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WO2000055206A1 - Stimulation de cellules endotheliales a l'aide d'un complexe de fibronectine et de facteur de croissance endotheliale vasculaire - Google Patents

Stimulation de cellules endotheliales a l'aide d'un complexe de fibronectine et de facteur de croissance endotheliale vasculaire Download PDF

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WO2000055206A1
WO2000055206A1 PCT/US2000/007183 US0007183W WO0055206A1 WO 2000055206 A1 WO2000055206 A1 WO 2000055206A1 US 0007183 W US0007183 W US 0007183W WO 0055206 A1 WO0055206 A1 WO 0055206A1
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vegf
binding
fibronectin
growth factor
fragment
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Errol S. Wijelath
Jacqueline Murray-Wijelath
William P. Hammond
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Hope Heart Institute
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Hope Heart Institute
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention pertains to complexes containing vascular endothehal growth factor (VEGF) in association with the adhesion protein fibronectin or fragments thereof, and to methods of administering the complexes in vitro or in vivo to promote or induce endothehal cell migration, angiogenesis and wound healing.
  • VEGF vascular endothehal growth factor
  • wound healing is complex and represents a serious medical problem affecting a large number individuals.
  • healing problems occur in dermal wounds such as decubitus ulcers, severe burns and diabetic ulcers and eye lesions including dry eye and corneal ulcer, as well as surgical wounds, and other wound related pathologies.
  • One important aspect of wound healing is the migration of new cells from tissues surrounding a wound site which leads to the generation of new tissue having the proper population of cell types and tissue organization.
  • central to the wound healing process is the growth of a functional blood supply system to cells in the healing zone. Without a regular flow of blood to the wound site there can be no substantial wound healing and restoration of health tissue.
  • Patent No. 5,880,090 and in the healing of ischemic areas of the heart.
  • Angiogenesis is the process by which new blood vessels are formed from a preexisting microvascular network, and takes place within the extracellular matrix. This process is crucial for embryogenesis or wound healing, as well as for supporting the growth of solid tumors. Steps involved in the angiogenic process include the proteolytic remodeling of local vascular basement membrane and extracellular matrix, migration of endothehal cells into the matrix, endothehal cell proliferation, and the formation of tubular capillaries and a new basement membrane. In contrast, vasculogenesis involves the formation of new blood vessels by the in situ differentiation of mesodermal precursors to endothehal cells. Both are complex processes that involve coordinated regulation of endothehal cell proliferation, migration and differentiation (Risau and Flamme, Ann. Rev. Cell Dev. Biol. 11:73-91 (1995); Folkman and Shing, J. Biol. Chem. 267: 10931-10934 (1992)).
  • VEGF vascular endothehal growth factor
  • FN fibronectin
  • VEGF has been shown to play a major role in vasculogenesis and angiogenesis by gene deletion studies (Ferrara et al, Nature 380:439-442 (1996); Carmeliet, et al., Nature 380:435-439 (1996)).
  • Targeted disruption of the gene encoding VEGF receptor FLK-1 in mice resulted in failure of blood-island formation and endothehal differentiation (Shalaby et al., Nature 376:62-66 (1995); Shalaby et al., Cell 89:981-990 (1997)).
  • deletion of the FLK-1 kinase domain resulted in impaired vasculogenesis and angiogenesis (Hiratsuka, et al., Proc.
  • FLK-1 is also the first endothehal receptor tyrosine kinase to be expressed in the hemangioblast (Choi et al., Development 125:725-732 (1998); Yamaguchi et al., Development 118:489-498 (1993)).
  • ECM-integrin interactions not only mediate cell adhesion but can directly activate specific signalling pathways or integrate with growth factor induced signals to potentiate cellular processes (Miyamoto et al., J. Cell. Biol. 135: 1633-1642 (1996); Lee et al, J Biol. Chem. 274:22401-22408 (1999); Eliceiri et al., J. Cell. Biol. 140:1255-1263 (1998)). Recent studies suggest that clustering of integrin and growth factor receptors is one potential mechanism through which signals integrate to amplify cellular processes (Jones et al., J. Cell. Biol.
  • Fibronectin exists as a soluble dimer, each subunit being a mosaic of repeating modules having a molecular weight of about 230 kDa. In its soluble state, it is nonreactive with adhesion receptors, but is highly reactive after the soluble form has polymerized onto specialized areas on cell surfaces or intracellular matrix. Fibronectin is believed to contribute to capillary formation both by supporting adhesion of endothehal cells that have migrated to the matrix, and by inducing the expression of other adhesion molecules needed for microtubule assembly (see, e.g., Nehls and Drenckhahn, Microvasc. Res. 50:311-322 (1995)).
  • Nehls and Drenckhahn (1995) describe an assay for quantifying endothehal cell migration and angiogenesis. This assay involves seeding endothehal cells on gelatin-coated microcarrier beads, then suspending the beads in a solution of fibrinogen. The fibrinogen is induced to polymerize, thereby entrapping the microcarriers in a three-dimensional fibrin matrix. These investigators observed that in response to either fibronectin, basic fibroblast growth factor or VEGF, cells migrated out of the matrix to form capillarylike structures.
  • VEGF vascular endothelial growth factor
  • Vuori et al. did not establish whether the synergistic effects of the PDGF/vitronectin composition was due to the direct binding of the two molecules into a single complex prior to binding of vitronectin to an integrin cell surface receptor.
  • Vuori et al. did not extend their studies to include a determination of whether other growth factors and extracellular matrix protein compositions, such as vascular endothehal growth factor and fibronectin might have effects on physiologically important cell migration events such as angiogenesis.
  • compositions and methods for both inducing and inhibiting angiogenesis are made available.
  • compositions effective in stimulating cell migration are isolated complexes containing fibronectin (FN) and bound vascular endothehal growth factor binding domain (VEGF).
  • the cell stimulatory composition comprises a peptide fragment of FN containing at least one VEGF binding site fused to a peptide encoding an arginine-glycine-aspartic acid (RGD) amino acid integrin binding domain, and bound VEGF.
  • the FN component of the composition usually contains an amino terminal 70 kDa peptide of FN.
  • the FN component of the fusion peptide may have a carboxy terminal 40 kDa peptide of FN.
  • the FN portion of the composition contains a fibronectin/vitronectin chimeric protein that is capable of stimulating cell migration.
  • the FN portion of the chimeric protein is capable of forming tight associations with VEGF and the vitronectin (VN) portion is capable of binding platelet derived growth factor-BB (PDGF-BB).
  • the chimeric FN/VN protein is preferably fused to a RGD integrin binding domain and bound with both VEGF and PDGF-BB.
  • the matrix provides a physical support for cell migration and angiogenesis, as well as providing a means to deliver the fibronectin/VEGF or fibronectin/VEGF/vitronectin/PDGF compositions to sites in a human or animal in need of wound healing and angiogenesis.
  • a matrix is fibrin glue, which when containing fibronectin/VEGF or fibronectin/VEGF/vitronectin/PDGF compositions may be applied to vascular grafts or other wound sites, such as burns, ulcers, and surgical wounds to faciliate healing.
  • these compositions can be used to induce angiogenesis at ischemic areas of the heart.
