extent (Fig. 0 13A). EDTA completely inhibits WT and SM-supported cell adhesion and GRGDSP peptide or anti-ανβ3 mAb LM609 inhibits cell adhesion, indicating that the cell adhesion involves integrin α νβ 3 . Anti-a6 mAb GoH3 also inhibited some cell adhesion, consistent with the results of the α6β 1 -HSPG co-receptor (Leu, S.-J., Lam, S. C. Τ· and Lau,

L. F. (2002) 5/0/. C/zem. 277, 46248-46255). Conversely, HUVEC adhesion supported by DM and TM can be completely inhibited by EDTA, GRGDSP peptide and LM609, but GoH3 does not. These results indicate that DM and TM can support the adhesion of activated HUVEC via α νβ 3 for use in 48 1356097, but do not have the ability to support-regulate cell adhesion. Example 18 CCN1 or Mutant Supports Migration of HUVECs Using a modified Boyden chamber assay, it was found that WT CCN1 and mutants can stimulate HUVEC migration at the aJrHSPG binding site (Fig. 13B) ). HUVEC migration, supported by CCN1 or mutants, was assessed using a Transwell chamber. 15 μg/ml of CCN1 WT or Mutant SM, 50 μg/ml of mutant DM or TM was immobilized on the lower surface of the transfer disk membrane, wherein the transfer disk membrane divided the reaction chamber into two zones. HUVECs were treated with 100 nM PMA for 30 minutes to activate the integrin receptor. It is indicated here that the cells are buffered with vehicle (not added), normal mouse IgG (100 μg/ml) (Sigma, St. Louis, MO), GoH3 (50 μg/ml) before being cultured in the upper reaction chamber. (Chemicon, Temecula, CA), LM609 (50 μg/ml) (Immunotech, Marseille, France) for an additional 30 minutes pre-incubation. The cells were allowed to migrate for 8 hours, and cells transferred to the lower reaction chamber were counted in 10 random high power fields. Cell migration supported by CCN1 and all mutants was completely inhibited by anti-ανβ3 mAb LM609, whereas anti-a6 mAb GoH3 had no effect. Thus, similar to WT, CCN1 mutants with a break in the HSPG binding position stimulate the migration of ανβ3-dependent HUVEC. Example 19 Enhancement of VEGF-induced DNA synthesis via integrin ανβ3 The 1356097 CCN1 mutant enhances growth factor-induced DNA synthesis without undergoing cell division by itself. See Kireeva, M. L., Mo, F.-E., Yang, G. P. and Lau, L. F. (1996) Mo/· Ce//. 5ζ·ο/· 16, 1326-1334. HUVECs were pre-incubated with vehicle buffer (not added), LM609 (25 μg/ml), GoH3 (25 μg/ml), or normal mouse IgG (25 μg/ml) for 1 hour. The cells were then treated with VEGF (5 ng/ml) and/or CCN1 or mutant protein (5 μg/ml each) in the presence of [3H]thymidine, and the combined effects were analyzed after 48 hours. The data shown is the average soil S.D. of the three measurements and is a representative of the three experiments. As predicted, treatment with VEGF (5 ng/ml) induced DNA synthesis in HUVEC, whereas addition of CCN1 or its mutant ij has no effect (Fig. 14). However, treatment of cells with CCN1, SM, DM or TM in the presence of VEGF enhances VEGF-induced DNA synthesis by ~2 fold. LM609 blocks this potentiation, thus reducing DNA synthesis to a degree that is induced by VEGF alone. Conversely, neither IgG nor GoH3 has any effect. Thus, similar to WT CCN1, SM, DM and TM enhance growth factor-induced DNA synthesis in endothelial cells via integrin ανβ3. Example 20 CCN1 Mutant Promotes HUVEC Survival CCN1 has previously been shown to promote endothelial cell survival via zygote ανβ3 in the absence of growth factors. See Leu, S.-J., Lam, S. C. T. and Lau, L. F. (2002) ·/· million/〇/. C/zew. 277, 46248-46255. In order to determine that the CCN1 mutant promotes cell survival, its effect on HUVEC inoculated with laminin was examined by TUNEL analysis (Fig. 1 5 50 1356097). The unfreshed HUVEC was attached to a coverslip precoated with 20 μg/ml laminin (LN) for 4 hours, followed by addition of serum, CCN1 or mutant protein (5 μg/ml each) for 16 hours. Cells were fixed and cell death was monitored by TUNEL assay. Here, it is indicated that a plurality of anti-CCN1 antibodies are pre-incubated with the test reagent for 3 minutes before the medium is added. When the cells were maintained in 20% serum, only a few apoptotic cells were detected, and in the medium without jk clear, > 70% of the cells were apoptotic. Adding 匚CN1, SM, DM, or TM can reduce ~40. /. The number of cells that are apoptotic. On the contrary, in the presence of the anti-CCN1 antibody, the effect of promoting cell survival is the activity of the CCN J polypeptide. DNA synthesis of treated HuveC under the same conditions was also monitored (data not shown). Consistent with the results of Figure 14, the rate of DNA synthesis was not affected by the presence of CCN1 or the mutation alone, indicating that the increase in non-apoptotic cells was not due to an increase in cell proliferation. These results show that the CCN1 mutant can still promote endothelial cell survival in the absence of growth factors. This effect is regulated by integrin 〇^03. Example 21 Tube formation CCN1 was also induced to induce tubular formation of cells upon activation in a collagen gel via integrin ανβ3. See hS.VS.C T. and Lau, L_F·(fine 2) j. coffee·cw 277, 46248 46255. To test the role of the CCN1 mutant, culture it in the war! Type collagen gel (2 mg/ml) pre-coated 24-well plate (this coating is in ccni (wt) or TM (20 μg/ml each).

