HK1041289B - Methods and compositions useful for modulation of angiogenesis using tyrosine kinase src - Google Patents

Description

Methods and compositions for modulating angiogenesis using tyrosine kinase SRC

Reference to related applications

This application claims the benefit of U.S. provisional application serial No. 60/087,220 filed on 29/5/1998, which is incorporated by reference as if it were all references cited herein.

Technical Field

The present invention relates generally to the field of medicine and in particular to methods and compositions for modulating tissue angiogenesis using protein tyrosine kinases Src, variants of Src, and nucleic acids encoding them.

Background

Angiogenesis is a process involving vascularization of tissue in which newly developed blood vessels grow into tissue and is also referred to as neovascularization. This process is mediated by endothelial cell and smooth muscle cell infiltration. This process is believed to proceed in any one of three ways: blood vessels grow from pre-existing blood vessels; the de novo development of blood vessels may be caused by precursor cells (angiogenesis); or the small vessels present may be enlarged in diameter. See alsoBlood et al biochemistry and biophysics *(Bioch.Biophvs.Acta)1032，89-118(1990)。

Angiogenesis is an important process in the growth of neonates, and is also important in the wound healing process and in the pathogenesis of a number of different clinical conditions including tissue inflammation, arthritis, tumor growth, diabetic retinopathy, macular degeneration caused by retinal neovascularization, and the like. These clinical manifestations associated with angiogenesis are referred to as angiogenic diseases. See Folkman et al science(Science),235: 442-447(1987). Angiogenesis is generally absent in adults or mature tissues, although it does occur during wound healing and during the corpus luteum growth cycle. See, e.g., Moses et al science(Science)，248：1408-1410(1990)。

Inhibition of angiogenesis has been proposed as a useful therapy for limiting tumor growth. It has been proposed to inhibit angiogenesis by: (1) inhibit the release of "angiogenic molecules" such as bFGF (basic fibroblast growth factor); (2) such as by using anti- β bFGF antibodies to inactivate angiogenic molecules; (3) use of vitronectin receptor alpha_vβ₃The inhibitor of (1); and (4) inhibiting the response of endothelial cells to the angiogenic stimulus. This latter strategy has received attention, and Folkman et al in cancer biology(Cancer Biology),3: 89-96(1992) several endothelial cell response inhibitors have been described, including collagenase inhibitors useful in inhibiting angiogenesis, basement membrane renewal inhibitors, static vascular steroids, angiogenesis inhibitors produced by fungi, platelet factor 4, thrombopoietin, arthritic drugs such as D-penicillamine and gold thiomalate, vitamin D₃Analogs, interferon-alpha, and the like. For additional suggested angiogenesis inhibitors, see Blood et al biochemistry and biophysics *(Bioch.Biophys.Acta)1032, 89-118 (1990); moses et al science(Science),248: 1408-1410 (1990); ingber et al laboratory researchStudy (A)(Lab.Invest.)59: 44-51 (1988); and U.S. patents 5,092,885, 5,112,946, 5,192,744, 5,202,352, 5,753,230, and 5,766,591. None of the angiogenesis inhibitors described in the above references include Src protein.

For angiogenesis to occur, endothelial cells must first degrade and pass through the vascular basement membrane in a manner similar to that used during tumor cell invasion and metastasis formation.

It has been reported that angiogenesis depends on the interaction between vascular integrins and extracellular matrix proteins. See Brook et al science(Science),264: 569-571(1994). Furthermore, it is reported that programmed cell death (apoptosis) of angiogenic vascular cells is triggered by this interaction, which is initiated by the vascular integrin α_vβ₃Certain antagonists of (a). See Brook et al cell(Cell),79: 1157-1164(1994). Further, the use of α has been reported_vβ₅The antagonist can inhibit matrix metalloproteinase-2 (MMP-2) and vitronectin receptor (alpha)_vβ₅) And thereby the enzymatic function of the protease can be inhibited. See Brook et al cell(Cell)，85：683-693(1996)。

Summary of the invention

The present invention relates to methods of modulating angiogenesis in a tissue using the tyrosine kinase Src, also referred to herein generally as Src.

The present invention concerns compositions and methods for modulating disease-associated angiogenesis in a tissue. Administering to the treated diseased tissue responsive to modulated angiogenesis a composition comprising an angiogenesis modulating amount of Src protein. Such compositions for providing Src protein may comprise a purified protein, a biologically active fragment of a protein, a recombinantly produced Src protein or protein fragment or fusion thereof, or a gene/nucleic acid expression vector for expressing Src protein.

If Src protein is inactivated or inhibited, the modulation process is inhibition of angiogenesis. If Src protein is active or activated, the modulation process is to enhance angiogenesis.

The tissue treated may be any tissue in need of modulation of angiogenesis. For angiogenesis inhibition, it is used to treat diseased tissue that develops unwanted neovascularization. Exemplary tissues include inflammatory tissues, solid tumors, metastases, tissues in which restenosis occurs, and the like.

For reinforcement, it is used to treat patients with ischemic limbs where there is poor circulation in the limb due to diabetes or other diseases. Patients with chronic wounds that do not heal and thus benefit from increased vascular cell proliferation and neovascularization may also be treated.

Particularly preferred is the use of Src proteins comprising a modified amino acid sequence as described herein. Several particularly useful modified Src proteins and their expression are described herein.

The invention also includes a pharmaceutical composition for stimulating angiogenesis in a target mammalian tissue comprising a viral or non-viral gene transfer vector comprising a nucleic acid and a pharmaceutically acceptable carrier or excipient; the nucleic acid has a nucleic acid fragment encoding a src protein with any amino acid residue other than tyrosine, serine or threonine at codon 527.

Also contemplated is a pharmaceutical composition for inhibiting angiogenesis in a target mammalian tissue comprising a viral or non-viral gene transfer vector comprising a nucleic acid and a pharmaceutically acceptable carrier or excipient; the nucleic acid has a nucleic acid fragment which encodes a src protein which does not have kinase activity.

Brief description of the drawings

In the accompanying drawings which form a part of the disclosure of the present specification:

FIG. 1 shows the cDNA sequence of chicken c-Src, which was first introduced in "cells" by Takeya et al(Cell),32: 881-890(1983) deleting the full coding sequence of the intron. This sequence can be found by the GenBank accession number J00844. This sequence contains 1759 nucleotides which start and end at the corresponding nucleotides 112 and 1713, respectively, of the protein coding portion.

FIG. 2 is an amino acid residue sequence of chicken c-Src encoded by the coding sequence shown in FIG. 1.

FIG. 3 is a drawing of a first journal of the national academy of sciences of the United states, by Braeuninger et al(Proc. Natl.Acad.Sci.USA),88: 10411 and 10415 (1991). This sequence can be found by the GenBank accession number X59932X 71157. The sequence contains 2187 nucleotides whose protein coding portions begin and end at the corresponding nucleotides 134 and 1486, respectively.

FIG. 4 is the amino acid residue sequence of human c-Src encoded by the coding sequence shown in FIG. 3.

FIG. 5 illustrates the activation of endogenous Src by bFGF or VEGF as described in example 4. The upper part of the figure shows the use of bFGF or VEGF for the multiplicative activation of endogenous c-SrcIn vitroAnd (4) kinase test results. The lower part of the figure is a kinase assay blot probed with an anti-Src antibody as a loading control for equal amounts of Src and IgG.

FIG. 6 illustrates the effect of retrovirus-mediated gene expression of c-Src A on angiogenesis in the chick chorioallantoic membrane (CAM) as described in example 4. 9 day old chicken CAMs were exposed to RCAS-Src A (active mutant c-Src) or control RCAS-GFP (Green fluorescent protein; a fluorescent indicator protein) retrovirus or buffer for 72 hours. As shown in fig. 6A, the representative micrograph (4x) of fig. 6B corresponding to the results of each treatment recorded with a stereomicroscope was used to quantify the level of angiogenesis.

FIG. 7 illustrates retroviral expression of c-Src A in the activation of vascular MAP kinase phosphorylation. FIG. 7A shows tissue extracts from 10 day old chicken CAMs that have been exposed to VEGF or PMA for 30 minutes or infected with c-Src A retrovirus for 48 hours. NT represents no treatment. Src immunoprecipitation was performed from equal amounts of total protein extract and using FAK-GST fusion protein as a substrateIn vitroImmune complex kinase assay, which was electrophoresed and transferred onto nitrocellulose. Aliquots of the whole tissue lysates described above were also assayed for endogenous ERK phosphorylation by immunoblotting using anti-phospho-ERK antibodies. FIG. 7B shows 10 day old CAMs infected with either mock RCAS or RCAS containing SRC A. After two days, the CAMs were dissected, stored cryogenically in OCT and cut into 4 μm pieces. Sections were immunostained with anti-phosphorylated ERK antibodies (New England Biolabs), washed and detected with goat anti-rabbit FITC conjugated secondary antibodies. The fluorescence image was captured on a static CCD camera (PrincetonInst).

Figure 8 illustrates the selective requirement for Src activity during VEGF but not bFGF-induced angiogenesis. 9 day old chicken CAMs were contacted with RCAS-Src251 or control RCAS-GFP retrovirus or buffer for 20 hours and then cultured for 72 hours in the presence or absence of bFGF or VEGF. Figure 8A angiogenesis levels were quantified as described above and representative micrographs were recorded using a stereomicroscope (6x) as shown in figure 8B. Figure 8C shows a blot probed with anti-Src antibody to demonstrate Src251 expression in transfected cells compared to mock treatment.

FIG. 9 illustrates the results of retroviral transport of RCAS-Src251 to human tumors. Fig. 9A is a micrograph showing human medulloblastoma fragments infected with RCAS-GFP (RCAS-green fluorescent protein) expressing GFP only in tumor vessels (arrows) according to the light section method using a Bio Rad laser focus scanning microscope (raster ═ 500 μm). FIG. 9B depicts data from tumors treated by topical administration of retrovirus, allowed to grow for 3 or 6 days after excision and determination of wet weight. Data are expressed as mean change in tumor weight (from 50mg tumor starting weight) +/-SEM of 2 replicates. Fig. 9C depicts a representative micrograph of a medulloblastoma surgically taken from an embryo (350 μm raster). The lower panel is a high magnification photograph of each tumor (350 μm raster) showing the vasculature of each tumor in particular. Arrows indicate vascular rupture in RCAS-Src251 treated tumors.

FIG. 10 is a schematic diagram illustrating a restriction enzyme map of the RCASBP (RCAS) vector construct.

Detailed Description

A. Definition of

Amino acid residues: amino acids formed by a polypeptide upon chemical digestion (hydrolysis) of its peptide bonds. The amino acid residues described herein are preferably in the "L" isoform. However, any L-amino acid residue may be substituted with a "D" isomeric residue, provided that the polypeptide retains the desired functional properties. NH (NH)₂Refers to the free amino group present at the amino terminus of the polypeptide. COOH refers to the state of retention of standard polypeptide names (in journal of biochemistry)(J.Biol.Chem.)243: 3552-59(1969) and at the carboxy terminus of the polypeptide as set forth in 37 CFR § 1.822(b) (2).

It should be noted that all amino acid residue sequences are represented herein by a general formula, the left and right side orientations of which are conventional orientations from the amino terminus to the carboxy terminus. Furthermore, the dashed line at the beginning or the end of the amino acid residue sequence represents a peptide bond to another sequence of one or more amino acid residues.

Polypeptide: refers to a linear arrangement of amino acid residues linked to each other by peptide bonds between the alpha-amino and carboxyl groups of adjacent amino acid residues.

Peptide: this term is used herein to refer to a linear arrangement of no more than about 50 amino acid residues joined to each other as in a polypeptide.

Cyclic peptide: refers to compounds having a ring structure that includes several amide bonds as in typical peptides. The cyclic peptide may be a "head to tail" homocyclic peptide; or it may contain a heterobond ring structure wherein the ring is closed by disulfide bridges, lactone bridges, thioesters, thioamides, guanidino groups, and the like.

Protein: refers to a linear arrangement of more than 50 amino acid residues that are linked to each other in a polypeptide.

Fusion protein: refers to a polypeptide comprising at least two distinct polypeptide domains operably linked by a typical peptide bond ("fused"), wherein the two domains correspond to peptides not found fused in nature.

Synthesizing a peptide: refers to a chemically generated chain of amino acid residues joined to each other by peptide bonds that is free of naturally occurring proteins and fragments thereof.

B. General discussion

The present invention relates generally to the discovery that angiogenesis mediated by the tyrosine kinase Src protein and can be modulated by providing activity to potentiate or inhibit angiogenesis or inactivating Src protein, respectively.