  • Fibronectin compositions are also provided which lack a RGD integrin binding domain but retain the capacity to tightly bind to VEGF.
  • the FN component of the composition contains an amino terminal 70 kDa peptide of FN.
  • the FN component of the composition contains a carboxy terminal 40 kDa peptide of FN.
  • inventive chimeric proteins are provided which contain fibronectin and vitronectin fragments lacking a RGD integrin binding domain, yet are still capable of binding tightly to VEGF and PDGF.
  • fibronectin and fibronectin/vitronectin compositions that bind VEGF, yet do not bind to integrin are useful as inhibitors of the physiological responses induced by VEGF and PDGF, in particular, angiogenesis, cell migration and cell differentiation.
  • a chimeric polypeptide in which a VEGF- binding peptide of fibronectin is covalently joined to one end of a linker peptide that includes a RGD peptide sequence capable of binding to an integrin receptor.
  • exemplary VEGF-binding peptides of FN are an amino terminal 70 kDa peptide and a carboxy terminal 40 kDa peptide.
  • the chimeric polypeptide additionally comprises the platelet-derived growth factor (PDGF)-BB binding fragment of vitronectin, which is joined to the carboxy-terminus of the RGD integrin-binding domain by additional amino acids flanking the integrin- binding region.
  • PDGF platelet-derived growth factor
  • the second chimeric protein is capable of binding to an integrin cell surface receptor protein as well as to both VEGF and PDGF-BB.
  • the peptide sequences specifying the VEGF- binding fragment of fibronectin/integrin binding domain fusion and the VEGF- binding fragment of fibronectin/integrin binding domain/PDGF-BB binding fragment of vitronectin fusion are repeated multiple times.
  • the above described chimeric proteins when bound to VEGF and PDGF-BB may also be incorporated into fibrin glue or other matrix formulations to facilitate deliver of these compositions to sites in need of healing or stimulation of angiogenesis.
  • RNA fragments from the amino or carboxy termini of fibronectin that contain VEGF binding regions and fibronectin/vitronectin fusion proteins that contain both VEGF and PDGF binding regions but do not contain a RGD integrin binding domain can be applied, for example, to inhibit endothehal cells from migrating or to inhibit cells from differentiating into endothehal cells.
  • the inhibitor FN peptide is a carboxy terminal 40 kDa peptide fragment.
  • the method uses an amino terminal FN fragment of 70 kDa.
  • a method for inhibiting VEGF receptor function is provided.
  • First a VEGF/fibronectin fragment complex is formed by contacting a carboxy terminal fragment of fibronectin with VEGF.
  • the FN fragment of the complex lacks a RGD integrin binding domain.
  • the carboxy terminal fragment is about 40 kDa in size.
  • the VEGF/fibronectin fragment complex is the contacted with a VEGF receptor to inhibit VEGF function.
  • Usual VEGF receptors are VEGFR-1 and VEGFR-2. Inhibition of VEGF receptor function prevents binding of VEGF to the receptor and the signaling of cell migration. In addition, inhibition of VEGF receptor function prevents VEGF mediated induction of MAPK kinase activity.
  • Agents capable of blocking the formation or activity in vivo of fibronectin/VEGF complexes and vitronectin PDGF complexes and or their association to cognate receptors can be used to inhibit the migration of cells as a means of inhibiting or preventing angiogenesis in tumors or to inhibit the formation of scar tissue or other undesired cell migration.
  • Such blocking agent include peptides that mimic the protein domains in fibronectin or VEGF and vitronectin or PDGF that mediate their respective associations, including synthetically modified peptides.
  • FIGURE 1 illustrates microvessel endothehal cell (MVEC) adhesion to each of several matrix proteins when cultured in the presence of 10 ng/ml VEGF.
  • MVEC microvessel endothehal cell
  • FIGURE 2 compares the effects on MVEC migration of several different adhesion proteins when tested alone or together with VEGF.
  • FIGURE 3 graphically illustrates measurements of MVEC migration in the presence of various concentrations of VEGF with or without the simultaneous addition of fibronectin to the medium.
  • FIGURE 4 A shows a diagrammatic representation of the fibronectin molecule (not to scale), with the text above the diagram denoting various binding regions, including regions that bind to the indicated matrix proteins.
  • FIGURE 4B illustrates the capacity of various fibronectin fragments to bind with VEGF in a filter-binding assay.
  • FIGURE 5 graphically presents SPR analysis of VEGF binding to the N-terminal 70 kDa FN fragment. Representative trace shows that maximal VEGF binding to the 70 kDa N-terminal FN fragment was achieved within 1 minute.
  • FIGURE 6A graphically illustrates the effect of VEGF/ECM protein complexes on endothehal cell migration. Data are presented as mean ⁇ standard error of the mean.
  • FIGURE 6B graphically illustrates the effect of ECM protein and VEGF on hematopopoietic CD34 + cell differentiation into endothehal cells.
  • FIGURE 7 graphically illustrates the effects of VEGF/FN (closed circles), VEGF/120 kDa FN fragment (triangles) and VEGF/vitronection (open circles) on MAPK activity.
  • FIGURE 8A graphically illustrates inhibition of VEGF-induced endothehal cell migration in the presence or absence of the amino terminal FN 70 KDa arid carboxy terminal FN 40 kDa fragments. Data are represented as the mean ⁇ standard error of the mean.
  • FIGURE 8B graphically illustrates FN fragment inhibition of 125 I-VEGF binding to VEGFR-1. Data are represented as the mean ⁇ standard error of the mean.
  • FIGURE 9 graphically illustrates measurements of MVEC migration in the presence of various fibrin glue compositions with or without fibronectin and VEGF.
  • A represents fibrin glue minus fibronectin
  • B represents fibrin glue minus fibronectin plus VEGF (50 ng/ml)
  • C represents fibrin glue plus fibronectin and VEGF (50 ng/ml);
  • compositions containing a complex formed between VEGF and an adhesion protein such as fibronectin, laminin or fibrin. It is shown here that an affinity exists between VEGF and fibronectin, such that when the two proteins are mixed together in solution, they form a tight complex that retains the physiological activity of VEGF.
  • This VEGF/FN complex is capable of stimulating the migration of vascular endothehal cells to a greater extent than when the cells are exposed to VEGF alone.
  • the effects of VEGF on endothehal cell migration can be enhanced by mixing the VEGF with fibrin, laminin or a mixture of fibronectin, fibrin and laminin.
  • inventive composition of VEGF and FN induces differentiation of CD34 + hematopoietic cells into endothehal cells as compared to VEGF alone or VEGF complexes with other extracellular matrix (ECM) proteins.
  • ECM extracellular matrix
  • the present invention provides an isolated complex of VEGF and fibrinogen.
  • isolated complex formed between VEGF and FN refers to any isolated complex whereby FN and VEGF are bound to each other and the complex is greater than about 50% pure. Usually the VEGF/FN complex is greater than 80% pure, and more often greater than 95% pure.
  • inventive isolated VEGF/VN complex can also be incorporated into a fibrin glue.
  • a fibrin glue is a biological adhesive consisting of highly concentrated human fibrinogen, thrombin and factor VIII (see, for example, Suzuki et al., Arch. Surg. 130:952-55 (1995)).