Is a (one) or exist in existence), will HUVEC 1356097

Treatment with vehicle buffer (not added) or stimulation with 5 nM PMA in serum-free medium. A second layer of gel of the same formulation was overlaid on the attached cells' and tube formation was assessed 16 hours later. It is indicated here that LM609 (40 μg/ml) or g〇H3 (40 μg/ml) is added before the cultivation.

Into the cell suspension. The results are representative of three individual experiments (magnification X 200). In the absence of CCN1, unstimulated HUVEC showed no tube formation' and 20 μg/ml of CCN1 could not induce tube formation _ (Fig. 16) ). However, PMA-activated HUVEC responds to WT CCN1 or TM stimulation by forming a tube. Add LM609 (40 μg/ml) to the cell suspension to inhibit tube formation before incubation.

GoH3 (40 μg/ml) has no effect. The same results were also observed in sm and DM (data not shown). These results show that the CCN1 mutant can induce ανβ3-dependent tube formation in activated endothelial cells. [Simplified illustration] Figure 1 confirms the purity of the protein of the recombinant CCN1 domain fragment and its ability to bind to cells. Recombinant CCN1 domain fragment is a six-histidine-tagged fusion protein by baculovirus_ expression system, and a schematic representation and separation of the complete CCN1 domain by diamond-separation: The domain fragment. The T1 sequence in the domain ΙΠ (TSP1 field) is indicated by a shaded area. 5. The recombinant CCN1 domain fragment and intact CCN1 (2 μg) were electrophoresed in a 15% SDS-polyacrylamide gel and detected by Gumar blue staining. C's immunostained the solubilized protein with multiple anti-ccNl antibodies. See Kireeva et al. (1996) Cei/. 52 1356097 16, 1326-13 34. The mark of molecular mass is indicated on the left in units of kDa. Maleic anhydride Reacti-Bind microtiter wells were coated with purified recombinant CCN1 domain fragment or bovine serum albumin (BSA) (50 μg/ml, 50 μg/well) at 4GC overnight and at 1% BSA blocked. Protein coating efficiency was detected by an enzyme-linked immunoassay (ELISA) using an anti-polyhistamine monoclonal antibody (mAb). £, the washed 1064SK fibroblasts were resuspended in serum-free medium and inoculated into wells previously coated with CCN1 (20 μg/ml) or designated domain fragments (5 μg/ml) ( There are 3 X 104 cells per well). The cells were allowed to adhere for 30 minutes at 37 GC. Adherent cells were fixed, stained with methylene blue, and quantified by absorbance at 620 nm. Data are the mean ± S.D. of three measurements. The D and E diagrams are representative of three experiments. Figure 2 demonstrates that domain III (TSP1 domain) of CCN1 supports fibroblast adhesion via integrin αόβι. Adhesion of fibroblasts was performed on microtiter wells coated with intact CCN1 (20 μg/ml) or Domain III fragments (50 μg/ml) as described in Figure 1. J, indicated that the cells were suspended in serum-free medium before vaccination, and the medium contained EDTA (2.5 mM), Mg++ (5 mM), Ca++ (5 mM), or Mn++ (0.5 mM). B, before cell seeding, 'with buffer buffer (not added), normal mouse IgG (loo microgram / ml), anti-ανβ3 mAb LM609 (50 μg / ml) (Chemicon-Temecula, CA), anti- -a6 mAb GoH3 (50 μg/ml) (Immunotech-Marseille, France), or anti-mAb P4C10 (1:50 ascites) (Life Ding 6〇1111〇1〇§168/0丨13.〇-:610〇 Pre-culture for 6 minutes. The data is the average of the second measured value S_D. and represents the three experiments 1356097