This finding is important because angiogenesis, i.e., the formation of new blood vessels, plays an important role in the progression of various diseases. If the tissue associated with the disease requires angiogenesis for tissue growth, it is desirable to inhibit angiogenesis and thus the growth of the diseased tissue. If the damaged tissue requires angiogenesis for tissue growth and healing, it is desirable to enhance or promote angiogenesis and thus tissue healing and growth.

Inhibiting angiogenesis can reduce the deleterious effects of disease if new blood vessel growth is the cause of or contributes to the development of a pathological condition associated with diseased tissue. By inhibiting angiogenesis, it is possible to interfere with the disease, ameliorate symptoms and in some cases cure the disease.

Examples of tissues associated with disease and neovascularization that benefit from the inhibitory modulation of angiogenesis include rheumatoid arthritis, diabetic retinopathy, inflammatory diseases, restenosis, and the like. If the unwanted tissue growth requires support for new blood vessel growth, then inhibiting angiogenesis can reduce the amount of blood supplied to the tissue and thereby reduce the quality of the tissue based on the amount of blood supplied. Examples include the growth of tumors, where neovascularization is continuously required to grow the tumor to a thickness of more than a few millimeters and form solid metastases.

If new blood vessel growth results in tissue healing, then enhanced angiogenesis aids in healing. Examples include treating patients with limb ischemia, where poor circulation in the limb is caused by diabetes or other diseases. Of further therapeutic interest are patients with certain wounds that do not heal and can therefore benefit from increased vascular cell proliferation and neovascularization.

The methods of the invention are effective, in part, because the therapy is highly selective for angiogenesis and has no other biological processes.

As mentioned above, angiogenesis includes many processes involving neovascularization of tissue, including "sprouting", angiogenesis or vessel enlargement, all of which are effected by Src protein. In addition to wound healing, luteal formation and embryogenesis, most angiogenic processes are thought to be associated with the progression of disease. Thus, the present treatment is selective for the disease and has no deleterious side effects.

Src protein

The tyrosine kinase Src protein used in the present invention may vary from application to application. The term "Src protein" or "Src" is used to refer to the various forms of tyrosine kinase Src protein in active or inactive form as described herein.

By "active Src protein" is meant the various forms of Src protein that potentiate angiogenesis. The assay for determining the degree of enhancement of angiogenesis is described herein but is not intended to be limiting. A protein is considered active if the level of angiogenesis is at least 10%, preferably 25% and more preferably 50% higher than the control level in the test system without src added. A preferred assay for determining the degree of enhancement is a CAM assay using the RCAS viral vectors described in the examples, wherein the angiogenic index is calculated by counting branch points. Preferred active Src proteins also exhibit tyrosine kinase activity. Exemplary active Src proteins are described in the examples and include Src-A.

"inactivated Src protein" refers to various forms of Src protein that inhibit angiogenesis. The assay to determine the degree of inhibition of angiogenesis is described herein but is not limiting. A protein is considered inactive if the level of angiogenesis is at least 10%, preferably 25% and more preferably 50% below the control level in the test system to which no exogenous Src has been added. A preferred assay for determining the degree of inhibition is the CAM assay using the RCAS viral vectors described in the examples, wherein the angiogenic index is calculated by counting branch points. Preferred inactivated Src proteins also exhibit reduced tyrosine kinase activity. Exemplary inactivated Src proteins are described in the examples and include Src-251.

The Src protein used in the present invention may be produced by any of various methods, including isolation from natural sources including tissues, production by recombinant DNA expression and purification, and the like. Src protein can also be produced "in situ" by introducing a gene therapy system into the tissue of interest and then expressing the protein in that tissue.

The gene encoding Src protein can be prepared by various methods known in the art and the present invention is not limited in this respect. For example, it is well known that the natural history of Src includes homologues from mammalian, avian, viral and like species, and that its gene can be conveniently cloned using cDNA cloning from any tissue expressing the protein. Preferred Src for use in the present invention are cellular proteins such as mammalian or avian homologs designated c-Src. Particularly preferred is human c-Src.

D. Recombinant DNA molecules and expression systems for expressing Src protein

Several nucleotide sequences are described which are particularly useful in the present invention. These sequences include sequences encoding the Src protein for use in the present invention, as well as various DNA fragments, recombinant DNA (rDNA) molecules, and vectors constructed for expression of the Src protein.

The DNA molecules (fragments) of the invention may thus contain sequences encoding the complete structural gene, structural gene fragments and transcription units as further described herein.

Preferred DNA fragments are nucleotide sequences encoding Src protein as described herein, or a biologically active fragment thereof.

Preferred amino acid residue sequences and nucleotide sequences of c-Src are described in the examples.

Preferred DNA fragments encode amino acid residue sequences that are substantially identical to, and preferably consist essentially of, the amino acid residue sequences corresponding to the Src proteins described herein, or portions thereof. Representative and preferred DNA fragments are further described in the examples.

The sequence of amino acid residues of a protein or polypeptide is directly related by the genetic code to the deoxynucleic acid (DNA) sequence of the structural gene encoding the protein. Thus, a structural gene or DNA segment can be defined in terms of the amino acid residue sequence, i.e., the amino acid residue sequence of the protein or polypeptide that it encodes.

An important and well-known feature of the genetic code is its redundancy. That is, for most amino acids used to make proteins, more than one coding nucleotide triplet (codon) may encode or specify a particular amino acid residue. Thus, a large number of different nucleotide sequences may encode a particular sequence of amino acid residues. Such nucleotide sequences are considered functionally equivalent, since they can lead to the production of identical sequences of amino acid residues in all organisms. In some cases, methylated variants of purines or pyrimidines may be introduced into a given nucleotide sequence. However, such methylation does not affect any form of coding relationship.

A nucleic acid is any polynucleotide or nucleic acid fragment which may be a polydeoxyribonucleotide or a polyribonucleotide, i.e.RNA or DNA or an analogue thereof. In a preferred embodiment, the nucleic acid molecule is in the form of duplex DNA fragments, i.e. DNA fragments, although for certain molecular biological methods single-stranded DNA or RNA is preferred.

DNA fragments are produced by a number of methods including chemical synthesis and recombinant means, preferably by cloning or by Polymerase Chain Reaction (PCR). By chemical techniques such as Matteucci et al, journal of the American chemical Association(J.Am.Chem.Soc.),103: 3185 DNA fragments encoding the Src protein portion may conveniently be synthesised by the phosphotriester method as described in 3191, 1981 or by using automated synthesis. In addition, longer DNA fragments can be conveniently prepared by well-known methods such as synthesis of a set of oligonucleotides defining the DNA fragment, followed by hybridization and ligation of the oligonucleotides to construct a complete fragment. Another method comprises the step of isolating preferred DNA fragments by PCR using a pair of oligonucleotide primers for use on a cDNA library believed to contain a Src protein-encoding component.

Of course, any desired modification can be readily made by chemical synthesis by substituting the appropriate base for that encoding the natural amino acid residue sequence. Such methods are well known and may be conveniently used to produce the various "modified" Src proteins described herein.

Furthermore, a DNA fragment consisting essentially of the structural gene encoding the Src protein may subsequently be modified, such as by site-directed mutagenesis or random mutagenesis, to introduce any desired substitutions.

1. Cloning of Src Gene

The Src gene can be cloned from genomic DNA or messenger rna (mrna) from a suitable source by various biochemical methods. Cloning of these genes can be performed as described in the examples and as a general method well known in the art.

The source of nucleic acid for cloning the Src gene suitable for use in the method of the invention may comprise genomic DNA or messenger rna (mrna) in the form of a cDNA library from tissues thought to express these proteins. The preferred tissue is human lung tissue, although any other suitable tissue may be used.

A preferred cloning method comprises the steps of preparing a cDNA library using standard methods and isolating a nucleotide sequence encoding Src by PCR amplification using paired oligonucleotide primers based on the nucleotide sequence described herein. Alternatively, desired cDNA clones can be identified and isolated from cDNA or genomic libraries by conventional nucleic acid hybridization methods using hybridization probes based on the nucleic acid sequences described herein. Other methods of isolating and cloning the src-encoding nucleic acid will be apparent to those skilled in the art.

2. Expression vector

Recombinant DNA molecules (rDNA) containing DNA fragments encoding Src protein can be produced as described herein. In particular, expressible rDNA can be produced by operably (in-frame, expressible) ligating a vector with a DNA fragment encoding src. Thus, a recombinant DNA molecule is a hybrid DNA molecule of nucleic acids that contains at least two nucleotide sequences that are not normally found together in nature.

As is well known in the art, the choice of a vector to which a DNA segment is operably linked depends directly on the functional properties desired, e.g., the expression of the protein and the host cell being transformed. Vectors suitable for use in the practice of the present invention are at least capable of directing replication and preferably also expression of the structural gene included in the vector DNA segment to which it is operably linked.

Prokaryotic and eukaryotic expression vectors are well known to those skilled in the art of vector constructionAnd is published by Ausebel et al in molecular biology, latest protocols(Current Protocols in Molecular Biology)Wiley and Sons, New York (1993) and by Sambrook et al in molecular cloning: laboratory Manual(Molecular Cloning：A Laboratory Manual)Described in cold spring lane laboratories (1989). Many of the general recombinant DNA methods referred to herein are also described in these references.

In one embodiment, suitable vectors include prokaryotic replicons, i.e., DNA sequences having the ability to replicate autonomously directly and extrachromosomally in prokaryotic host cells, such as bacterial host cells transformed therewith, to maintain the recombinant DNA molecule. Such replicons are well known in the art. In addition, those embodiments that include prokaryotic replicons also include genes whose expression confers resistance to bacterial hosts transformed therewith. Typical bacterial anti-agent genes are those that confer resistance to ampicillin or tetracycline.

Those vectors that include prokaryotic replicons may also include prokaryotic promoters capable of directing the expression (transcription and translation) of the structural genes in bacterial host cells transformed therewith, such as E.coli. A promoter is an expression control element formed from a DNA sequence that allows binding of RNA polymerase and translation. Promoter sequences compatible with bacterial hosts are typically carried in plasmid vectors containing convenient restriction sites for insertion of the DNA fragments of the invention. Typical such plasmid vectors are: pUC8, pUC9, pBR322 and pBR329 commercially available from Biorad Laboratories, (Richmond, Calif.); pRSET commercially available from Invitrogen (San Diego, CA) and pPL and pKK223 commercially available from Pharmacia, Piscataway, N.J.

Expression vectors compatible with eukaryotic cells, preferably those compatible with vertebrate cells, can also be used to form the recombinant DNA molecules of the invention. Eukaryotic expression vectors are well known in the art and they are available from several commercial sources. Generally, such vectors containing convenient restriction sites are provided for insertion of the desired DNA fragment. Typical such vectors are pSVL and pKSV-10(Pharmacia), pBPV-1/pML2d (Internatinal Biotechnologies, Inc.), pTDT1(ATCC, #31255), pRc/CMV (Invitrogen, Inc.), preferred vectors described in the examples, and similar eukaryotic expression vectors.

Particularly preferred systems for gene expression in the context of the present invention comprise a gene transport component, i.e.a component having the ability to transport a gene to a tissue of interest. Suitable vectors are "infectious" vectors such as recombinant DNA viruses, adenoviruses or retroviral vectors that are genetically engineered to express the desired protein and have the characteristic of infecting a preselected target tissue. Particularly preferred is the replication competent avian sarcoma virus (RCAS) described herein.

Mammalian cell lines that use recombinant viruses or viral elements to direct expression can be genetically engineered. For example, when using an adenoviral expression vector, the coding sequence for the polypeptide can be linked to an adenoviral transcription/translation control complex such as the late promoter and tripartite leader sequence. Then can pass throughIn vitroOrIn vivoRecombination inserts this chimeric gene into the adenovirus genome. Insertion into a non-essential region of the viral genome (e.g., the E1 or E3 region) results in the viability of the recombinant virus and the ability to express polypeptides in the infected host (see, e.g., Logan et al, Proc. Natl. Acad. Sci. USA, Inc.)(Proc.Natl.Acad.Sci.USA),81: 3655-3659(1984)). Alternatively, the vaccinia virus 7.5K promoter can be used (see, e.g., Mackett et al, Proc. Natl. Acad. Sci. USA, Inc.)(Proc.Natl.Acad.Sci.USA),79: 7415-7419 (1982); mackett et al journal of virology(J.Virol.),49: 857-864 (1984); panicali et al, "proceedings of the national academy of sciences of the United states of America(Proc. Natl.Acad.Sci.USA),79: 4927-4931(1982)). Of particular interest are bovine papilloma virus-based vectors that have the ability to replicate as an extrachromosomal element (Sarver et al, cell molecular biology)(Mol.Cell.Biol.),1: 486(1981)). Shortly after this DNA has entered the target cell, the plasmid replicates at approximately 100 and 200 copies/cell. Plug-inTranscription of the incoming cDNA does not require integration of the plasmid into the host chromosome, thereby resulting in high levels of expression. These vectors can be used for stable expression by including a selectable marker such as a novel gene in the plasmid. Alternatively, the retroviral genome may be modified for use as a vector capable of introducing and directing expression of a nucleotide sequence encoding a polypeptide in a host cell (Cone et al, Proc. Natl. Acad. Sci. USA)(Proc.Natl. Acad.Sci.USA),81: 6349-6353(1984)). Inducible promoters including, but not limited to, the metallothionein IIA promoter and heat shock promoters may also be used to achieve high levels of expression.