  • the fibrinogen is mixed with the other two components during the application of the glue, so that fibrin forms in situ upon exposure to the thrombin and factor VIII.
  • Additional biologically active molecules such as the VEGF complexes of the present invention, can be incorporated into such a glue. This mode of use will ensure the slow and continuous release of the VEGF complexes at the site of glue application.
  • the fibrin glue also provides an easy to use delivery vehicle for directing the inventive compositions to sites in need of wound healing or angiogenesis. Yet a further advantage of the fibrin glue compositions is that the glue matrices provide a temporary support structure onto which migrating cells can attach themselves.
  • vascular endothelial growth factor/adhesion protein complexes of the present invention may also be incorporated into other types of matrices other than fibrin glue, including, but not limited to matrices made from hyaluronic acid, chondroitin sulphate, agarose and collagen.
  • matrix refers to any biocompatible solid or non-solid support which functions as a scaffold for tissue repair or angiogenesis and is complexed or conjugated with fibronectin and VEGF.
  • Suitable matrix materials for use in the practice of the invention include, for example, hyaluronic acid, chondroitin sulfate, heparin, heparin sulfate, polylactate, polyglycolic acid, starch and collagen.
  • Effective concentrations of fibronectin and VEGF for incorporation into a matrix will vary depending upon the composition of the matrix and the particular purpose. Such concentrations may be determined using assays and techniques disclosed herein as well as other assays and methods that are well known in the art. For example, a variety of matrices can be assembled, each containing a different ratio of fibronectin to VEGF. Each combination is then evaluated for its ability to support cell attachment and migration using in vitro and in vivo assays.
  • the biologically active molecules of the present invention will be present in the above matrix compositions in a concentration range of about 50 ng/ml to about 1 mg/ml.
  • chimeric polypeptides containing a peptide fragment of fibronectin which is capable of binding to VEGF that is covalently joined at its carboxyl terminus to a RGD peptide capable of binding to an integrin receptor are disclosed herein.
  • An additional chimeric polypeptide contains the fibronectin/integrin receptor binding site fusion but further includes a peptide fragment of vitronectin which is capable of binding to platelet-derived growth factor-BB (PDGF-BB) that is covalently joined at its amino terminus to said fibronectin/integrin receptor binding site fusion protein.
  • PDGF-BB platelet-derived growth factor-BB
  • PDGF-BB is a particular isoform of platelet-derived growth factor that has been shown to have a synergistic effect upon cell migration and wound healing when combined with vitronectin, an integrin ligand (See U.S. Patent No. 5,654,267).
  • inventive chimeric polypeptides in some embodiments of the invention, also include an arginine-glycine-aspartic acid (RGD) amino acid sequence capable of binding to an integrin receptor.
  • RGD arginine-glycine-aspartic acid
  • the RGD sequence has been shown to be essential for binding to ⁇ ⁇ integrin and other integrin molecules. Pierschbacher et al.
  • a chimeric peptide of the present invention that is useful in stimulating cell migration and cell differentiation includes a RGD integrin binding peptide and is attached covalently at one end to a VEGF-binding fragment derived from fibronectin.
  • the chimeric peptides are composed of a 70 kDa fragment from the amino terminus of FN or a 40 kDa fragment from the carboxy terminus of FN.
  • the chimeric protein additionally comprises a PDGF-BB binding region of vitronectin that is covalently joined to the VEGF-binding fragment of fibronectin and the RGD integrin binding peptide sequence.
  • Chimeric molecules may include several iterations and combinations of these three motifs.
  • such chimeric peptides may contain multiple copies of each of the three binding domains.
  • the amino acids surrounding the RGD peptide may be selected such that the inventive chimeric proteins will bind to ⁇ ⁇ , ⁇ ⁇ ⁇ 3 , and a s ⁇ ⁇ or any other integrin that will promote angiogenesis.
  • inventive fibronectin/vitronectin chimeric proteins bound to VEGF and PDGF can also be incorporated into fibrin flue and other types of matrices in order to facilitate delivery of the compositions to tissue sites in need of stimulation of cell migration and angiogenesis.
  • a method for promoting angiogenesis whereby cells are contacted with a composition comprising an isolated complex formed between VEGF and fibronectin.
  • the above described chimeric proteins having a RGD integrin binding site can also be used to practice the inventive method.
  • the angiogenesis promoting composition further contains a matrix such as fibrin glue.
  • the subject invention further provides a method of inhibiting the physiological responses of vascular endothehal cells to vascular endothehal growth factor and/or platelet derived growth factor.
  • cells are contacted with a portion or fragment of the fibronectin molecule that binds VEGF but is lacking the RGD amino acid domain necessary for binding to an integrin receptor.
  • LDV integrin binding site does not affect the inhibitory activity of the VEGF/FN fragement complex.
  • endothehal cells are exposed to a portion or fragment of the vitronectin molecule that contains a PDGF-BB-binding domain capable of binding PDGF-FF but is lacking the RGD integrin binding domain.
  • the two growth factor-binding domains, both lacking their respective RGD integrin receptor binding regions, are fused together to form a chimeric protein capable of binding to both VEGF and PDGF-BB.
  • a method of inhibiting VEGF receptor function is provided.
  • First a VEGF/fibronectin fragment complex is formed by contacting a carboxy terminal fragment of fibronectin with VEGF.
  • the carboxy terminal fragment is about 40 kDa in size.
  • the VEGF/fibronectin fragment complex is the contacted with a VEGF receptor to inhibit VEGF function.
  • Usual VEGF receptors are VEGFR-1 and VEGFR-2. Inhibition of VEGF receptor function prevents binding of VEGF to the receptor and the signaling of cell migration. In addition, inhibition of VEGF receptor function prevents VEGF mediated induction of MAPK kinase activity.
  • VEGF receptors typically are class III receptor-type tyrosine kinases characterized by having several, typically 5 or 7, immunoglobulin-like loops in their amino-terminal extracellular receptor ligand-binding domains (Kaipainen et al., J. Exp. Med. 178:2077-2088 (1993)).
  • VEGF receptors include FLT-1 homolog from human (Flt-1 homolog gene sequenced by Shibuya et al. (Oncogene 5:519-524 (1990)); KDR, described in PCT/US92/01300, filed Feb. 20, 1992, and in Terman et al. (Oncogene 6: 1677-1683 (1991)); and FLK-1 (murine Flk-1 gene sequenced by Matthews et al. (Proc. Natl. Acad. Sci. 88:9026-9030 (1991)).
  • the KDR receptor from humans is also known as VEGFR-1 (Neufeld et al. FASEB J. 13:9-22 (1999)).
  • VEGF receptor refers to any mammalian protein having substantially the same amino acid sequence as VEGFR-1, VEGFR-2, FLK-1 and FLT-1. "Substantially the same” amino acid sequence is defined herein as a sequence with at least 70%, preferably at least about 80%, and more preferably at least about 90% homology to another amino acid sequence, as determined by the FASTA search method in accordance with Pearson and Lipman ((1988) Proc. Natl. Acad. Sci. 85:2444-2448).
  • VEGFR-1 is a human form of a VEGF receptor having MW 180 kDa.
  • FLK-1 is the murine homolog of VEGFR-1.
  • FLT-1 an additional murine form of VEGF receptor different from, but related to, the VEGFR-l/FLK-1 receptor.