Figure 3 demonstrates that the recombinant GST-T1 fusion protein can support-dependent. 'J. Fibrillary cell adhesion 4, and the microtiter well is 200 μg/ml of recombinant GST-peptide fusion protein (the sequence is shown in The coating was applied. The protein coating was carried out overnight at 4QC, followed by blocking with 1% BSA. The adhesion of fibroblasts was evaluated as described in Figure 1. 5. The cells were suspended in serum-free medium. The medium contains EDTA (2.5 mM), Mg++ (5 mM), Ca++ (5 mM), or Mn++ (0.5 mM) and is inoculated with Chusamine-sulfur S-transferase (GST) (50 μg/ml), GST. -T1 (50 μg/ml), or CCN1 (1 μg/ml) coated microtiter wells. c, inoculated with cells 将' with vehicle buffer (not added), normal mouse IgG (1 〇〇μg/ml), anti-ανβ3 mAb LM609 (50 μg/ml), anti-_a6 mAb GoH3 (50 μg/ml), or anti-seven mAb selected lean strain P4C10 (1:50 ascites) pre-culture 6〇 Minutes. The data is the average soil SD of three measurements and is representative of three experiments. Figure 0 Figure 4 confirms that the synthesized T1 peptide can support αόβι_dependence. Cell adhesion < attached. 乂, at 4 ° C, the microtiter wells were coated with synthetic τΐ, Τ2, Τ3, or T4 peptide (〇.2 mM) overnight, followed by 1% BSA blocking Adhere the fibroblasts to the well-coated wells for 2 min at 37QC. 5. Prior to inoculation to the T1-coated wells, place the cells with vehicle buffer (not added) or indicated The monoclonal antibody was pre-incubated for 6 min. The data is the average soil SD of the three-human measured value and is a representative of three experiments. Figure 5 demonstrates that the soluble T1 peptide can inhibit "I•dependent cell adhesion. The titration plate wells were coated with CCN1 〇μg/ml), CCN2 (2 micro 54 1356097 g/ml), or CCN3 (5 μg/ml), and blocked with 1% BSA. The washed fibroblasts were loaded. Buffer (not added) or pre-treated with soluble guanidine, T2, T3, or T4 peptide (0.2 mM) for 30 minutes and then seeded in the indicated CCN protein coated wells. 5 and C' Shown in inoculating to fibronectin (fN, 2 μg/ml), vitronectin (VN, 0.4 μg/ml), type I collagen ( Various concentrations of T1 peptide were added to the cell suspension before 层.5 μg/ml), laminin (LN, 5 μg/ml) or CCN1 (1 μg/ml) coated wells. Recombinant rats The CCN1-like protein is purified from serum-free insect cell whole nutrient medium using the baculovirus expression system described above. See Kireeva et al. (1996) A/W· Ce//· 5/〇/· 16, 1326-1334. Rat type I collagen, vitronectin, laminin, and fibronectin were purchased from Collaborative Biomedical (Bedford, ΜΑ). Cell adhesion was assessed as described in Figure 1. The data is the average of the secondary measurements; t S.D. and is a representative of three experiments.