Currently, the long-term survival of athymic mice with human ovarian cancer has been studied for Thymidine Kinase (TK) gene therapy driven by the Cytomegalovirus (CMV) promoter and the Rous Sarcoma Virus (RSV) promoter. The cytocidal efficacy of adenovirus-mediated CMV promoter-driven herpes simplex virus TK gene therapy was found to be 2-10 fold more effective than RSV-driven therapy. (Tong et al, 1999 hybridoma(Hybridoma)18(1): 93-97). Also described is the design of chimeric promoters for use in carrying out gene therapy requiring low levels of expression followed by inducible high levels of expression. (Suzuki et al, 1996 "Gene therapy in humans")(Human Gene Therapy)7：1883-1893)。

For the production of recombinant proteins in high yield over a long period of time, stable expression is preferred. Instead of using an expression vector containing a viral origin of replication, a host cell can be transformed with the cDNA and a selectable marker under the control of appropriate expression control elements (e.g., promoter and enhancer sequences, transcription terminators, polyadenylation sites, etc.). As described above, the selectable marker in the recombinant plasmid confers resistance to selection and allows the cell to stably integrate the plasmid into its chromosome and grow to form foci that can be subsequently cloned and expanded into a cell line.

For example, after introduction of exogenous DNA, engineered cells can be grown in enriched media for 1-2 days and then switched to selectionIn a sexual medium. A number of selection systems can be used, including but not limited to herpes simplex thymidine kinase (Wigler et al cell)(Cell),11: 223(1977)), hypoxanthine guanine phosphoribosyl transferase (Szybalska et al, proceedings of the national academy of sciences of the United states of America)(Proc.Natl.Acad.Sci.USA),48: 2026(1962)) and adenine phosphoribosyl transferase (Lowy et al cell(Cell),22: 817(1980)) genes, which can be used for tk, respectively^-、hgprt^-Or aprt^-A cell. Furthermore, genes that provide resistance to antimetabolites can be used as a basis for selection; for example, dhfr (Wigler et al, Proc. Natl. Acad. Sci. USA) for resistance to methotrexate(Proc. Natl.Acad.Sci.USA),77: 3567 (1980); o' Hare et al, proceedings of the national academy of sciences of the United states(Proc.Natl.Acad.Sci.USA),78: 1527(1981), gpt to confer mycophenolic acid resistance (Mullingan et al, proceedings of the national academy of sciences USA)(Proc. Natl.Acad.Sci.USA),78: 2072(1981), neo (Colberre-Garapin et al, J. Molec. biol.) to confer resistance to the aminoglycoside G-418(J.Mol. Biol),150: 1(1981)) and hygro (Santerre et al Gene) conferring resistance to hygromycin(Gene),30: 147 (1984)). Recently, alternative genes have been described that allow cells to use indole in place of trpB for tryptophan and histidinol in place of histidine (Harman et al Proc. Natl. Acad. Sci. USA)(Proc.Natl.Acad.Sci.USA),85: 804(1988)) and ODC (Ornithine decarboxylase) conferring resistance to the ornithine decarboxylase inhibitor 2- (difluoromethyl) -DL-ornithine DFMO (McConlogue L., latest Communications in Molecular Biology, Cold spring Lane laboratory Ed (1987)).

The major vector of interest for human gene therapy is derived from retroviruses (Wilson, 1997, immunological clinical trials)(Clin.Exp.Immunol.)107 (supplement 1): 31-32; bank et al, 1996 "biological assay(Bioessays)18(12)：999-1007; robbins et al, 1998 pharmaceutical therapy(Pharmacol.Ther.)80(1): 35-47). The therapeutic potential of gene transfer and antisense therapies has stimulated the development of many vector systems for the treatment of various tissues. (vascular System: see Stephan et al, 1997 "basis of clinical medicine(Fundam.Clin.Pharmacol.)11(2): 97 to 110; feldman et al 1997 cardiovascular research(Cardiovasc.Res.)35(3): 391-404; vassalli et al, 1997 cardiovascular research(Cardiovasc.Res.)35(3): 459-69; baek et al, 1998 circulation study(Circ.Res.)82(3): 295-305; kidney: see Lien et al, 1997, International journal of Kidney augmentation(Kidney Int.Suppl.)61: s85-8; liver: see Ferry et al, 1998 "human Gene therapy(Hum.Gene Ther.)9(14): 1975-81; muscles, see Marshall et al, 1998 "latest views on genetic development(Curr.Opn.Genet.Dev.)8(3): 360-5). In addition to these tissues, a key target for human gene therapy is cancer, i.e., the tumor itself or related tissues. (Runnebaum, 1997 anticancer research)(Anticancer Res.)17 (4B): 2887-90; spear et al, 1998 Neurovirology(J.Neurovirol.)4(2)：133-47)。

Specific examples of viral gene therapy vector systems that are readily adaptable for use in the methods of the present invention are briefly described below. The gene transport of retroviruses has now been reviewed by Federspiel and Hughes (1998, methods in cell biology(Methods in Cell Biol.)52: 179-214), which describe, inter alia, the Avian Leukemia Virus (ALV) retrovirus family (Federspiel et al, proceedings of the national academy of sciences of the United states of America)(Proc.Natl.Acad.Sci. USA)93: 4931 (1996); federspiel et al, proceedings of the national academy of sciences of the United states(Proc. Natl.Acad.Sci.USA),91: 11241(1994)). Retroviral vectors, including ALV and Murine Leukemia Virus (MLV), are further described by Svoboda (1998, Gene)(Gene)206：153-163)。

Modified retroviral/adenoviral expression systems may be conveniently adapted for use in carrying out the methods of the invention. For example, Karavana et al, in "oncology/hematology margin research(Crit. Rev.in Oncology/Hematology)28: murine Leukemia Virus (MLV) systems are reviewed in 7-30. Von Seggern and Nemerow in Gene expression System(Gene Expression Systems)(Fernandez &The adenovirus expression system is reviewed in Hoeffler, Academic Press, San Diego, CA, 1999, Chapter 5, pp 112-157).

Protein expression systems have been shown to have potent effects both in vivo and in vitro. For example, efficient gene transport to human squamous cell carcinoma by herpes simplex virus type 1 (HSV) amplicon vectors has been described. (Carew et al, 1998 journal of American science)(Am.J.Surg.)176: 404-408). Herpes simplex viruses have been used for gene transport to the nervous system. (Goins et al, 1997, neurovirology(J.Neurovirol.)3 (supplement 1): s80-8). Targeted suicide vectors using HSV-TK have been tested on solid tumors. (Smiley et al, 1997 "human Gene therapy(Hum.Gene Ther.)8(8): 965-77). Herpes simplex type 1 viral vectors have been used in cancer gene therapy for colon cancer cells. (Yoon et al, 1998 < annual review of surgery >)(Ann.Surg.)228(3): 366-74). Hybrid vectors have been developed for extending the length of transfection time, including HSV/AAV (adeno-associated virus) hybrids for treating liver cells. (Fraefel et al, 1997 molecular drugs)(Mol.Med.)3(12)：813-825)。

Vaccinia virus has been developed for use in human gene therapy because of its large genome. (Peplinski et al "American tumor surgery clinical Standard" of(Surg.Oncol.Clin. N.Am.)7(3): 575-88). Vaccinia virus lacking thymidine kinase expressing purine nucleoside pyrophosphatase has been described for use as a vector for tumor-targeted gene therapy. (Puhlman et al, 1999 "human Gene therapy(Human Gene Therapy)10：649-657)。

Adeno-associated type 2 viruses (AAV) have been described for use in human gene therapy, however, AAV requires a helper virus, such as an adenovirus or herpes virus, for optimal replication and packaging within mammalian cells. (Snoeck et al, 1997 experiment in nephrology(Exp. Nephrol.)5(6): 514-20; rabinowitz et al, 1998 "recent opinion on biotechnology")(Curr.Opn.Biotechnol.)9(5): 470-5). However, packaging of infectious recombinant AAV in vitro has been described, making this system extremely valuable. (Ding et al, 1997 Gene therapy(Gene Therapy)4: 1167-1172). AAV-mediated transfer of the ecotropic retroviral receptor cDNA has been demonstrated to allow ecotropic retroviral transduction of defined and major human cells. (Qing et al, 1997 J.Virol. J.(J.Virology)71(7): 5663-5667). Gene therapy for cancer using AAV vectors expressing human wild-type p53 has been demonstrated. (Qazilbash et al, 1997 Gene therapy(Gene Therapy)4: 675-682). Methods for transferring genes into vascular cells using AAV vectors have also been demonstrated. (Maeda et al, 1997 cardiovascular Studies(Cardiovasc. Res.)35(3): 514-521). AAV has been shown to be useful as a suitable vector for liver-directed gene therapy. (Xiao et al, 1998 journal of virology(J.Virol.)72(12): 10222-6). AAV vectors have been demonstrated to be useful for gene therapy of brain tissue and the central nervous system. (Chamberlin et al, 1998 "brain Studies")(Brain Res.)793: (1-2): 169 to 75; during et al, 1998 Gene therapy(Gene Therapy)5(6): 820-7). AAV vectors were also compared to adenovirus vectors (AdV) for their pulmonary gene therapy and metastasis to human cystic fibrosis epithelial cells. (Teramoto et al, 1998 journal of virology(J.Virol.)72(11)：8904-12)。

Chimeric AdV/retroviral gene therapy vector systems can incorporate useful amounts of each virus to produce non-integrative AdV, making it functionally integrative through intermediate production by retroviral producer cells. (Feng et al, 1997 InternationalBiotechnology(Nat. Biotechnology)15(9): 866 to 70; bilbao et al, 1997, J FASEB(FASEB J)11(8): 624-34). This newly generated potent gene therapy vector has been used for targeted cancer gene therapy. (Bibao et al, 1998 Experimental drug biological Advances(Adv.Exp.Med.Biol.)451: 365-74). A single injection of AdV expressing p53 inhibited the growth of subcutaneous tumor nodules in human prostate cancer cells. (Asgari et al, 1997J. International cancer)(Int.J.Cancer)71(3): 377-82). AdV-mediated gene transfer has been described for wild-type p53 in patients with advanced non-small cell lung cancer. (Schuler et al, 1998 "Gene therapy in humans(Human Gene Therapy)9: 2075-2082). This same cancer has been the subject of treatment for AdV vector-mediated p53 gene replacement therapy. (Roth et al, 1998 Oncology study)(Semin. Oncol.)25(3 supplement 8): 33-7). AdV-mediated gene transfer of p53 inhibits endothelial cell differentiation and angiogenesis in vivo. (Riccionii et al, 1998 Gene therapy(Gene Ther.)5(6): 747-54). Adenovirus-mediated expression of the melanoma antigen gp75 is also described as an immunotherapy for metastatic melanoma. (Hirschowitz et al, 1998 Gene therapy(Gene Therapy)5: 975-983). AdV aids in infecting human cells with an environmentally friendly retrovirus and increases the efficacy of the retroviral infection. (Scott-Taylor et al, 1998 Gene therapy(Gene Ther.)5(5): 621-9). AdV vectors have been used in vascular smooth muscle cells (Li et al, 1997, J. Chinese pharmaceuticals (English edition))(Chin.Med.J.(Engl))110(12): 950-4), squamous cell carcinoma cells (Goebel et al, 1998 "otorhinolaryngology department of head and neck surgery")(Otolarynol Head Neck Surg)119(4): 331-6), esophageal cancer cells (Senmaru et al, 1998 journal of International cancer(Int.J.Cancer)78(3): 366-71), mesangial cells (NaHman et al, 1998 journal of drug research(J. Investig.Med.)46(5): 204-9), glial cells (Chen et al, 1998 cancer research(Cancer Res.)58(16): 3504-7) and gene transfer to animal joints (Ikeda et al, 1998 journal of rheumatology)(J. Rheumatol.)25(9): 1666-73). Recently, catheter-based pericardial gene transfer mediated by AcV vectors has been demonstrated. (March et al 1999 "clinical cardiology(Clin.Cardiol.)22(1 supplement 1): i23-9). Operating the AdV system with appropriately controlled genetic elements requires AdV-mediated regulatable target gene expression in vivo. (Burcin et al, 1999)PNAS(USA)96(2)：355-60)。