  • the human homolog to the murine FLT-1 receptor is VEGFR-2.
  • Other VEGF receptors that can be inhibited by certain compositions of the present invention include those that can be cross-link labeled with VEGF, or that can be co-immunoprecipitated with VEGFR-1 (MW 180 kDa).
  • VEGF receptors have molecular weights of approximately 170 kDa, 150 kDa, 130-135 kDa, 120-125 kDa and 85 kDa. See, for example, Quinn et al. (Proc. Nat. Acad. Sci 90:7533-7537 (1993); Scher et al. (J. Biol. Chem. 271 :5761-5767 (1996)).
  • the VEGF receptor is usually bound to a cell, such as an endothehal cell.
  • the VEGF receptor may also be bound to a non-endothelial cell, such as a tumor cell.
  • the VEGF receptor may be free from the cell, preferably in soluble form.
  • All of the physiologically inhibitory fibronectin fragments, vitronectin fragments and FN/VN chimeric fragment proteins are capable of binding to VEGF, PDGF-BB or both VEGF and PDGF-BB molecules respectively, thereby preventing the VEGF and/or PDGF-BB molecules from binding to their biologically active receptors on the cell surface and initiating their cognate biological responses.
  • all of the inhibitory VN and VN/PDGF-BB proteins lack a RGD integrin binding site.
  • a carboxy terminal VEGF binding domain of FN is provided which contains a VEGF binding domain capable of binding to VEGF but does not contain a RGD integrin binding domain.
  • the carboxy terminal fibronectin fragment used as an inhibitor of VEGF responses has a molecular weight of about 40 kDa.
  • the fibronectin and vitronectin fragments can be used to inhibit endothehal cell migration and proliferation.
  • Example 1 Adhesion of MVECs to Various Matrix Proteins
  • 48-well plates were precoated with various matrix proteins by adding 250 ⁇ l of phosphate buffered saline (PBS) containing 2 ⁇ g/ml of each test protein, then incubating overnight at 4°C. Prior to the experiments, the PBS and test protein solution was removed from the plates, and the plates were washed with 500 ⁇ l of MCDB 131 culture medium obtained from Clonetics (San Diego, CA).
  • PBS phosphate buffered saline
  • MVECs microvessel endothehal cells
  • FBS fetal bovine serum
  • Example 2 Stimulation of MVEC Migration by VEGF and Matrix Proteins Experiments were conducted to determine the capacity of various proteins, including several extracellular matrix proteins, to induce the migration of MVECs in the presence of 10 ng/ml of VEGF. Cells were isolated and cultured as described in Example 1. To address this question, the chemotactic activity of MVECs was measured in Transwell migration assays in the presence of VEGF and one of the following proteins: albumin; fibronectin; vitronectin; fibrin; laminin; collagen I; or collagen IV.
  • Transwells Costar Corp., Cambridge, MA
  • Transwells When the Transwell is inserted into a well, cells in the upper compartment can be induced to migrate through the membrane by chemo-attractants present in the lower chamber.
  • Transwells used in these assays had 8 ⁇ m pore membranes.
  • MVECs Primary cultures of MVECs were isolated as described in Example 1. Monolayers of cultured MVECs were trypsinized, then washed twice in MCDB 131 containing 0.25% BSA (assay medium), and resuspended at lxlO 6 cells/ml in assay medium.
  • BSA assay medium
  • Each of the proteins to be tested (0.25 ml of 2 ⁇ g/ml in assay medium) was added to the wells of 24-well tissue culture plates (microwells), and incubated for 20 minutes at 37°C. Control wells received 0.25 ml of the same assay solution without the test protein. A solution containing VEGF (0.5 ml of a 10 ng/ml solution) was then added to the wells, and incubated for a further 10 minutes to permit the spontaneous formation of protein/VEGF complexes. One hundred ⁇ l of the MVEC suspension (at lxl 0 6 cells/ml) were added to each Transwell, and the Transwells were inserted into the microwells. Cell migration was measured after 6 hours of incubation at 37°C.
  • the non-migrant cells were removed from the upper face of the Transwell membrane with a cotton swab, and the migrant cells, i.e., those attached to the lower face of the membrane, were fixed and stained with 0.1% crystal violet.
  • Dye was eluted with 10%> acetic acid, and the numbers of cells were quantified by reading the absorbence at 600 nm.
  • FIGURE 2 shows that when fibrin, laminin or fibronectin was present together with VEGF in the lower chamber, cell migration was stimulated to a degree significantly higher than was observed when these proteins or VEGF were added alone.
  • fibronectin was used together with VEGF, the amount of migration observed exceeded the amount that would have been expected if the effects of the different molecules were merely additive (see FIGURE 2) This observation suggests that VEGF acts synergistically with fibronectin in stimulating cell migration
  • fibronectin The ability of fibronectin to stimulate cell migration was investigated further by determining whether the combined effects of fibronectin and VEGF on MVEC migration was dependent on VEGF concentration
  • VEGF concentration The results of a dose-response test, graphically illustrated in FIGURE 3, showed that in the presence of a fixed concentration of fibronectin (2 ⁇ g/ml), the amount of cell migration increased with increasing VEGF concentration up to a VEGF concentration of about 50-60 ng/ml This result suggests that the enhanced cell migration observed in the presence of fibronectin and VEGF may result from the formation of a specific complex
  • Example 3 Binding of VEGF to Extracellular Matrix Proteins
  • the proteins tested were fibronectin, vitronectin, laminin, fibrinogen, fibrin, collagen I, collagen IV and collagen IV
  • To prepare the nitrocellulose membrane 5 micrograms of each protein were loaded under vacuum onto nitrocellulose membranes using a slot-blot apparatus The membranes were then incubated for one hour at room temperature with a solution of 20 ng/ml VEGF in PBS After the binding step, the membranes were washed with PBS to remove unbound VEGF, and bound VEGF was quantified by incubating the filters with antibody against VEGF
  • Example 4 Binding of PDGF-BB to Extracellular Matrix Proteins
  • plasma proteins were tested for their ability to bind PDGF-BB in the same fashion as described in Example 3 for VEGF. Proteins tested were vitronectin (as a positive control) plasminogen, fibronectin, fibrinogen, and serum albumin. Five micrograms of each protein were loaded under vacuum onto nitrocellulose membranes using a slot-blot apparatus. The membranes were then incubated for one hour at room temperature with radiolabeled PDGF-BB (NEN Life Science, Boston, MA) in PBS.
  • FIGURE 4 A presents a diagrammatic representation of the fibronectin protein molecule (not to scale), with the various protein domains denoted, including regions that bind to the cellular matrix proteins.
  • the fibronectin fragments that were tested for their capacity to bind to VEGF are illustrated in FIGURE 4B, and were obtained from Sigma (St. Louis, MO) or Life Technologies (Grand Island, NY). Each fragment was bound to a nitrocellulose membrane as described above in Example 3.
  • the relative binding observed with each fragment is shown in FIGURE 4B, using a minus sign or one or more plus signs to indicate the degree of binding that was observed.
  • the strongest VEGF binding was observed using a carboxy terminal 40 kDa fragment.
  • An amino terminal 70 kDa fragment also exhibited strong binding to VEGF.