Figure 6 demonstrates that the TTSWSQCSKS sequence in T1 contains important determinants of α6βι-dependent cell adhesion. The alanine substitution at a specific position in the T1 sequence in the T1GST fusion protein is carried out as described in "Materials and Methods". A wild type T1 fusion protein (GST_T1_WT), a hybrid variant (GST-Tl-SCram) or an alanine substituted mutant was applied to the microtiter well at a protein concentration of 200 μg/ml. Adhesion of fibroblasts as assessed as described after blocking with i% bsA. The result is the average soil S.D. of the three measured values and is a representative map of three experiments. Figure 7 demonstrates the affinity purification of integrin~βι from the fibroblast lysate in the GST-Ti·coupled Affi_gel (4). The cell surface protein of fibroblast 55 1356097 is labeled with a radioactive dish by the lactoperoxidase-glucose oxidase method described in "Materials and Methods". The labeled cells are dissolved in an affinity column containing Affi gel agar containing 20 mM octyl grape (4) and starting buffer 胄 cell lysate (shelved by mixing with GST) U map) or by affinity column (4) of Am. gel t-lips coupled with GST_T1. After washing with starting buffer (block 2-4), the column is eluted with 〇.35]^ (Block 5_8). Analyze the protein in the extract in 7% of the non-reducing conditions in the polyacrylamide gel and detect it by autoradiography. ° Collect the GST_T1 column. High-salt extract and immunoprecipitation with anti-(G〇H3), or anti-_av(P3G8) mAb. The immunoprecipitated protein was analyzed under non-reducing conditions. The molecular quality symbol is indicated by coffee. On the left, the results are representative of the second experiment. Figure 8 demonstrates that T1 peptide in the collagen gel matrix can block ccni_-induced endothelial tube formation. Unstimulated HUVECs are cultured in CCN1 absent (not added) Or coated with a pre-type collagen gel (2 mg/ml) (5 μg/ml) 24 • well plate, and as "materials and methods" of the the second layer of the gel was overlaid on the cells adhere. Named here, the cell suspension was cultured for 3 minutes before the culture. Tube formation was assessed 16-20 hours later. The result is a representative of three individual experiments (magnification X 1 〇〇). Figure 9 shows the structure and performance of CCN1 and mutants. a, a representation of wild-type CCN1 (WT) and a mutant, which has a K23 9E point mutation (SM) in τι, and a fragment (dm) '56 1356097 or ΤΙ in H1 and H2, Mutants (TM) are found in both HI ' and H2. Each construct has a similar Ν-end secretion message and a c-terminal FLAG epitope marker. The recombinant protein is expressed in insect cells via a baculovirus vector. The sequence of the wild type T1, H1, and H2 and the change of the specific amino acid in the mutant were also shown. B ' fibers were cultured in microtiter wells coated with gst, GST-T1 conjugate, or GST-T1 (K239E) peptide fusion protein (504 g/ml each). The cells were allowed to adhere for 2 minutes at 37 °C. After washing, the adherent cells were fixed, stained with methylene blue, and quantified by absorbance at 620 nm. c, showing 〇〇/〇〇 SDS-PAGE stained with Gumasi blue, wherein the FLAG affinity-purified recombinant protein (2 pg each) was separated by electrophoresis. The symbol of molecular mass (kDa) is shown on the left. The gel was immunosuppressed with multiple anti-CCN1 antibodies and is shown in the lower panel. Figure 10 demonstrates that fibroblast adhesion supported by the mutant SM is heparin sensitive. A, 1064SK human fibroblasts were cultured in microtiter wells 'the microtiter wells were coated with the indicated amount of recombinant WT CCN1, or SM canine variants' and evaluated for cell adhesion as described in Figure 1 . B, the fibroblast cell line was untreated or passed through the liver before adhering to the microtiter wells coated with WT CCN1 (2 μg/ml), SM (2 μg/ml) or VN (0.5 μg/ml). Treatment with enzyme I (2 units/ml) or chondroitinase ABC (2 units/ml). The soluble heparin referred to herein is present in the medium in an amount of 丄 micrograms/ml. The data shown is the mean ± S.D. of three measurements and is a representative of three experiments. Figure 11 demonstrates that DM supports ajr-regulated cell adhesion. a. Culture the 57 1356097 fibroblasts in an indicated amount of Dni or tm coated microtiter wells and assess cell adhesion. Β, before culture, cells and 4 μg/ml of integrin ανβ3 (LM609) as function_blocked mAb, integrin a6 (GoH3), or integrin β, subunit (p4C1〇, i:5) The ascites was pre-incubated for 1 hour at room temperature. Cell adhesion was assessed as described above. The data shown is the mean ± s. D. of the second measured value and is a representative of the three experiments. Figure 12 demonstrates the effect of CCN1 mutation on MAPK activation and gene expression. A, MAPK activation. The i 〇64SK fibroblasts were deficient in serum and resuspended in a medium of 6×1 〇5 cells/ml in the medium without sputum. The cells were cultured as shown in WT CCN1 (1 μg/ml), SM (10 μg/ml), DM (250 μg/ml), or laminin (LN, 10 μg/ml). ML / culture fox (35 mm cultured solitary) in 1-5 hours. The clarified lysate was subjected to immunoturbation on 1% SDS-PAGE as an antibody against multiple 'anti-M A P K antibody' or anti-di-acidification activity p42/p44 MAPK. B, the regulation of gene expression. Primary human dermal fibroblasts were deficient in serum for 24 hours before treatment with WTCCN1 (WT), mutant protein (SM, DM and TM) or BSA control (B) at 10 μg/ml for 24 hours, then isolated Total cellular RNA. The expression of Feg/ and MMP-1 was evaluated by RNA disruption (20 μg total RNA in each column) followed by electrophoresis, and the performance of glyceraldehyde 3-phosphate dehydrogenase was monitored as a control. Figure 13 demonstrates that the adhesion and migration of HUVEC can be supported by integrin ανβ3, a CCN1 mutant. A, HUVEC, which was separated in 2.5 mM EDTA and resuspended in serum-free medium, was adhered to