Alphavirus vectors have been developed for use in the practice of human gene therapy using packaging cell lines suitable for transformation with expression cassettes suitable for use with vectors derived from Sindbis virus and Semliki forest virus. (Polo et al 1999 proceedings of the national academy of sciences of the United states of America)(Proc.Natl.Acad.Sci.USA)，96：4598-4603). Non-cytopathic flavivirus replicon RNA based systems have also been developed. (Varnavski et al, 1999 virology(Virology)255(2): 366-75). Suicide HSV-TK genes containing the Sindbis viral vector have been used for cell-specific targeting directed into tumor cells. (Iijima et al, 1998 journal of International cancer(Int.J.Cancer)80(1)：110-8)。

Retroviral vectors based on human foamy (foamy) virus (HFV) have also shown promise as gene therapy vectors. (Trobridge et al, 1998 "human Gene therapy(Human Gene Therapy)9: 2517-2525). Foamy virus vectors have been designed for suicide gene therapy. (Nestler et al, 1997 Gene therapy(Gene Ther.)4(11): 1270-7). Recombinant murine cytomegalovirus and promoter systems are also used as vectors for high level expression. (Manning et al, 1998J methods of virology(J. Virol.Meth.)73(1): 31-9; tong et al, 1998 hybridoma(Hybridoma)18(1)：93-7)。

It is possible to transfer a gene into an undivided cell by producing a vector based on Sendai virus. (Nakanishi et al1998 journal of controlled Release(J.Controlled Release)54(1)：61-8)。

In other efforts to potentially transform non-dividing somatic cells, lentiviral vectors have been investigated. Cystic fibrosis gene therapy using vectors based on replication-defective Human Immunodeficiency Virus (HIV) has been described. (Goldman et al, 1997 "human Gene therapy(Human Gene Therapy)8: 2261-2268). Sustained expression of genes transported into the liver and muscle by lentiviral vectors is also demonstrated. (Kafri et al, 1997, International genetics)(Nat.Genet.)17(3): 314-7). However, concerns about safety are major and development of improved vectors is also rapidly underway. (Kim et al, 1998 journal of virology(J.Virol.)72(2): 994-1004). The HIV LTR and Tat assays yield important information about the structure of the developed vector genome. (Sadaie et al, 1998 journal of pharmaceutical virology(J.Med.Virol.)54(2): 118-28). Thus, the genetic need for an effective HIV-based vector is now better understood. (Gasmi et al, 1999 journal of virology(J.Virol.)73(3): 1828-34). Self-inactivating vector or conditional packaging cell lines have been described. (e.g., Zuffery et al, 1998 journal of virology(J.Virol.)72(12): 9873-80; miyoshi et al, 1998 journal of virology(J.Virol.)72(10): 8150-7; dull et al, 1998 journal of virology(J.Virol.)72(11): 8463-71; and Kaul et al, 1998 virology(Virol ogy)249(1): 167-74). Efficient transduction of human lymphocytes and CD34+ cells by HIV vectors has been demonstrated. (Douglas et al 1999 human Gene therapy(Human Gene Ther.)10(6): 935 to 45 parts of; miyoshi et al, 1999 science(Science)283 (5402); 682-6). Efficient transduction of non-dividing human cells by Feline Immunodeficiency Virus (FIV) lentiviral vectors has been demonstrated, which minimizes safety concerns associated with the use of HIV-based vectors. (Poeschla et al, 1998 Natural drugs (Nature Medicine)4(3): 354-357). Productive infection of human blood mononuclear cells by FIV vectors has been demonstrated. (Johnston et al, 1999 journal of virology(J.Virol.)73(3)：2491-8)。

Although many viral vectors are difficult to control and the ability of the inserted DNA is limited, these limitations and drawbacks have been investigated. For example, in addition to simple viral packaging cell lines, parvoviral vectors derived from human herpesvirus, herpes simplex type 1 virus (HSV-1) and EB virus (EBV) have been developed to simplify the manipulation of genetic material and the production of viral vectors. (Wang et al, 1996 journal of virology(J.Virology)70(12): 8422-8430). It has been previously demonstrated that ligating plasmids can simplify the process of inserting foreign DNA into helper-independent retroviral vectors. (1987, J.Viridology(J. Virology)61(10)：3004-3012)。

Although several non-viral vectors have been described, viral vectors are not the only tool for carrying out gene therapy. Targeted non-viral gene delivery vectors based on the use of epidermal growth factor/DNA polyplex (EGF/DNA) have been shown to produce efficient and specific gene delivery. (Cristiano, 1998 anticancer research)(Anticancer Res.)18: 3241-3246). Gene therapy of the vascular system and CNS has been demonstrated using cationic liposomes. (Yang et al, 1997J Neurotrauma(J.Neurotrauma)14(5): 281-97). Transient gene therapy for pancreatitis was also accomplished using cationic liposomes. (Denham et al, 1998 "annual review of surgery(Ann.Surg.)227(6): 812-20). Chitosan-based vector/DNA complexes for gene delivery have proven effective. (Erbacher et al, 1998 drug research(Pharm. Res.)15(9): 1332-9). Non-viral DNA transport vectors based on the terplex system have been described. (Kim et al, 1998, 53 (1-3): 175-82). Viral particle coated liposome complexes are also used to achieve gene transfer. (Hirai et al, 1997 biochem. biophysics researchCommunication (science and technology)(Biochem.Biophys.Res. Commun.)241(1)：112-8)。

Gene therapy for cancer has been demonstrated using a non-viral T7 vector encoding a thymidine kinase gene directly injected into the tumor. (Chen et al, 1998 "human Gene therapy(Human Gene Therapy)9: 729-736). The preparation of plasmid DNA is important for gene transfer by direct injection. (Horn et al, 1995, human Gene therapy(Hum.Gene Ther.)6(5): 656-73). Modified plasmid vectors are particularly useful for direct injection. (Hartikka et al, 1996 "human Gene therapy(Hum.Gene Ther.)7(10)：1205-17)。

Thus, various gene transfer/gene therapy vectors and constructs are well known in the art. These vectors may be conveniently used in the method of the present invention. Many equivalent vectors for use in the practice of the present invention may be produced by appropriate manipulation using recombinant DNA/molecular biology techniques for inserting an operably linked src (active or inactive) into a selected expression/transport vector.

E. Method for modulating angiogenesis

The present invention provides a method for modulating angiogenesis in a tissue associated with a disease process or condition and thereby contributing to a condition in the tissue that is dependent on angiogenesis. In general, the method comprises the step of administering to a tissue associated with a disease process or condition a composition comprising an angiogenic modulating amount of Src protein or a nucleic acid vector expressing active or inactive Src.

As described herein, any of the various tissues or organs comprised of organized tissues may sustain angiogenesis in tissue diseases including skin, muscle, intestine, connective tissue, joints, bone, etc., where blood vessels may invade upon angiogenic stimulation.

Ideally, the patient to be treated in accordance with the present invention in its many embodiments is a human patient, although it is to be understood that the mechanism of the present invention indicates that the present invention is effective for all mammals, and therefore the term "patient" is meant to include all mammals. In the context of the present invention, mammals are understood to include any mammalian species, in particular agricultural and domestic mammalian species, in which treatment of tissue associated with diseases involving angiogenesis is desired.

Thus, the method comprises the step of administering to the patient a therapeutically effective amount of a pharmacologically acceptable composition comprising Src protein or a DNA vector for expressing Src protein in the practice of the method of the invention.

As further described herein, the dosage range in which Src protein is administered depends on the form of the protein and its efficacy. The dose is large enough to produce the desired angiogenesis and amelioration of symptoms of disease mediated by angiogenesis. However, the dose should not be so large as to cause adverse side effects such as hyperviscosity syndrome, pulmonary edema, congestive heart failure, and the like. Generally, the dosage varies with the age of the patient, the disease condition, the sex of the patient and the extent of the disease in the patient and can be readily determined by one skilled in the art. The dosages can also be adjusted by the individual clinician according to all complications.

A therapeutically effective amount is an amount of Src protein or nucleic acid encoding (active or inactive) Src protein sufficient to produce a detectable angiogenesis modulation in the treated tissue, i.e., an amount that modulates angiogenesis. Modulation of angiogenesis can be determined by CAM detection assays as described herein or by other methods known to those skilled in the art.

The Src protein or nucleic acid vector expressing the Src protein may be administered parenterally by injection or by continuous infusion over a period of time. Although tissue to be treated can generally be contacted in vivo by systemic administration, and therefore most often they are treated by intravenous administration of therapeutic compositions, other tissues and modes of delivery are contemplated. Thus, the compositions of the present invention may be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, transdermally and may be delivered by peristaltic means.

Therapeutic compositions containing Src protein or a nucleic acid vector expressing Src protein can be administered routinely by intravenous administration, such as, for example, by injection of unit doses. The term "unit dose" when used in reference to a therapeutic composition of the present invention refers to physically discrete units suitable as unitary dosages for the subject to be treated, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a desired diluent, i.e., carrier or excipient.

In a preferred embodiment, the agent is administered intravenously in a single dose. Topical administration can be accomplished by the following steps: direct injection or by using anatomical separation compartments, microcirculation to isolate the target organ system, reperfusion in the circulatory system, or temporary closure of the target area of the vascular system associated with diseased tissue on a catheter basis.

The composition is administered in a manner compatible with the dosage form and in a therapeutically effective amount. The amount and timing of administration will depend upon the subject being treated, the capacity of the subject system to utilize the active ingredient, and the extent of the desired therapeutic effect. The precise amount of active ingredient to be administered will depend on the judgment of the practitioner and will be specific to each individual. However, suitable dosage ranges for systemic administration are disclosed herein and depend on the route of administration. Suitable dosing regimens may also vary, but are generally repeated at 1 hour or more intervals following initial administration by subsequent injection or other mode of administration. On the other hand, it is of interest to maintain sufficientlyIn vivoContinuous intravenous infusion of blood levels within a specified range of therapy.

1. Inhibition of angiogenesis

Inhibition of angiogenesis is important in a variety of diseases known as angiogenic diseases. Such diseases include, but are not limited to: inflammatory diseases, such as immune or non-immune inflammation; chronic articular rheumatism and psoriasis; diseases associated with inappropriate or inappropriate vascular invasion, such as diabetic retinopathy, neovascular glaucoma, restenosis, capillary hyperplasia in atherosclerotic plaques and osteoporosis; and cancer-related diseases such as solid tumors requiring neovascularization to maintain tumor growth, solid metastases, angiofibromas, retrocrystalloid fibroplasia, hemangiomas, Kaposi's sarcoma, and the like.

Thus, methods of inhibiting disease-associated angiogenesis in tissues can ameliorate the symptoms of the disease and can cure the disease, depending on the disease condition. In one embodiment, the invention concerns the inhibition of angiogenesis in a tissue that is itself associated with a disease condition. The degree of angiogenesis in a tissue and thus the degree of inhibition achieved by the present method can be assessed by different methods.

Thus, in a related embodiment, the tissue treated is an inflamed tissue and the angiogenesis inhibited is angiogenesis of the inflamed tissue in which neovascularization of the inflamed tissue is present. In such cases, the present method is concerned with the inhibition of angiogenesis in arthritic tissues (such as in patients with chronic articular rheumatism), in immune or non-immune inflammatory tissues, in psoriatic tissues, and the like.

In another related embodiment, the tissue treated is retinal tissue of a patient with a retinal disease such as diabetic retinopathy, macular degeneration or neovascular glaucoma and the angiogenesis inhibited is angiogenesis of retinal tissue in which retinal tissue neovascularization is present.

In another related embodiment, the tissue treated is tumor tissue of a patient having a solid tumor, a metastasis, a skin cancer, a breast cancer, a hemangioma or angiofibroma, and the like and the angiogenesis inhibited is angiogenesis of tumor tissue in which neovascularization of the tumor tissue is present. Typical solid tumor tissues that may be treated by the methods of the invention include lung, pancreas, breast, colon, larynx, ovary and similar tissues. Inhibition of tumor tissue angiogenesis is a particularly preferred embodiment, as neovascularization plays an important role in tumor growth. In the absence of tumor tissue neovascularization, the tumor tissue does not acquire the required nutrients, slows growth, otherwise stops growth, regresses and eventually necroses that cause tumor death.