  • Two smaller fragments derived from the amino terminal 70 kDa fragment also bound VEGF, though not as strongly.
  • the 120 kDa fragment from the middle portion of fibronectin exhibited the least amount of binding. From these results, it appears that their are two VEGF-binding domains located in fibronectin.
  • One VEGF binding domain is located near the amino terminus of the molecule, and is entirely contained within the 70 kDa fragment shown in FIGURE 4B.
  • the other VEGF binding domain is located on the carboxy terminal portion of fibronectin.
  • FN fragments were coupled to CM5 dextran chips by amine coupling chemistry according to Biacore Inc. instructions.
  • the reference cell serving as a control
  • the reference cell had immobilized mouse IgG, and all binding curves were subtracted for non-specific binding to that reference.
  • VEGF was used at two different concentrations (1.3 ⁇ M and 2.6 ⁇ M). Using the SPR method VEGF was found to bind to the 70 kDa peptide (FIGURE 5). Binding was maximal within 1 minute. Similarly, VEGF also bound to the carboxy terminal 40 kDa peptide. As expected, VEGF binding was not observed with the 120 kDa peptide (data not shown).
  • Washed platelets were prepared as described in Patel et al. (Thromb Haemost 79:177-185 (1998)). For activation studies, platelets were resuspended in the presence of 1.5 mM calcium and at a count of 30 xlO /ml. One ml of platelets were stimulated with either saline (resting) or thrombin (1 U/ml) for 10 min. Whole cell lysates were also prepared from unstimulated platelets by lysing them with immunoprecipitation buffer. Supernatants were immunoprecipitated with an antibody to FN (Chemicon International Inc., Temecula, CA). Following SDS- PAGE and immunoblotting, VEGF was detected with a polyclonal antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA) by chemiluminescence. Immunocytochemical labelling of platelets.
  • Washed human platelets treated with 1 ⁇ M PGEi were fixed on slides with 4% (v/v) paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 10 min and permeabilised with 0.01% (v/v) triton X-100 for 5 min. Platelets were incubated with antibodies to FN (Chemicon) and VEGF. For positive identification of ⁇ - granules, an antibody to P-selectin (CD62P, Chemicon International Inc.) was used. Secondary antibodies used were goat anti-rabbit or goat anti-mouse IgG conjugated to fluorescein or Cy3 respectively (Chemicon). Labelled platelets were then mounted in fluoromount (BDH) or PBS/glycerol and visualised using a Nikon UFX- DX confocal microscope. Results Confocal microscopic analysis of resting platelets demonstrated that both
  • VEGF and FN co-localised in the ⁇ -granules as confirmed by P-selectin staining (data not shown).
  • whole cell lysates and supernatants of resting and activated platelets were examined for the presence of VEGF/FN complexes.
  • Immunoprecipitation of supernatants from activated platelets with antibodies to FN resulted in significant co-precipitation of VEGF compared to resting platelets (data not shown).
  • a minor amount of co-precipitation of VEGF from the resting supernatant was observed which was due to low level platelet activation during the isolation procedure.
  • VEGF was not immunoprecipitated with an antibody to FN. These results indicate that platelet activation is necessary for VEGF/FN complex formation and demonstrate that activated platelets are a potential source of VEGF/FN complexes.
  • Example 7 Identifying the Vitronectin Domain that Mediates Binding to PDGF-BB
  • the T7SelectTM Phage System (from Novagen Inc., Madison, WI) is used to determine the minimal VN amino acid sequence that will support high affinity PDGF-BB binding.
  • the T7SelectTM Phage System is a clone selection technique in which a foreign peptide is expressed as a fusion with a bacteriophage coat protein. Peptides up to 50 amino acids long are displayed in high copy numbers on the surface of the phage (about 400 polypeptides per phage).
  • a biopanning procedure using microtiter plates coated with PDGF-BB is used to select phage displaying the VN peptide that binds to PDGF-BB.
  • Phage binding to PDGF-BB are eluted, amplified, and taken through additional cycles and amplification to successively enrich the pool of phage in favor of high affinity binding to PDGF-BB.
  • individual clones are characterized by DNA sequencing.
  • a series of deletions is then created in the VN peptide to determine the minimum DNA sequence that will support PDGF-BB binding. Each deletion is tested for the capacity to bind to PDGF-BB using a slot-blot and solid phase assay methods described in more detail below.
  • PDGF-BB binding site on VN This method is described in more detail in the Novagen, Inc., T7SelectTM System Manual which is herein incorporated by reference Briefly, this system consist of three parts 1) Cloning of peptide coding sequences into T7 vectors, packaging the recombinant molecules into phage, and amplifying the phage to prepare for biopanning, 2) Performing several rounds of biopanning and then amplifying the phage that bind to the target PDGF-BB, 3) Characterization of phage that bind PDGF-BB by DNA sequencing
  • the VN cDNA is well characterized and numerous binding sites for a number of molecules have been determined
  • the DNA sequences coding for between 20-45 amino acids over the entire VN cDNA sequence are amplified using PCR primers
  • the 20-45 amino acid regions are amplified such that each peptide coding region overlaps with adjacently amplified coding regions
  • PCR primer pairs used for the DNA amplification of each 20-45 peptide region of VN are designed such that the 5' end of one PCR primer has an EcoRI restriction endonuclease site, while the other PCR primer has an Hind III restriction endonuclease site at its 5' terminus
  • DNA fragments are digested with EcoRI and Hind III and then covalently joined to the EcoRI and Hind III arms of the T7 bacteriophage vector using DNA ligase Ligation reactions are added to T7 packaging extracts for in vitro packaging An aliquot of in vitro packaged phage is diluted and plated onto bacteria to determine the number
  • peptides are custom made and tested for PDGF-BB binding capacity using the slot blot and solid phase binding assays described below Slot-blot assay: Slot-blot binding assays are performed using nitrocellulose membranes (Life Technologies, Grand Island, NY) pre- wetted in PBS in a slot blot apparatus (Hoeffer Scientific Instruments, Piscataway, NJ) under vacuum. 500 ng of the test VN peptide is made up in PBS to a final volume of 250 ml then applied to the membrane under vacuum. After application, the membrane is rinsed with distilled water.
  • the membranes are incubated with 50 ml of a 2% BS A/PBS solution at 37°C for one hour with shaking.
  • the nitrocellulose membranes are then incubated with PDGF BB (20 ng/ml) in PBS containing 0.1% BSA at 37°C for one hour with shaking.
  • the membrane is incubated with a monoclonal antibody to PDGF-BB (from R & D System, Minneapolis, MN) in 1% BS A/PBS for 30 minutes at 37°C with shaking followed by incubation with goat anti-mouse IgG conjugated to horseradish peroxidase (Bio-Rad Laboratories, Inc., Hercules, CA) in 1% BSA/TBS for 20 minutes at 37°C with shaking. Following washing, the nitrocellulose membrane is developed using Super Signal chemiluminescent reagent (Bio-Rad Laboratories, Inc.). Control peptides consist of non-binding PDGF-BB peptides.