5B 1356097 Μμg/ml of CCN1 wild-type CCN1 (WT) or SM, or 50 μg/ml of DM or TM coated wells. It is indicated here that the cells are EDTA (5 mM), GRGDSP peptide (RGDS, 0.2 mM), anti-ανβ3 mAb LM609 (40 μg/ml), anti-a6 mAb GoH3 (40 μg/ml) before culture. Handle for 60 minutes. The adhesion of the cells was measured as described above. B. The HUVEC migration supported by CCN1 or mutant was assessed using a Transwell chamber. 15 μg/ml of CCN1 WT or mutant SM, 50 μg/ml of mutant DM or TM was immobilized on the lower surface of the transfer disk membrane, wherein the transfer disk membrane divided the reaction chamber into two zones. HUVECs were treated with 100 nM PMA (Sigma, St. Louis, MO) for 30 minutes to activate the integrin receptor. It is indicated here that before incubation in the upper reaction chamber, the cells are buffered with vehicle (not added), normal mouse IgG (1 μg/ml), GoH3 (50 μg/ml), LM609 (50 μg/ ML) extra pre-culture for 30 minutes. The cells were allowed to migrate for 8 hours, and cells transferred to the lower reaction chamber were counted in 1 random high magnification region. The data shown is the mean ± S. D· of three measurements and is a representative of three experiments. Figure 14 demonstrates that CCN1 mutants enhance VEGF-induced DNA synthesis through integrin ανβ3. HUVECs were pre-incubated with vehicle buffer (not added), LM609 (25 μg/ml), GoH3 (25 μg/ml), or normal mouse IgG (25 μg/ml) for 1 hour. The cells were then treated with VEGF (5 μM/ml) and/or CCN1 or mutant protein (5 μg/ml each) in the presence of [3h] thymidine, and the combined effects were analyzed after 48 hours. The data shown is the average of three measurements S.D. and is a representative of three experiments. 59 1356097 Figure 15 demonstrates that CCN1 mutations promote survival of HUVECs. Serum-deficient HUVEC was attached to 20 μg/ml laminin (LN) · pre-coated coverslips for 4 hours, followed by serum, CCN1 or mutant egg-white matter (5 μg/ml each) for 16 hours. . Cells were fixed and cell death was monitored by TUMEL® analysis. Here, it is indicated that a plurality of anti-CCN1 antibodies are preincubated with the test reagent for 3 minutes before the medium is added. The data shown is the average soil S.D. of the three measurements and is a representative of three experiments. Figure 16 demonstrates that CCN1 mutants can embolize integrin ανβ3_dependent endothelial tube formation. Pre-coated in a 24-well plate pre-coated with type I collagen gel (2 mg/ml) (the coating is either CCN1 (WT) or tm (20 μg/ml each) is absent (-) or present The following was performed, HUVEC was treated with vehicle buffer (not added) or stimulated with 5 nM PM A in serum-free medium. A second layer of gel of the same formulation was applied to the attached cells, and the tube formation effect was evaluated after 16 hours. Here, LM609 (40 μg/ml) or G〇H3 (before culture) was used. Add 4 μg/ml to the cell < suspension. The result is a representative map of three individual experiments (magnification χ 2〇〇). Ω 1356097 Table I·peptide sequences used in GST-peptide fusion proteins peptides residues residues

P B I (I)