In other words, the present invention provides a method of inhibiting tumor neovascularization by inhibiting tumor angiogenesis according to the present methods. Similarly, the present invention provides a method of inhibiting tumor growth by performing a method of inhibiting angiogenesis.

The present method is also particularly effective against the formation of metastases because (1) formation of metastases requires vascularization of the original tumor so that the metastasized cancer cells can leave the original tumor and (2) their establishment in secondary sites requires neovascularization in order to maintain the growth of the metastases.

In a related embodiment, the invention concerns the practice of the present method in combination with other therapies such as conventional chemotherapy directed to solid tumors and for controlling the establishment of metastases. Administration of the angiogenesis inhibitor is generally carried out during or after chemotherapy, however, it is preferred to inhibit angiogenesis after the tumor tissue has undergone a chemotherapy regimen in response to toxic insult by inducing restoration of angiogenesis through the supply of blood and nutrients to the tumor tissue. Furthermore, it is preferred to administer the angiogenesis inhibition method after surgery in which a solid tumor has been excised as a precancerous tumor.

Since the present method is used to inhibit tumor neovascularization, the method can also be used to inhibit tumor tissue growth, inhibit metastasis formation, and regress established tumors.

Restenosis is a process of Smooth Muscle Cell (SMC) migration and proliferation into tissue at the site of percutaneous transluminal coronary angioplasty that prevents successful angioplasty. The migration and proliferation of SMC's during this restenosis process can be considered to be the angiogenic process inhibited by the present method. Accordingly, the present invention also concerns a method of inhibiting restenosis by inhibiting angiogenesis in a patient following angioplasty according to the present method. To inhibit restenosis, the inactivated tyrosine kinase is typically administered after angioplasty, because the coronary wall is compromised by restenosis, for about 2 to about 28 days, and more typically about the first 14 days after the surgery.

The present method for inhibiting angiogenesis associated with a disease condition in a tissue and thereby also for carrying out a method of treatment of a disease involving angiogenesis comprises the step of contacting the tissue at which angiogenesis occurs or is at risk of developing angiogenesis with a composition comprising a therapeutically effective amount of an inactivated Src protein or a vector expressing the protein.

Inhibition of angiogenesis and tumor regression occurred the first 7 days after initial contact with the therapeutic composition. The additional or prolonged contact with the inactivated Src protein is preferably carried out for 7 days to 6 weeks, preferably about 14 days to 28 days.

2. Enhancement of angiogenesis

In situations where it is desirable to promote or enhance angiogenesis, it is useful to administer an active Src protein to a tissue. The route and timing of administration may be similar to the methods described above for inhibition.

F. Therapeutic compositions

The present invention concerns therapeutic compositions for use in practicing the methods of treatment described herein. The therapeutic compositions of the present invention comprise a pharmacologically tolerable carrier and a Src protein or a carrier capable of expressing a Src protein as described herein dissolved or dispersed therein as an active ingredient. In a preferred embodiment, the therapeutic composition is not immunogenic when administered to a mammalian or human patient for therapeutic purposes.

As used herein, the terms "pharmaceutically acceptable", "pharmacologically tolerable" and grammatical variations thereof, as they refer to compositions, carriers, diluents and agents, are used interchangeably and represent substances that can be administered or administered to a mammal without the occurrence of undesirable pharmacological effects such as nausea, dizziness, gastrointestinal upset and the like.

The formulation of pharmaceutical compositions containing an active ingredient dissolved or dispersed therein is well known in the art and is not limited based on the formulation. Such compositions are typically prepared as liquid solutions or suspensions for injection, although solid dosage forms suitable for preparation as solutions or suspensions prior to use in liquid form may also be prepared. The preparation can also be emulsified or made into liposome composition.

The active ingredient may be mixed with pharmaceutically acceptable and compatible excipients in amounts suitable for use in the methods of treatment described herein. For example, suitable excipients are water, saline, dextrose, glycerol, ethanol, and the like, and mixtures thereof. In addition, if desired, the compositions may contain minor amounts of auxiliary substances which enhance the effectiveness of the active ingredient, such as wetting or emulsifying agents, pH buffering agents, and the like.

The therapeutic compositions of the present invention may include pharmaceutically acceptable salts of any salt-forming component therein. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the polypeptide) formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or organic acids such as acetic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like.

Pharmacologically tolerated carriers for active ingredients are well known in the art. Examples of liquid carriers are sterile aqueous solutions containing no material, including the active ingredient and water, or containing a buffer, such as sodium phosphate at physiological pH, physiological saline, or both (such as phosphate buffered saline). Still further, the aqueous carrier may contain one or more buffer salts as well as salts such as sodium chloride and potassium chloride, dextrose, polyethylene glycol, and other solutes.

The liquid composition may also contain a liquid phase including water and not including water. Exemplary such additional liquid phases are glycerol, vegetable oils such as cottonseed oil, and water-oil emulsions.

Therapeutic compositions comprising an angiogenesis modulating amount of Src protein of the invention, or a substantially recombinant DNA expression vector expressing an effective amount of Src protein, are generally formulated to contain at least 0.1 weight percent Src protein based on the weight of the total therapeutic components. The weight percentages are the weight ratio of Src protein to total composition. Thus, for example, 0.1 weight percent is 0.1g Src protein per 100g total composition. For DNA expression vectors, the amount administered will depend on the nature of the expression vector, the tissue being treated, and similar considerations.

G. Article of manufacture

The present invention also concerns an article of manufacture for providing a labelled container of a Src protein of the invention. The article of manufacture comprises a packaging material bearing suitable indicia for the condition being treated and a medicament contained within the packaging material.

The agent in the article of manufacture is any of the compositions of the present invention suitable for providing Src protein and formulated into a pharmaceutically-acceptable dosage form as described herein in accordance with the disclosed indications. Thus, the composition may contain Src protein or a DNA molecule capable of expressing Src protein. The article of manufacture contains an amount of the agent sufficient for the treatment of the diseases described herein, which may be a unit dose or multiple doses.

The packaging material carries indicia indicating the use of the agent contained therein, e.g., to aid in the treatment of diseases by inhibiting or enhancing angiogenesis and similar diseases disclosed herein. The indicia may further include instructions and related information needed for sale. The packaging may comprise a container for storing a medicament.

The term packaging material as used herein refers to a material such as glass, plastic, paper, foil, etc. which is capable of retaining a medicament in a holding device. Thus, for example, the packaging material may be a plastic or glass vial, a layered enclosure, and a container for containing pharmaceutical compositions including medicaments.

In a preferred embodiment, the packaging material includes a marking which is a positive indication of the content of the article and the use of the medicament contained therein.

Examples

The following examples relating to the present invention are illustrative and should not, of course, be taken as limiting the invention in particular. Further, such variations of the invention that are currently known or later developed within the purview of one skilled in the art are considered to be within the scope of the invention as hereinafter claimed.

Preparation of c-Src expression constructs

To prepare an expression construct for modulating angiogenesis by the method of the invention, the c-Src cDNA is manipulated and inserted into the expression construct/vector.

FIG. 1 shows the cDNA sequence (SEQ ID NO.: 2) encoding the wild-type (i.e. endogenous) chicken c-Src, and FIG. 2 shows the encoded amino acid residue sequence (SEQ ID NO.: 3). The encoded protein sequence was translated from nucleotide 112-1713 of the cDNA. The nucleic acid sequence (SEQ ID NO.: 4) and the encoded amino acid residue (SEQ ID NO.: 5) sequences corresponding to the human c-Src cDNA are shown in FIGS. 3 and 4, respectively. In the case of the human protein sequence, the coding sequence begins at nucleotide 134-1486 of the cDNA.

Wild-type and a large number of mutant c-Src cDNAs were prepared. The use of Kaplan et al in the journal of the European Association of molecular biology(EMBO J.)13: 4745 directed mutagenesis as described in 4756(1994) to prepare mutant c-Src constructs. Mutant c-Sr encoding mutant c-Src protein for use in the method of the present inventionc constructs such as Kaplan et al are described in the same references mentioned above. Kaplan et al describe various mutant c-Src constructs and encoded proteins useful in the practice of the present invention. For example, Kaplan et al, in FIG. 1 thereof, depict several products of the chicken c-Src allele, including SrcA and Src 251.

Two classes of c-Src that act to modulate angiogenesis are described. As mentioned above, one class contains Src molecules that increase angiogenesis and are thus considered active proteins. In the present invention, wild-type Src as well as various mutants that induce angiogenesis were confirmed. In this context according toIn vivoOne preferred mutation of wild type c-Src that contributes to the ability to induce vascular growth and thereby increase tumor weight is a Src a mutant with a point mutation at amino acid (aa) residue 527 where tyrosine 527 is changed to phenylalanine. This site is usually the site for down-regulation by the c-Src kinase known as the kinase CSK. The protein is inactivated when CSK can phosphorylate aa527 in wild type src. However, in mutant Src a, the regulated tyrosine is converted to phenylalanine, thereby conferring constitutive (i.e., permanent) activity to the protein without inactivating it through phosphorylation.

It was also demonstrated that the mutation in src has a reverse modulatory effect on angiogenesis, thereby inhibiting angiogenesis rather than stimulating it. Such mutations are referred to as inactivating src mutations. Proteins with mutations that provide this inhibitory activity are also referred to as dominant negative Src proteins, i.e., they inhibit neovascularization, including that caused by endogenous activity of Src and by increased Src activity stimulated by growth factors. Thus, certain mutants of the wild-type c-src of the invention block vascular growth in respect thereof and, for example, thereby reduceIn vivoThe ability to weight the tumor also plays a dominant negative role.

Such preferred inhibitory c-Src proteins include Src251, which expresses only the first 251 amino acids of Src. This construct lacks the complete kinase domain and is therefore referred to as the "kinase dead" src protein. The second construct is a Src (K295M) mutant with the lysine amino acid residue at position 295 mutated to methionine. Point mutations in this kinase domain prevent ATP binding and also block kinase-dependent Src function associated with vascular and tumor cell signaling and proliferation.

For example, for mutations at residue 527, so long as the resulting mutant amino acid residue is not tyrosine, serine, or threonine, it is contemplated that the presence of another amino acid at the desired site will result in a Src protein having the desired activity, promoting angiogenesis modulating activity.

In the case of point mutations, any mutant which results in the desired inhibitory or stimulatory activity is of interest for use in the present invention. Also contemplated are fusion protein constructs that incorporate the desired src protein (mutant or fragment thereof) with expressed amino acid tags, epitopes, fluorescent proteins, or other such proteins or peptides, provided that the desired modulation of the src protein is intact.

TABLE I

Effect of Src/mutant Src function on angiogenesis

Stimulation of c-Src + Activity

SrcA (T527F) + stimulation of Activity

Src527 (point) + Activity stimulation

Src 251-inactivation inhibition

Src (truncate) -inactivation inhibition

Src (K295M) -inactivation inhibition

Src295 (Point) -inactivation inhibition

One preferred expression construct for use in the present invention is the RCASBP (A) construct (SEQ ID NO).: 1). This expression vector is based on a series of replication competent avian sarcoma viruses containing enhanced Bryan Polymerase (BP) with improved titers and is specific for type A envelope glycoproteins expressed on normal avian cells (reviewed in Methods in Cell Biology, 52: 179-214 (1997); see also Hughes et al, 1987, J. Virol)(J.Virol.)61：3004-3012；Fekete&Cepko, 1993 cytomolecular biology(Mol.Cellular Biol.)13(4): 2604-2613; itoh et al, 1996 development(Development)122: 291-300; and Stott et al 1998 biotechnological(BioTechniques)24: 660-666). The full sequence of RCASBP (A) (SEQ ID NO: 1) is given in the accompanying sequence Listing and the restriction map of this construct is depicted as FIG. 10, referred to herein as RCAS.