  • Solid-phase binding assay Microtiter plates are coated with PDGF-BB binding peptides (50 ⁇ l; 1 ⁇ g/ml) in 0.1 M bicarbonate buffer (pH 9) overnight at
  • the microtitre plates is blocked with 20 mM Hepes (pH 7.4) containing 0.137 mM NaCl, 1 mM MgCl 2 and 0.1% BSA (binding buffer) at 37°C for 30 minutes.
  • 2 ng of 1 5 I-PDGF-BB (NEN Life Science, Boston, MA) in binding buffer containing 1 ⁇ g of unlabeled PDGF is added to the microtitre plates and incubated for one hour at room temperature. After washing the plates, they are incubated with 0.1M NaOH for 30 minutes and the amount of bound 125 I-PDGF-BB determined by using a gamma counter.
  • the specificity of binding is determined by performing control experiments with 2 ng of l25 I-PDGF-BB in a binding buffer containing 500 ng of unlabeled PDGF-BB.
  • the peptides containing a PDGF-BB binding domain are tested for their ability to inhibit the migration-enhancing activity exhibited by the vitronectin/PDGF complexes in the Transwell assay.
  • the cell migration assay of Example 2 is used to perform experiment in which varying amounts of the VN-PDGF-BB binding domain peptide is mixed with vitronectin in the microwells prior to addition of PDGF-BB. By preventing the formation of vitronectin/PDGF complexes, the binding domain peptide abolishes in a dose-dependent manner the enhanced cell migration seen in the Transwell experiments of Example 2.
  • fibronectin protein capable of physiologically inhibiting the cell migration inducing activity of VEGF and PDGF the following assay is performed.
  • Human microvessel endothehal cells are cultured in 24-well plates until confluent in MCDB 131 medium containing 5% FBS. After washing the cells twice with PBS, the cells are incubated in sodium bicarbonate free-MCDB 131 containing 10 mM Hepes buffer and 0.5% BSA with 1 ng of 125 I-VEGF or 1 5 I-PDGF-BB (purchased from NEN Life Science, Boston, MA) in the presence and absence of the fibronectin or vitronectin fragments to be tested for inhibition of VEGF or PDGF-BB binding to cells.
  • the binding assay sample is incubated for one hour on ice.
  • the cells are then washed with PBS to remove unbound 125 I-growth factor.
  • the cells are then treated with 0.1 M sodium hydroxide and the amount of radioactive growth factor bound to the cells determined by use of a gamma radiation counter.
  • FN and VN fragments that prevent the binding of VEGF or PDGF, respectively, to microvessel endothehal cells are suitable as a cell migration inhibitory substance.
  • FIGURE 6A shows the effects of VEGF/ECM protein complexes on endothehal cell migration.
  • HMVEC cells plated onto vitronectin coated transwells were exposed to BSA, VEGF, or VEGF with ECM proteins or FN peptides. The number migrating cells were terminal after 6 hours. Endothehal cells that were exposed to VEGF/FN complexes exhibited a synergistic effect on cell migration.
  • VEGF/FN complexes increasing cell migration by more than 2.5 fold compared to VEGF/collagen I or VEGF/vitronectin combinations (FIGURE 6A).
  • endothehal cells were exposed to VEGF and the FN 120 kDa peptide, endothehal migration was similar to that observed with VEGF alone.
  • Human foetal liver CD34 + cells (Poietic Technologies Inc., Gaithersburg, MD) were then seeded (1x10 cells) in MCDB-131 medium supplemented with VEGF (10 ng/ml), bFGF (1 ng/ml), IGF-1 (1 ng/ml), Flt-3 ligand (10 ng/ml) and 5% foetal bovine serum. Endothehal cell colonies were identified by immunostaining for von Willebrand Factor (DAKO Corp., Carpinteria, CA) and CD31 (Pharmigen, San Diego, CA).
  • FIGURE 6B endothehal cell colonies derived from CD34 + cell differentiation were observed for all the VEGF/extra cellular matrix protein combinations tested.
  • CD34 + cells were incubated on VEGF/FN coated plates, there was more than a 5-fold increase in endothehal colonies, indicating that FN was the preferred extra cellular matrix protein. Plates coated with the VEGF/ 120 kDa peptide and VEGF were not as effective as the intact FN molecule plus VEGF at stimulating the differention of CD34 + cells into endothehal cells.
  • Example 10 Effects of VEGF/FN Complex On Integrin/VEGF Receptor Interaction
  • HMVEC Human microvessel endothehal cells
  • MCDB 131 media (Clonetics) supplemented with 0.1% BSA were plated on FN, collagen or 120 kDa internal cell binding FN fragment coated plates containing VEGF (20 ng/ml) for 1 h.
  • Cells were lysed with lysis buffer (20 mM HEPES, pH 7.5, containingl50 mM n-hexyl- ⁇ -D-glucopyranoside, 0.5% Brij 35, 0.2% NP-40, 100 mM NaCl, 5%> glycerol, 0.1% BSA and protease inhibitors) and immunoprecipitated with antibodies to ⁇ 5 ⁇ l, ⁇ v or ⁇ 2 ⁇ l integrin (Chemicon). After SDS-PAGE and immunoblotting, membranes were probed with antibodies to VEGFR-1 (Santa Cruz Biotechnology Inc.) or VEGFR-2 (R&D Systems, Minneapolis, MN). Positive protein bands were visualised by chemiluminescence.
  • lysis buffer 20 mM HEPES, pH 7.5, containingl50 mM n-hexyl- ⁇ -D-glucopyranoside, 0.5% Brij 35, 0.2% NP-40, 100 mM NaCl, 5%> glycerol
  • MAPK mitogen-activated protein kinase
  • FIGURE 7 shows that when endothehal cells were plated on VEGF/FN complexes, but not with VEGF/vitronectin or VEGF/120 kDa peptide, a sustained activation of MAPK kinase activity was observed. HMVEC lysates were assayed at the time points indicated in FIGURE 7 for MAPK activity. The data presented in FIGURE 7 demonstrate that an intact FN molecule is required to mediate the VEGF- induced VEGFR-l/ ⁇ 5 ⁇ ⁇ association and its subsequent prolonged activation of
  • Example 11 VEGF Binding Domain Fragments Inhibit VEGF Activity
  • proteolytic fragments of extracellular matric proteins and blood-derived proteins such as endostatin and angiostatin can inhibit endothehal cell migration (Ji et al., Faseb. J. 12: 1731-1738 (1998); Yamaguchi et al., EMBO J. 18:4414-4423 (1999)). Therefore, the effects of the amino terminal FN 70 kDa and the carboxy terminal 40 kDa peptides on VEGF stimulated migration of endothehal cells were tested using the methods of Example 2. Endothehal cells were exposed to VEGF (20 ng/ml) for 6 hours in the presence or absence of the carboxy terminal 40 kDa and amino terminal 70 kDa FN peptide fragments.
  • the carboxy terminal FN 40 kDa fragment was found to inhibit VEGF- induced cell migration by over 75% (FIGURE 8A). Similarly, the carboxy terminal FN 40 kDa peptide also had an inhibitory effect (27%) on CD34 + cell differentiation (data not shown). No significant effects were observed with the amino terminal FN 70 kDa fragment (FIGURE 8A).
  • FIGURE 8B shows that FN 70 kDa and 40 kDa peptides are capable of inhibiting the binding of 125 I-VEGF to VEGFR-1.