12 3 4 I Σ I I

LAI>S TCPAACHCPIiEAPKCAPGV(31<VRDCI AFOVGIiVRDGCGCCKVCAKQIiNED CKVCAXQLNEDCSRTQP KBDCSKTQPCDETKGIiSCNFGASSTA£iK6lC 21-49 40-53 53-69 61-91

C

{ID

1 2 3 4 5 6 VVVVVV CRAQSSORPCEYNSRiyQHSBSFQPK <»3Ρ〇ΡΗΟΚΒ<}(:Τ<:ΙΠ0Αν6<;ΙΡΙ^Ρ〇ΕΙι3Ιι 〇Ε8Ρς2ΡΝΟΚΗςε!Τ(!ΙΙ3αΑν〇Ο:ΙΡΙ»ΰΡ0&1ι3Ζ«ΡϊΤΣιΟ<:- PNPB NCKHQCTCIDGAVGCIPLCP ΙιβΙιΡΝΙιβΟΡΝΡΚΙινκνΒΟΟεΟΕΕ^ναΟΕΒδΙΚ Ι)ε〇8:ε£ϊ3βΣι02>(2Β:01ιΙι6Ιι1)Α8ΕνΈ:1ιΤΙΙΗΝΕ1ιΙΑΙΟΚ- 058Ι<Κ^ΡνΡβΤΕΡΚνΊ<ΡΚΡ1ΗΑΗΟ〇Κ 91-116 110-140 1X0-149 116-135 138-169 1ί4-226

P S T (III) T1T2T3T4

aQKCIVQTTSWSQCSKS SWSQCSKSCGTOISTRVTHD GXSTRVTXn)NPBCRLVKETR RL VKETRICEVRPCGQP VVS S IiK 224-240 233-252 244-263 257-279 61

Claims (1)

(Amended in August 2011) I wn , , ie§»i · 1356097 1. An isolated CCN1 fragment, wherein the fragment is selected from the group consisting of: (a) Amino acid sequence of murine CCN1 Position residues 231-240 (SEQ ID ΝΟ: 1); and (b) amino acid sequence of human CCN1, position 233-242, thiol (SEq ID ΝΟ: 1). 2. A pharmaceutical composition comprising a cCN1 fragment as claimed in claim i, and a pharmaceutically acceptable adjuvant, diluent, or carrier. 3. An antibody' which can be specifically bound to the CCN1 fragment of item i of the scope of the patent application. 4 · A variety of medical composition > including such as the application of genuine South 丨 — - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 5. Use of a pharmaceutical composition according to item 2 or 4 of the above-mentioned patent scope for the preparation of a medicament for regulating CCN1 in a patient. 6. If the application for patent application 帛5 is used, it is a drug related to neoplasia or angiogenesis, and a drug for treating a disease or symptom associated with neovascularization. The use of the drug of the second aspect of the aforementioned patent application, or the pharmaceutical composition of the Anfc, for the treatment of cancer. 1356097 i "I i·· r",., L_l /,乂·:.. □ ι:ϋ i \ Ο K Π I Λ ~ M ^— E3 ( af * 〇Μ n .· ”· rti o Mias BSA M CCNI B 5 OS V 5 0 n /1 No added ϋ □ □ I14C El (...IP □ IM (ID tel BSA CCNI domain m 1356097 Λ B {§ V - tf, — ‘ o c ~ 02 y I: mouth not I: 劾丨丨 □ ly<; □ Rt; IJS □ l.MOl)') 1IMC 10 (;ST GSI-H CCN1 /vAr λ rsi brother J Figure 1356097 s b. -4 u τι □ Τ2 E2TJ ΠΤ4 rwm BSA Rl· i i ! CCM CCN2 CCN3 β OS V 2 ϋ : .-<1 \/ n dam% 3 4 ^ rET^ F^v^]i Il 1_ 2 (9) 300 400 11 (u,i) 1356097 Section 8 1356097 A 0.5 1.6 1 1.5 2 4 μg / liter L2 [Hi!!1.11 Mi赖I Ιϊοί'λΊ'ι'^^ΛΒί' b(4) 1356097 A ο u Figure 11 1356097 A os< o □ EDTA □ RGDS LM6O9 SGoH , Jo o 6 0 o 2 o BSA WT SM DM TM B BSA WT SM DM TM 1356097 6 ΏΜώ'Χ. Anti-CCN1 BSA 20% WT SM DM TM Dish clear 15th B 1356097 ΡΜΛ. (,oH3 Figure 16