The original Src251 construct was subcloned by NIH in dr. harbord varmus' laboratories by dr. pam schwartberg. Briefly, cloning of the Src cDNA sequence to facilitate its expression is accomplished by inserting a linker containing a Not I-BstB1-Not I restriction site into the unique Not I site in the 5' end of Src 251. Src carries a unique Cla I site at the 3' end. Digestion of Src251 with BstB1 and Cla I yields a BstB1-Cla I fragment, which is then ligated to the Cla I site on RCASBP (A). The overhang of BstB1 required ligation with a Cla I overhang that was not re-cut with Cla I. Src constructs suitable for use in the practice of the invention are conveniently obtained in the above vector by first digesting the RCAS vector containing Src251 (in the DAM + background) with Not I and Cla I for insertion of similarly digested Src cDNA. Thus, this original RCASBP (A) construct containing Src251 is further used as described above and Kaplan et al (1994, journal of the European Association of molecular biology)(EMBO J.)13(20): 4745-4756) all other Src constructs described by Src251 construction were subcloned into RCASBP (A) by the Not I-Cla I fragment generated. To generate the desired c-src mutation in the cDNA, standard site-directed mutagenesis procedures well known to those skilled in the art are applied. PCR primers designed to introduce the desired mutation are also designed with restriction sites to facilitateFollowed by a cloning step. The entire fragment of the nucleic acid sequence encoding Src is deleted from the nucleic acid construct by PCR amplification techniques based on the well-known cDNA sequences of chicken, human and similar homologs of Src and subsequent formation of a new construct.

In one embodiment of the invention, the 3' PCR primers used to amplify the src nucleic acid also encode in-frame sequences. This primer was used to add a 9E10-myc epitope tag to the carboxy terminus of the subsequent Src construct.

The following amino acids were added after amino acid 251 of Src in order to generate a vector construct containing a 9E10-myc epitope tag: VDMEQKLIAEEDLN (SEQ ID NO: 6). Two independent PCRs were performed for each construct and similar results were obtained. All mutant constructs constructed by PCR were also sequenced by PCR to confirm the cloned putative DNA sequence. Wild-type and mutant Src cDNAs for use in the expression systems of the invention are also commercially available from commercial birds as well as human Src and Upstate biotech laboratories, Lake plasmid, NY, of several kinase death and activation mutants.

Still another expression vector for expressing a Src protein of the invention includes adenoviral vectors as described in U.S. Pat. nos. 4,797,368, 5,173,414, 5,436,146, 5,589,377, and 5,670,488. Another method for transporting Src-modulated proteins includes transporting Src cDNA using a non-viral vector system as described in U.S. Pat. No. 5,675,954 and transporting the cDNA itself as naked DNA as described in U.S. Pat. No. 5,589,466. The delivery of the constructs of the invention is also not limited to the topical administration of viral vectors as described in the following CAM assay systems. For example, viral vector formulations are also administered by intravenous injection for systemic transport into vascular beds. These vectors can also be directed to sites of increased vascularization by local injection into the tumor, as an example.

To pairIn vitroOf interest for the expressed protein is its transport after expression and purification of the selected Src protein by the method used for transporting the protein or polypeptide. One such method includes, for example, U.S. Pat. Nos. 4,356,167, 5,580,575. 5,542,935 and 5,643,599. Other vectors and protein delivery systems for expressing and/or delivering the Src protein of the invention are well known to those skilled in the art.

2. Characterization of untreated chick chorioallantoic Membrane (CAM)

Preparation of CAM

Angiogenesis can be induced on the chick chorioallantoic membrane (CAM) after normal embryo angiogenesis leads to mature vessel formation. Induced angiogenesis has been shown to occur in nature by Leibovich et al(Nature)329: 630(1987) and Ausprunk et al, J. American Pathology(Am.J.Pathol.)79: 597(1975) or tumor fragments. CAMs are prepared from chicken embryos which are subsequently used to induce angiogenesis and its inhibition. 10-day-old chicken embryos were obtained from McIntyre Poultry (Lakeside, Calif.) and cultured at 37 ℃ and 60% humidity. A small hole was made through the shell of the egg placed directly on the edge of the egg on the balloon using a small tool drill (Dremel, Division of Emerson Electric co. A second hole was drilled on the broad side of the egg in the area of the embryo vessel not previously determined by inspection of the egg for light transmission. Negative pressure was applied to the original wells, resulting in a CAM (chorioallantoic membrane) that was pulled out of the eggshell membrane and created a false air pocket on the CAM. A1.0 centimeter (cm) by 1.0cm square opening was punched into the shell of the egg on the dropped CAM using a small grinding wheel (Dremel). The small opening allows direct access to the CAM below.

The resulting CAM preparations were then used 6 days of embryogenesis, which was marked by active neovascularization, with no additional treatment of the CAM reflecting the pattern used to assess the effect on embryonic vascularization or use at 10 days of embryonic vascularization where angiogenesis had declined. The latter preparation is thus used in the present invention to induce reinitiated angiogenesis in response to cytokine treatment or tumor exposure as described below.

Assay for CAM angiogenesis

A. Growth factor-induced angiogenesis

Angiogenesis has been shown to be induced by cytokines or growth factors.

Angiogenesis was induced by placing 5 millimeter (mm) x 5mm Whatman filter discs (Whatman filter paper No. 1) saturated with Hanks balanced salt solution (HBSS, GIBCO, Grand Island, NY) containing 2 micrograms/milliliter (μ g/ml) of recombinant basic fibroblast growth factor (bFGF) or Vascular Endothelial Growth Factor (VEGF) (Genzyme, Cambridge, MA) on the CAM of 9 or 10 day old chicken embryos in the area without blood vessels and then sealing the openings with tape. Other concentrations of growth factors are also effective in inducing blood vessel growth. For assays to assess angiogenesis inhibition by intravenous administration of antagonists, angiogenesis was first induced in fibroblast growth medium with 1-2 μ g/ml bFGF or VEGF. Angiogenesis was monitored by microscopy after 72 hours.

Embryo angiogenesis

The CAM preparations used to evaluate the effect of angiogenesis inhibitors on the formation of naturally occurring embryonic neovascularization were embryos from 6-day-old embryonated chicken as described above. During this development phase, the blood vessels are allowed to re-grow and thus provide a useful system for assessing the angiogenic modulation by the Src protein of the invention. The CAM system was prepared as described above except that the assay was performed at day 6 embryos instead of day 9 or 10 embryos.

4. Modulation of angiogenesis as determined by CAM detection assay

To assess the effect of Src protein on angiogenesis, the following assay was performed on 10-day-old chicken CAM preparations. 5 μ g of the RCAS construct prepared as described in example 1 was transfected into the chicken immortalized fibroblast cell line DF-1 (gift from Doug Foster, U.S. of Minn.). This cell line, as well as the primary chicken embryo fibroblast cell line, was able to produce viruses, although the DF-1 cell line produced higher titers. In serum-free CLM medium [ composition: using DMSO, folic acid, and riceF-10 medium substrate supplemented with amino acids and MEM vitamin solution]Wherein virus supernatant is collected from a subconfluent DF-1 producer cell line. 35ml of virus supernatant was concentrated by ultracentrifugation at 22,000rpm for 2 hours at 4 ℃. These concentrated virus pellets were resuspended in 1/100 original volume in serum-free CLM medium, aliquoted and stored at-80 ℃. Titers were estimated by infection on primary chicken embryo fibroblasts cultured for 48-72 hours by sequential dilution of a control viral vector having a nucleotide sequence encoding Green Fluorescent Protein (GFP) called RCAS-GFP. The titer of the virus stock solution obtained after concentration is usually more than 10⁸I.u./ml. To perform the CAM detection assay using the virus stock solution, a 6mm diameter Whatman filter disc of cortisone acetate soaked for 30 minutes with 3mg/ml cortisone acetate in 95% ethanol was prepared. The filter discs were dried in a laminar flow hood hyperstatic bench and then soaked with 20 μ Ι of virus stock solution/disc for 10 minutes. These pieces were placed on the CAM of 9 or 10 day old chicken embryos and sealed with cellophane tape and incubated at 37 ℃ for 18-24 hours. The mock PBS or growth factor was then added to the CAM in a 20. mu.l volume of the appropriate virus stock solution at a concentration of 5. mu.g/ml as an additional virus boost to the CAM tissue. After 72 hours, CAMs were collected and examined for changes in angiogenic index as determined by double-blind counting of the number of fulcrums in the sub-disc CAM. For the kinase detection assay, the sublaminated tissue was collected in RIPA, homogenized with an automated grinder and Src immunoprecipitated from an equal amount of total protein and performed using FAK-GST fusion protein as substrateIn vitroA kinase detection assay. For immunofluorescence studies, CAM tissue was frozen with cryo-OCT under slides, cut into 4 μm pieces, fixed in acetone for 1 minute, cultured in 3% normal goat serum for 1 hour, and subsequently cultured in primary rabbit anti-phospho ERK antibody as described above (Eliceiri et al, J. Cell. biol.,(J.Cell.Biol.)140: 1255-1263(1998)), washed in PBS and detected with a fluorescent secondary antibody.

A. Activation of endogenous Src by bFGF or VEGF

To evaluate the growth factorThe following assays were performed to determine the effect of the factor on modulating angiogenic Src activity. Tissue extracts of 10 day old chicken CAMs that had been exposed to bFGF or VEGF (2 μ g/ml) for 2 hours were lysed. Immunoprecipitation of endogenous Src from an equivalent amount of total protein and its performance using FAK-GST fusion protein as a substrateIn vitroImmune complex kinase detection test, electrophoresis and transfer to nitrocellulose.

The results of the assay are shown in figure 5, where there is a significant increase in Src activity during the increase in gel density with bFGF or VEGF treatment compared to untreated (mock) samples that exhibited baseline Src activity in the CAM assay. bFGF and VEGF, which resulted in an approximately 2-fold increase in endogenous Src activity, were present in the CAM. The above kinase assay blots were also probed with an anti-Src antibody as a loading control with equal Src and IgG content.

B. Effect of retrovirus-mediated Src A Gene expression on angiogenesis in Chicken CAM

The following assays were performed to evaluate the effect of mutant Src proteins on angiogenesis in CAM preparations. To perform this assay, 9-day-old chicken CAMs were contacted with retroviral-expressing RCAS-Src a or RCAS-GFP or buffer for 72 hours following the protocol described above.

The results of the experiment are shown in fig. 6, where the level of angiogenesis was quantified as described above. A representative micrograph (4x) was recorded with a stereomicroscope as shown in fig. 6B. The baseline endogenous Src activity has a angiogenic index of about 50. In contrast, the RCAS treated with RCAS-Src a expressed from a retroviral vector having a point mutation at amino acid residue 527 from tyrosine to phenylalanine resulted in an enhanced (evoked) angiogenic index of angiogenesis of about 90. The increase in Src-a mediated angiogenesis is evident in the photograph shown in figure 6B.

Retroviral expression of src a activates vascular MAP kinase phosphorylation

The effect of Src a on vascular MAP kinase phosphorylation compared to the growth factors VEGF and PMA was also assessed following the experimental procedures described above and herein. Tissue extracts of 10 day old chicken CAMs exposed for 30 minutes to VEGF or PMA (another comparable concentration of mitogen) were compared to those infected with Src a expressing retrovirus for 48 hours. Src was then (than) immunoprecipitated from an equivalent amount of total protein extract and subjected to an in vitro immune complex kinase detection assay using FAK-GST fusion protein as substrate, electrophoresed and transferred to nitrocellulose.

The results of the assay are shown in fig. 7A, where untreated cams (nt) exhibit baseline endogenous Src-mediated phosphorylation of vascular MAP kinase. VEGF and PMA resulted in an approximately 2-fold increase over baseline. In contrast, Src a increased about 5-10 fold over the activity observed with the untreated sample.

The extent of endogenous ERK phosphorylation was also determined on aliquots of the above whole tissue lysates by immunoblotting with anti-phospho-ERK antibodies as shown in figure 7B. To perform this assessment, 10 day old CAMs were infected with either mock RCAS or SRC a expressing RCAS. After 2 days, the CAMs were dissected, cryo-preserved in OCT and cut into 4 μm pieces. Sections were immunostained with anti-phosphorylated ERK antibodies (New England Biolabs), washed and detected with goat anti-rabbit FITC conjugated secondary antibodies. The fluorescence images were captured on a stabilized CCD camera (Princeton Inst). The micrographs show enhanced immunofluorescence of the Src a treated preparations compared to the mock control.

D. Selective requirement for Src activity during VEGF, but not bFGF induced angiogenesis

To evaluate the effect of Src modulating activity on growth factor-induced angiogenesis, the following assays were performed. 9 day old chicken CAMs are contacted with retroviral preparations expressing dominant negative Src mutants called Src251 or Src 295M as described above. RCAS-Src251 or control RCAS-GFP retrovirus or buffer CAMS were treated for 20 hours and then incubated for 72 hours with or without bFGF or VEGF.

The levels of angiogenesis quantified as described above are shown in figure 8A. A representative micrograph (6x) shown in figure 8B was recorded using a stereomicroscope. Figure 8C illustrates blots probed with anti-Src antibodies to confirm Src251 expression in transfected cells compared to mock treatment.