  • Recombinant VEGFR-1 immobilized on microtiter plates were incubated with 125 I-VEGF and increasing concentrations of the 70 kDa and 40 kDa FN fragments.
  • 125 I-VEGF binding to recombinant VEGFR-1 was inhibited by nearly 80%, whereas the amino terminal FN 70 kDa fragment inhibited 125 I-VEGF binding by only 20%.
  • VEGFR-1 phosphorylation experiments were performed by exposing HMVEC cells to VEGF (20ng/ml) with or without the 70 kDa and 40 kDa FN peptides (100 ng/ml) for 5 minutes. Cell lysates were then immuno precipitated with anti-phosphotyrosine antibodies followed by immunoblotting with monoclonal VEGFR-1 antibody. Phosphorylation of VEGFR-1 was greatly reduced when endothehal cells were stimulated with VEGF in the presence of the carboxy terminal FN 40 kDa peptide. In contrast, phosphorylation of VEGFR-1 was only slightly decreased when treated with VEGF and the amino terminal 70 kDa peptide (data not shown).
  • Example 12 Fibrin Glue Preparations Containing Fibronectin and VEGF Fibrin glue preparations were made by first mixing 15 mg/ml fibrinogen with
  • fibrin glue composition 0.5 mg/ml fibronectin and 8 mg/ml calcium chloride.
  • fibrinogen may be used in a range of 10 to 150 mg/ml
  • fibronectin in a range of 0.25 to 100 mg/ml
  • thrombin in a range of 20 to 500 units.
  • the biologically active molecules of the present invention may be added to the above fibrin glue mixture in a concentration range of 50 ng/ml to 1 mg/ml prior to addition of the gel initiation components.
  • Example 2 To test the effectiveness of fibrin glue compositions containing fibronectin and VEGF the cell migration assay described in Example 2 was used with the following modifications. MVECs were labeled with 3 H-thymidine for 24 hours prior to assay. The Transwells were pre-coated on the underside with fibrin glue compositions as described above. The cell migration assays were performed for 24 hours in the presence of hydroxy urea to block cell proliferation but allow cell migration. After 24 hours of incubation each fiberin glue sample was removed and solubilized in 0.1 M NaOH. The amount of cell migration and adhesion was quantified by measuring the amount of labeled 3 H-thymidine present within each solubilized glue sample in a scintillation counter.
  • the bar graph represented in FIGURE 9 graphically illustrates measurements of MVEC migration in the presence of various fibrin glue compositions with or without fibronectin and VEGF.
  • the brackets at the top of each bar in the graph shown in FIGURE 9 represent one standard deviation.
  • the results show that the fiberin glue composition containing both fibronectin and VEGF (bar C) promotes greater cell migration then do glue compositions that contain no fibronectin or VEGF (bar A) or contain only VEGF (bar B).
  • chimeric proteins of the present invention can be constructed using the polymerase chain reaction (PCR) based methods as described by Higuchi (PCR Technology: Principles and Applications for DNA Amplification, Stockton Press, New York, p. 61-70 (1989)) and Pont Kingdom (Biotechniques, 16:1010-1011
  • protein domains capable of binding to an integrin receptor should be understood to include a RGD amino acid sequences as well as the amino acid residues flanking this site that help determine whether a ligand containing a RGD integrin binding site will bind to bind to oc J j , ⁇ u ⁇ 3 , ⁇ ⁇ and or any other integrin that will promote angiogenesis.
  • FN fibronectin
  • PCR primers A and B are used to amplify a FN VEGF-binding fragment molecule that contains unique restriction endonuclease enzyme sites at both ends, and in addition, contains the coding region for an integrin receptor binding domain fused in frame with the FN VEGF-binding fragment.
  • the nucleotide sequence at the 3' end of primer A is selected such that it contains a sequence that is identical to a 15 to 30 base sequence located at the 5' end of the FN VEGF-binding fragment coding strand (See Kornblihtt et al., Proc. Natl. Acad. Sci. U.S.A. 80:32118-3222 (1983)) for the complete nucleotide sequence of fibronectin).
  • Primer A is complementary to sequences on the template strand of the FN VEGF-binding fragment.
  • the 5' end of primer A is selected such that it encodes a restriction endonuclease that is unique to the PCR amplified FN VEGF-binding fragment DNA molecule.
  • This restriction enzyme site is useful for the directional cloning of the amplified fragment into a plasmid or viral vector suitable for expressing the FN VEGF-binding fragment/integrin binding domain fusion protein within the desired host organism.
  • Suitable protein expression vectors are well known in the art of molecular biology as reflected in Hitzeman et al. (U.S. Patent No. 5,618,676) and references cited therein.
  • primer A which will function to accomplish the construction of the inventive chimeric proteins is: 5' -CAG GCT CAG CAA ATG GTT CA- 3' [SEQ ID 1].
  • Primer B is selected such that it is complementary at its 3' end to a 20 to 60 nucleotide sequence located at the 3' end of the FN VEGF-binding fragment coding strand.
  • the middle region of primer B contains nucleotide bases selected to encode the amino acid sequence for RGD.
  • the 5' end of primer B contains a restriction endonuclease site that is unique to the final PCR amplification product. Again, this added restriction enzyme site aids in the subsequent cloning of the amplified DNA fragment into a suitable expression vector.
  • primer B is: 5' -TGA GCT TGG ATA GGT CTG TGT TCA CTG AGC GCC CCT AC A CAA GTG ATA C- 3 '[SEQ ID 2].
  • PCR method can be used.
  • two different DNA products are independently amplified from two different DNA molecules containing either the coding region for the FN VEGF-binding fragment or the VN PDGF-binding fragment
  • the first product is amplified with PCR primers C and D that are complementary to nucleotide sequences flanking the coding region of the FN VEGF-binding fragment
  • the nucleotide sequence of the 3' end of primer C is selected such that it is identical in sequence to a 15 to 20 nucleotide sequence located at the 5' end of the FN VEGF-binding fragment coding strand
  • the 5' end of primer C is selected such that it encodes a restriction endonuclease that is unique to the PCR amplified FN VEGF- binding fragment DNA molecule This restriction enzyme site is useful for the directional cloning of the amplified fragment into a plasmid or viral vector suitable for expressing the FN VEGF-binding fragment/RGD integrin binding domain fusion protein within the desired host organism
  • the nucleotide sequence represented as SEQ ID 1 is one possible embodiment of primer C
  • the nucleotide sequence of primer D is selected such that it is complementary at its 31' end to a 15 to 20 nucleotide sequence located at the 3' end of the FN VEGF- binding fragment coding strand
  • the middle region of primer D contains nucleo
  • the 5' terminal sequence of primer E contains 15 to 30 nucleotides that are complementary to the 5' terminal sequence of primer D and are further selected to maintain an open reading frame with the nucleotides complementary to the N- terminal coding region of the VN PDGF-binding fragment
  • the nucleotide sequence of the 3' terminus of primer E is selected such that it has 15 to 30 nucleotides that are identical to sequences located at the 5' end of the VN PDGF-binding fragment coding strand
  • One possible embodiment of primer E is 5' -CAA GTG ACT CGC GGG GAT GTG TTC ACT ATG ATG GCA CCC CGC CCC TCC TTG AC- 3' [SEQ ID 4]
  • the nucleotide sequence of primer F is selected such that the bases near its 5' terminus encode a restriction endonuclease enzyme cutting site that is unique within the DNA sequence which encodes the final FN VEGF
  • primer F The sequence of the 3' region of primer F is selected such that it is complementary to a 15 to 20 nucleotide sequence located at the 5' end of the VN PDGF-binding fragment coding strand.
  • One possible embodiment of primer F is: 5'- CAG ATG GCC AGG AGC TGG GCA-3' [SEQ ID 5].
  • the resulting target PCR product contains the coding region of the FN VEGF-binding fragment fused in-frame with a RGD integrin receptor-binding domain, which is in turn fused in-frame with the VN PDGF-binding fragment coding sequence.
  • Unique restriction endonuclease sites are additionally contained at either end of the second PCR amplification product that can be digested with appropriate restriction endonuclease enzymes to facilitate the directional cloning of the second amplified DNA fragment into a plasmid or viral vector which is suitable for expressing the FN VEGF-binding fragment/integrin receptor binding domain/VN PDGF-binding fragment fusion protein within the desired host organism.
  • Chimeric proteins containing multiple copies of the above described fusion proteins may be made using a wide variety of gene cloning techniques that are well known in the art.
  • artisans will well appreciate the importance of constructing gene fusions encoding these multimer proteins in such fashion as to maintain a proper protein translation reading frame at each of the junctions between the DNA sequences encoding a copy of the fibronectin/integrin receptor binding site fusion protein or fibronectin/ RGD integrin receptor binding site/vitronectin fusion protein.
  • Chimera consisting of FN VEGF-binding fragment and vitronectin (VN) PDGF- binding fragment
  • a two step PCR amplification method similar in strategy to that described for the chimeric protein containing a FN VEGF-binding fragment and a VN PDGF-binding fragment both fused to a peptide sequence capable of binding to a RGD integrin receptor is used.
  • two different DNA products are independently amplified from two different DNA molecules containing either the coding region for the FN VEGF-binding fragment or the VN PDGF-binding fragment.
  • the first product is amplified with PCR primers C and G that are complementary to nucleotide sequences flanking the coding region of the FN VEGF-binding fragment.
  • the nucleotide sequence of primer G is selected such that it is complementary at its 3' end to a 15 to 20 nucleotide sequence located at the 3' end of the FN VEGF- binding fragment coding strand.
  • the 5' end of primer G contains a 10 to 20 nucleotide sequence that is complementary to the 5' end of primer H (primer H is described below).
  • primer G is: 5' -TCA TAG TGA ACA CAG TCA CTT GTG AGC TTG GAT AGG TCT GT- 3' [SEQ ID 6],
  • Primers H and F are used to amplify a second PCR product from the coding region of a VN PDGF-binding fragment coding sequence (see Jenne et al., EMBO J. 4:3153-3157 (1985)) for the complete nucleotide sequence of vitronectin).
  • the 5' terminal sequence of primer H contains 15 to 30 nucleotides that are complementary to the 5' terminal sequence of primer G and are further selected to maintain an open reading frame with the nucleotides complementary to the N- terminal coding region of the VN PDGF-binding fragment.
  • the nucleotide sequence of the 3' terminus of primer H is selected such that it has 15 to 30 nucleotides that are identical to sequences located at the 5' end of the VN PDGF-binding fragment coding strand.
  • One possible embodiment of primer H is: 5' -CAA GTG ACT GTG TTC ACT ATG ATG GCA CCC CGC CCC TCC TTG AC- 3' [SEQ ID 7].
  • the resulting target PCR product contains the coding region of the FN VEGF-binding fragment fused in-frame with the VN PDGF-binding fragment coding sequence.
  • Unique restriction endonuclease sites are additionally contained at either end of the second PCR amplification product that can be digested with appropriate restriction endonuclease enzymes to facilitate the directional cloning of the second amplified DNA fragment into a plasmid or viral vector which is suitable for expressing the FN VEGF-binding fragment/VN PDGF-binding fragment fusion protein within the desired host organism.
  • Example 14
  • the endothehal migration assay described in Example 12 and a chick embryo chorioallantoic membrane (CAM) assay are used to determine the effectiveness of the chimeric proteins in promoting angiogenesis.
  • the CAM model is frequently used to test for angiogenic and anti-angiogenic factors.
  • the assay is well known in the art and is performed as described by Dammacco et al. (Experimental Hematology, 26: 1215-1222 (1998)).
  • the chimeric proteins of the present invention are incorporated into a matrix, such as provided in Example 8, in order to facilitate the therapeutic delivery of these proteins to in vivo sites in need of stimulation or inhibition of endothehal cell migration, angiogenesis and wound healing. While the preferred embodiment of the invention has been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention.

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Abstract

La présente invention concerne des complexes isolés contenant le facteur de croissance VEGF en association avec la fibronectine de protéine d'adhésion ou de fragments de cette dernière et des procédés d'administration desdits complexes in vitro ou in vivo afin de favoriser ou d'induire la migration des cellules endothéliales, l'angiogénèse et la cicatrisation des blessures.
PCT/US2000/007183 1999-03-18 2000-03-17 Stimulation de cellules endotheliales a l'aide d'un complexe de fibronectine et de facteur de croissance endotheliale vasculaire Ceased WO2000055206A1 (fr)

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US7112654B2 (en) 2000-02-04 2006-09-26 Supratek Pharma Inc. Ligand for vascular endothelial growth factor receptor
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KR101179678B1 (ko) 2003-02-05 2012-09-04 퀸스랜드 유니버시티 오브 테크놀로지 성장인자 복합체 및 세포 이동 및 성장의 조절
US20130004543A1 (en) * 2003-02-05 2013-01-03 Queensland University Of Technology Fibronectin: growth factor chimeras
WO2004069871A1 (fr) * 2003-02-05 2004-08-19 Queensland University Of Technology Complexes a facteurs de croissance et modulation de la migration et de la croissance cellulaires
US8871709B2 (en) 2003-02-05 2014-10-28 Queensland University of Technolgy Synthetic chimeric proteins comprising epidermal growth factor and vitronectin
US9090706B2 (en) * 2003-02-05 2015-07-28 Queensland University Of Technology Fibronectin: growth factor chimeras
KR101930916B1 (ko) 2009-11-30 2018-12-19 티슈 테라피스 리미티드 피브로넥틴：성장 인자 키메라
EP2576790A4 (fr) * 2010-06-03 2014-03-19 Univ Queensland Chimères vitronectine:facteur de croissance des kératinocytes
US9862750B2 (en) 2012-06-11 2018-01-09 University Of Newcastle Upon Tyne Recombinant polypeptide
CN114557908A (zh) * 2022-03-22 2022-05-31 芜湖英特菲尔生物制品产业研究院有限公司 一种具有皮肤屏障修复功效的纤连弹性蛋白组合物冻干微芯及其制备方法
CN114557908B (zh) * 2022-03-22 2023-11-17 芜湖英特菲尔生物制品产业研究院有限公司 一种具有皮肤屏障修复功效的纤连弹性蛋白组合物冻干微芯及其制备方法

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