The results of the above experiments indicate that bFGF and VEGF treated CAMS induced angiogenesis in the presence of the RCAS-GFP control exceeded Src-mediated baseline angiogenesis observed using either mock or untreated CAM preparations. The expressed dominant negative mutant Src251 was effective in inhibiting VEGF-induced angiogenesis back to baseline levels, but not in inhibiting bFGF-mediated angiogenesis. The micrograph shown in fig. 8B graphically confirms the data shown in fig. 8A. Thus, retrovirus-expressed Src251 is a potent angiogenesis inhibitor when VEGF is used to induce angiogenesis.

It is contemplated in the present invention that Src protein of the invention be administered with other models of angiogenesis as described in the examples below.

5. Regression of tumor tissue growth with Src modulators determined by in vivo rabbit eye model experiments

The effect of Src modulators on growth factor-induced angiogenesis can be observed in the natural transparent structures typically exemplified by the cornea of the eye. New blood vessels grow from the limbus, where they are rich in blood supply, to the center of the cornea, where they normally do not supply blood. Angiogenic stimulants such as bFGF induce new blood vessel growth from the limbus of the cornea when applied to the cornea. Angiogenesis antagonists applied to the cornea inhibit the growth of new blood vessels from the corneal limbus. Thus, the cornea undergoes angiogenesis by endothelial cell invasion from the corneal limbus into the tough, easily observable collagen-encapsulated corneal tissue. The rabbit eye model test thus provides a means for direct observation of irritation and inhibition of angiogenesis after direct implantation of the compound into the cornea of the eyeIn vivoAnd (4) modeling.

A. In vivo rabbit eye model test

1) Growth factor-induced angiogenesis

In thatIn vivoAngiogenesis was induced in a rabbit eye model assay with the growth factors bFGF or VEGF and described in the following section.

a. Preparation of Hydron particles containing growth factor and monoclonal antibody

For example, D' Amato et al in the proceedings of the national academy of sciences of the United states(Proc.Natl.Acad.Sci. USA),91: 4082-. Each granule contained 650ng of growth factor alone combined with sucralfate (Carafet, MarionMerrell Dow Corporation) to stabilize the growth factor and ensure its slow release into the surrounding tissue. Furthermore, hydro particles containing the desired Src-expressing retrovirus as described above were prepared. The granules were cast in specially prepared teflon shuttle cores with a 2.5mm core penetration surface. About 12. mu.l of the cast material was placed in each bobbin and polymerized overnight in a sterile fume hood. The particles are then sterilized by ultraviolet irradiation. The effect of Src protein was then assessed as described above.

6. A chimeric mouse: in vivo regression of tumor tissue growth with Src modulators as determined in human trials

In vivo chimeric mice were generated by replacing part of the skin from SCID mice with human neonatal foreskin: a human model. For example, Yan et al in "J.Clin. Res."(J.Clin.Invest.)91: 986. sup. 996(1993)In vivoChimeric mice: a human model. In summary, 2cm were surgically removed from SCID mice (6-8 weeks old)²And replaced with a human foreskin. Mice were anesthetized and scraped from 5cm on each side of the lateral abdominal region²The hair was removed from the area. Preparation of 2cm by taking full thickness skin down to fascia²Two annular graft beds. Full-thickness human skin grafts of the same size derived from human neonatal foreskin were placed on the wound bed and sutured in place. The graft was covered with a Band-Aid sutured to the skin. Will alsoThe microporous cloth belt is used for covering wounds.

The immunoreactivity of the M21-L human melanoma cell line or MDA23.1 breast cancer cell line (ATCC HTB 26; tissue sections using mAb LM609 was alpha_Vβ₃Negative) were used to form human solid tumors on human skin grafts on SCID mice. Will be 5X 10⁶Single cell suspensions of M21-L or MDA23.1 cells were injected intradermally into human skin grafts. Mice were then observed for 2-4 weeks to allow measurable growth of human tumors.

After establishment of measurable tumors, retroviral preparations of the present invention or PBS were injected into the tail vein of mice. After a period of 2-3 weeks, tumors were excised and analyzed by weight and histology. The effect of the Src proteins expressed by the invention on this tumour is then assessed.

7. In vitro regression of human tumor tissue growth with Src modulators as determined by CAM assay

Tumor growth is dependent on angiogenesis (Folkman, 1992; Weidner et al, 1991; Brooks et al, 1994 b). Indeed, recent reports suggest that tumor growth is sensitive to the anti-angiogenic effects of VEGF receptor antagonists (Kim et al, 1993). We therefore examined whether the inhibition of angiogenesis by transport kinase-deficient Src251 would affect the growth of human medulloblastoma (DAOY), a highly angiogenic tumor known to produce VEGF and minimal bFGF (data not shown).

Day 3 and 6 DAOY medulloblastoma growth assays were performed in chicken CAMs as described mainly above (Brooks et al, 1994). Washing 5X 10 cultured in RPMI 1640 containing 10% fetal bovine serum⁶DAOY cells were seeded onto CAM of day 10 embryos to generate DAOY tumor fragments. After 7 days, 50mg of the tumor fragments were dissected and reseeded on another 10-day embryo and cultured for an additional 3 or 6 days by topical administration (25. mu.l) of the control RCAS-GFP retrovirus, RCAS-Src251 or mock treatment. Using whole tissue focused imaging of infected tumors as a guide, we were able to use this local approach to determine the surrounding and internal tumor fragmentsSignificant expression of the RCAS construct was present. Tumor resection and weighing were performed in a double-blind fashion to remove only readily defined solid tumor masses (Brooks et al, 1994). Wet tumor weights after 3 or 6 days were compared to the original weight and the percent change in tumor weight for each group was determined.

These tumors were prone to growth on the CAM and generated active angiogenesis (fig. 9), allowing us to selectively target avian-derived tumor vasculature by using a bird-specific RCAS retrovirus.

FIG. 9 depicts the results showing retroviral transport of RCAS-Src251 to human tumors growing on chicken CAM, which regresses tumor growth. FIG. 9A shows human medulloblastoma grown on chicken embryonic CAM as described above. Retroviruses containing RCAS-GFP or RCAS-Src251 were topically applied to pre-established tumors greater than 50 mg. Representative micrographs of medulloblastoma fragments infected with GFP-expressing RCAS-GFP showed definitive expression in tumor vessels (arrows) detected by optical sectioning using a Bio Rad laser focus scanning microscope (raster 500 μm). FIG. 9B shows results from tumors treated as described above, which were grown for 3 or 6 days after tumor resection and determination of wet weight. Data are expressed as mean change in tumor weight (original weight from 50mg tumor) +/-SEM of 2 replicates. RCAS-Src251 had a significant effect on tumor growth after 3 days (, P < 0.002) and also after 6 days (, P < 0.05). Figure 9C shows a representative stereomicrograph of a medulloblastoma surgically removed from the embryo recorded with an Olympus stereomicroscope (raster: 350 μm). (lower panel) high magnification micrographs of each tumor specifically show the vasculature of each tumor (raster 350 μm). Arrows indicate vascular rupture in RCAS-Src251 treated tumors.

The results indicate that the transport of Src 251-containing RCAS to pre-established medulloblastoma produces selective viral expression in tumor-associated vessels (fig. 9A) and this ultimately leads to regression of these tumors over a 6-day interval (fig. 9B). Importantly, tumor-associated vessels were severely disrupted in animals treated with Src 251-containing virus and were in lower numbers than tumor vessels in control animals (fig. 9C). The fact that RCAS-GFP infected tumors showed that GFP was localized only in the tumor vasculature suggests that the anti-tumor effects observed with retrovirus transported Src251 were due to its anti-angiogenic properties.

The foregoing embodiments and the accompanying description are illustrative and are not to be construed as limiting. The invention is also not limited in scope to the deposited cell lines, as the deposited embodiments are intended to be illustrative of one aspect of the invention. Any functionally equivalent cell line is within the scope of the invention. The deposit of material does not constitute an admission that the written description contained herein is not sufficient to enable the practice of any aspect of the invention, including the best mode, nor is it intended to limit the scope of the claims to the particular interpretation represented thereby. Indeed, various modifications of the invention, including those illustrated and described herein, which would be obvious to those skilled in the art, are intended to be within the scope of the appended claims.

Sequence listing

<110> Scripps institute, etc

<120> methods and compositions for modulating angiogenesis using tyrosine kinase SRC

<130>TSRI 651.1

<140> is still unknown

<141> to be determined

<150>60/087,220

<151>1998-05-29

<160>6

<170> PatentIn version 2.0

<210>1

<211>11627

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: avian sarcoma virus-based RCASBP (A)

<220>

<221> misc _ feature

<222>(7649)..(11258)

<223> pBR322 sequences

<220>

<221>LTR

<222>(7166)..(7494)

<223> upstream

<220>

<221>LTR

<222>(1)..(101)

<223> upstream (start coding at upstream R)

<220>

<221> misc _ feature

<222>(11394)..(11623)

<223>U3

<220>

<221> misc _ feature

<222>(1)..(21)

<223>R

<220>

<221> misc _ feature

<222>(22)..(101)

<223>U5

<220>

<221> misc _ feature

<222>(102)..(119)

<220>

<221>LTR

<222>(7166)..(7494)

<223> downstream

<220>

<221> misc _ feature

<222>(7166)..(7393)

<223>U3

<220>

<221> misc _ feature

<222>(7394)..(7414)

<223>R

<220>

<221> misc _ feature

<222>(7415)..(7494)

<223>U5

<220>

<221> misc _ feature

<222>(7154)..(7165)

<223>PPT

<220>

<221> misc _ feature

<222>(388)..(391)

<223> splice Donor (AGGT)

<220>

<221> misc _ feature

<222>(5074)..(5077)

<223> env splice Acceptor (AGGC)

<220>

<221> misc _ feature

<222>(6982)..(6985)

<223> Clal splice acceptor (AGGA)

<220>

<221> Gene

<222>(372)..(902)

<223>gag p19

<220>

<221> Gene

<222>(909)..(1094)

<223>gag p10

<220>

<221> Gene

<222>(1095)..(1814)

<223>gag p27

<220>

<221> Gene

<222>(1843)..(2108)

<223>gag p12

<220>

<221> Gene

<222>(2109)..(2480)

<223>gag p15

<220>

<221> misc _ signal

<222>(2481)..(2483)

<223> gag termination

<220>

<221> Gene

<222>(2501)..(4216)

<223>pol RT

<220>

<221> Gene

<222>(4217)..(5185)

<223>pol IN

<220>

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<222>(5186)..(5188)

<223> pol termination

<220>

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<222>(5244)..(6263)

<223>env gp85

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<222>(6264)..(6878)

<223>env gp37

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<222>(6879)..(6881)

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<223> in Dam + strain, Clal site/Clal site in gag was methylated and not cleaved.

<400>1

gccatttgac cattcaccac attggtgtgc acctgggttg atggccggac cgttgattcc 60

ctgacgacta cgagcacctg catgaagcag aaggcttcat ttggtgaccc cgacgtgata 120

gttagggaat agtggtcggc cacagacggc gtggcgatcc tgtctccatc cgtctcgtct 180

atcgggaggc gagttcgatg accctggtgg agggggctgc ggcttaggga ggcagaagct 240

gagtaccgtc ggagggagct ccagggcccg gagcgactga cccctgccga gaactcagag 300

ggtcgtcgga agacggagag tgagcccgac gaccacccca ggcacgtctt tggtcggcct 360

gcggatcaag catggaagcc gtcattaagg tgatttcgtc cgcgtgtaaa acctattgcg 420

ggaaaatctc tccttctaag aaggaaatcg gggccatgtt gtccctgtta caaaaggaag 480

ggttgcttat gtctccctca gatttatatt ctccggggtc ctgggatccc atcactgcgg 540

cgctctccca gcgggcaatg gtacttggaa aatcgggaga gttaaaaacc tggggattgg 600

ttttgggggc attgaaggcg gctcgagagg aacaggttac atctgagcaa gcaaagtttt 660

ggttgggatt agggggaggg agggtctctc ccccaggtcc ggagtgcatc gagaaaccag 720

ctacggagcg gcgaatcgac aaaggggagg aggtgggaga aacaactgtg cagcgagatg 780

cgaagatggc gccagaggaa gcggccacac ctaaaaccgt tggcacatcc tgctatcatt 840

gcggaacagc tgttggctgc aattgcgcca ccgccacagc ctcggcccct cctccccctt 900

atgtggggag tggtttgtat ccttccctgg cgggggtggg agagcagcag ggccagggag 960

ataacacgtc tcggggggcg gagcagccaa gggaggagcc agggcacgcg ggtcaggccc 1020

ctgggccggc cctgactgac tgggcaaggg taagggagga gcttgcgagt actggtccgc 1080

ccgtggtggc catgcctgta gtgattaaga cagagggacc cgcctggacc cctctggagc 1140

caaaattgat cacaagactg gctgatacgg tcaggaccaa gggcttacga tccccgatca 1200

ctatggcaga agtggaagcg ctcatgtcct ccccgttgct gccgcatgac gtcacgaatc 1260

taatgagagt gattttagga cctgccccat atgccttatg gatggacgct tggggagtcc 1320

aactccagac ggttatagcg gcagccactc gcgacccccg acacccagcg aacggtcaag 1380

ggcgggggga acggactaac ttggatcgat taaagggctt agctgatggg atggtgggca 1440

acccacaggg tcaggccgca ttattaagac cgggggaatt ggttgctatt acggcgtcgg 1500

ctctccaggc gtttagagaa gttgcccggc tggcggaacc tgcaggtcca tgggcggaca 1560

tcacgcaggg accatctgag tcctttgttg attttgccaa tcggcttata aaggcggttg 1620

aggggtcaga tctcccgcct tccgcgcggg ctccggtgat cattgactgc tttaggcaga 1680

agtcacagcc agatattcag cagcttatac gggcagcacc ctccacgctg accaccccag 1740

gagagataat caaatatgtg ctagacaggc agaagattgc ccctcttacg gatcaaggca 1800

tagccgcggc catgtcgtct gctatccagc ccttagttat ggcagtagtc aatagagaga 1860

gggatggaca aactgggtcg ggtggtcgtg cccgagggct ctgctacact tgtggatccc 1920

cgggacatta tcaggcacag tgcccgaaaa aacgaaagtc aggaaacagc cgtgagcgat 1980

gtcagctgtg tgacgggatg ggacacaacg ctaaacagtg taggaagcgg gatggcaacc 2040

agggccaacg cccaggaaga ggtctctctt cggggccgtg gcccggccct gagcagcctg 2100

ccgtctcgtt agcgatgaca atggaacata aagatcgccc cttggttagg gtcattctga 2160

ctaacactgg gagtcatcca gtcaaacaac gttcggtgta tatcaccgcg ctgttggact 2220

ccggagcgga catcactatt atttcggagg aggattggcc tactgattgg ccggtggtgg 2280

acaccgcgaa cccacagatc catggcatag gagggggaat tcccatgcga aaatcccggg 2340

atatgataga ggtgggggtt attaaccgag acgggtcgtt ggagcgaccc ctgctcctct 2400

tccccgcagt cgctatggtt agagggagta tcctaggaag agattgtctg cagggcctag 2460

ggctccgctt gacaaattta tagggagggc cactgttctc actgttgcgc tacatctggc 2520

tattccgctc aaatggaagc cagaccgcac gcctgtgtgg attgaccagt ggcccctccc 2580

tgaaggtaaa cttgtaggcc taacgcaatt agtggaaaaa gaattacagt taggacatat 2640

agagccctca cttagttgtt ggaacacacc tgtttttcgt gatccggaag gcttccgggt 2700

cttatcgctt attgcatgat ttgcgcgctg ttaacgccaa gcttgtccct tttggggccg 2760

tccaacaggg ggcgccagtt ctctccgcgc tcccgcgtgg ctggcccctg atggtcctag 2820

acctcaagga ttgcttcttt tctatccctc ttgcggaaca agatcgcgaa gcttttgcat 2880

ttacgctccc ctctgtgaat aaccaggccc ccgctcgaag attccaatgg aaggtcttgc 2940

cccaagggat gacctgttct cccactatct gtcagttggt agtgggtcag gtgctcgagc 3000

ccttgcgact caagcaccca gctctgcgca tgttgcatta tatggacgat cttttgctag 3060

ccgcctcaag tcatgatggg ttggaagcgg cagggaagga ggttatcggt acattggaaa 3120

gagccgggtt cactatttcg ccggataaga tccagaggga gcccggagta caatatcttg 3180

ggtacaagtt aggcagtacg tatgtagcac ccgtaggctt ggtagcagaa cccaggatag 3240

ccaccttgtg ggatgttcaa aagctggtgg ggtcacttca gtggcttcgc ccagcgttag 3300

ggatcccgcc acgactgatg ggtccctttt atgagcagtt acgagggtca gatcctaacg 3360

aggcgaggga atggaatcta gacatgaaaa tggcctggag agagatcgta cagcttagca 3420

ctactgctgc cttggaacga tgggaccctg cccagcctct ggaaggagcg gtcgctagat 3480

gtgaacaggg ggcaataggg gtcctgggac agggactgtc cacacaccca aggccatgtt 3540

tgtggttatt ctccacccaa cccaccaagg cgtttactgc ttggttagaa gtgctcaccc 3600

ttttgattac taagctacgc gcttcggcag tgcgaacctt tggcaaggag gttgatatcc 3660

tcctgttgcc tgcatgcttc cgggaggacc ttccgctccc ggaggggatc ctgttagcac 3720

ttagggggtt tgcaggaaaa atcaggagta gtgacacgcc atctattttt gacattgcgc 3780

gtccactgca tgtttctctg aaagtgaggg ttaccgacca ccctgtgccg ggacccactg 3840

tctttaccga cgcctcctca agcacccata aaggggtggt agtctggagg gagggcccaa 3900

ggtgggagat aaaagaaata gttgatttgg gggcaagtgt acaacaactg gaggcacgcg 3960

ctgtggccat ggcacttctg ctgtggccga caacgcccac taatgtagtg actgactctg 4020

cgtttgttgc gaaaatgtta ctcaagatgg gacaggaggg agtcccgtct acagcggcgg 4080

cttttatttt agaggatgcg ttaagccaaa ggtcagccat ggccgccgtt ctccacgtgc 4140

ggagtcattc tgaagtgcca gggtttttca cagaaggaaa tgacgtggca gatagccaag 4200

ccacctttca agcgtatccc ttgagagagg ctaaagatct tcataccgct ctccatattg 4260

gaccccgcgc gctatccaaa gcgtgtaata tatctatgca gcaggctagg gaggttgttc 4320

agacctgccc gcattgtaat tcagcccctg cgttggaggc cggggtaaac cctaggggtt 4380

tgggacccct acagatatgg cagacagact ttacgcttga gcctagaatg gctccccgtt 4440

cctggctcgc tgttactgtg gacaccgcct catcagcgat agtcgtaact cagcatggcc 4500

gtgttacatc ggttgctgca caacatcatt gggccacggc tatcgccgtt ttgggaagac 4560

caaaggccat aaaaacagat aacgggtcct gcttcacgtc cagatccacg cgagagtggc 4620

tcgcgagatg ggggatagca cacaccaccg ggattccggg aaattcccag ggtcaagcta 4680

tggtagagcg ggccaaccgg ctcctgaaag ataagatccg tgtgctcgcg gagggggacg 4740

gctttatgaa aagaatcccc accagcaaac agggggaact attagccaag gcaatgtatg 4800

ccctcaatca ctttgagcgt ggtgaaaaca caaaaacacc gatacaaaaa cactggagac 4860

ctaccgttct tacagaagga cccccggtta aaatacgaat agagacaggg gagtgggaaa 4920

aaggatggaa cgtgctggtc tggggacgag gttatgccgc tgtgaaaaac agggacactg 4980

ataaggttat ttgggtaccc tctcggaaag ttaaaccgga tgtcacccaa aaggatgagg 5040

tgactaagaa agatgaggcg agccctcttt ttgcaggcat ttctgactgg ataccctggg 5100

aagacgagca agaaggactc caaggagaaa ccgctagcaa caagcaagaa agacccggag 5160

aagacaccct tgctgccaac gagagttaat tatattctca ttattggtgt cctggtcttg 5220

tgtgaggtta cgggggtaag agctgatgtc cacttactcg agcagccagg gaacctttgg 5280

attacatggg ccaaccgtac aggccaaacg gatttttgcc tctctacaca gtcagccacc 5340

tccccttttc aaacatgttt gataggtatc ccgtccccta tttccgaggg tgattttaag 5400

ggatatgttt ctgatacaaa ttgcaccacc ttgggaactg atcggttagt ctcgtcagcc 5460

gactttactg gcggacctga caacagtacc accctcactt atcggaaggt ctcatgcttg 5520

ttgttaaagc tgaatgtctc tatgtgggat gagccacctg aactacagct gttaggttcc 5580

cagtctctcc ctaacattac taatattgct cagatttccg gtataaccgg gggatgcgta 5640

ggcttcagac cacaaggggt tccttggtat ctaggttggt ctagacagga ggccacgcgg 5700

tttctcctta gacacccctc tttctctaaa tccacggaac cgtttacagt ggtgacagcg 5760

gataggcaca atctttttat ggggagtgag tactgcggtg catatggcta cagattttgg 5820

aacatgtata actgctcaca ggtggggcgg cagtaccgct gtggtaatgc gcgcacgccc 5880

cgcacgggtc ttcctgaaat ccagtgtaca aggagaggag gcaaatgggt taatcaatca 5940

caggaaatta atgagtcgga gccgttcagc tttacggtga actgtacagc tagtagtttg 6000

ggtaatgcca gtgggtgttg cggaaaagca ggcacgattc tcccgggaaa gtgggtcgac 6060

agcacacaag gtagtttcac caaaccaaaa gcgctaccac ccgcaatttt cctcatttgt 6120

ggggatcgcg catggcaagg aattcccagt cgtccggtag ggggcccctg ctatttaggc 6180

aagcttacca tgttagcacc taagcataca gatattctca aggtgcttgt caattcatcg 6240

cggacaggta taagacgtaa acgaagcacc tcacacctgg atgatacatg ctcagatgaa 6300

gtgcagcttt ggggtcctac agcaagaatc tttgcatcta tcctagcccc gggggtagca 6360

gctgcgcaag ccttaagaga aattgagaga ctagcctgtt ggtccgttaa acaggctaac 6420

ttgacaacat cactcctcgg ggacttattg gatgatgtca cgagtattcg acacgcggtc 6480

ctgcagaacc gagcggctat tgacttcttg ctcctagctc acggccatgg ctgtgaggac 6540

gttgccggaa tgtgctgttt caatttgagt gatcagagtg agtctataca gaagaagttc 6600

cagctaatga aggaacatgt caataagatc ggcgtggata gcgacctaat tggaagttgg 6660

ctgcgaggac tattcggggg aataggagaa tgggccgttc atttgctgaa aggactgctt 6720

ttggggcttg tagttatttt gttgctagta gtgtgcctgc cttgcctttt gcaaatgtta 6780

tgcggtaata ggagaaagat gattaataac tccatcagct accacacgga atataagaag 6840

ctgcaaaagg cctgtgggca gcctgaaagc agaatagtat aaggcagtac atgggtggtg 6900

gtatagcgct tgcgagtcca tcgagcaagg caggaaagac agctattggt aattgtgaaa 6960

tacgcttttg tctgtgtgct gcaggagctg agctgactct gctggtggcc tcgcgtacca 7020

ctgtggcatc gatgcgatgt acgggccaga tatacgcgta tctgagggga ctagggtgtg 7080

tttaggcgaa aagcggggct tcggttgtac gcggttagga gtccccttag gatatagtag 7140

tttcgctttt gcatagggag ggggaaatgt agtcttatgc aatactcttg tagtcttgca 7200

acatggtaac gatgagttag caacatgcct tacaaggaga gaaaaagcac cgtgcatgcc 7260

gattggtgga agtaaggtgg tacgatcgtg ccttattagg aaggcaacag acgggtctga 7320

catggattgg acgaaccact gaattccgca ttgcagagat attgtattta agtgcctagc 7380

tcgatacaat aaacgccatt tgaccattca ccacattggt gtgcacctgg gttgatggcc 7440

ggaccgttga ttccctgacg actacgagca cctgcatgaa gcagaaggct tcatttggtg 7500

accccgacgt gatagttagg gaatagtggt cggccacaga cggcgtggcg atcctgtctc 7560

catccgtctc gtctatcggg aggcgacttc gatgaccctg gtggaggggg ctgcggctta 7620

gggaggcaga agctgagtac cgtcggaggg gatccacagg acgggtgtgg tcgccatgat 7680

cgcgtagtcg atagtggctc caagtagcga agcgagcagg actgggcggc ggccaaagcg 7740

gtcggacagt gctccgagaa cgggtgcgca tagaaattgc atcaacgcat atagcgccag 7800

cagcacgcca tagtgactgg cgatgctgtc ggaatggacg atatcccgca agaggcccgg 7860

cagtaccggc ataaccaagc ctatgcctac agcatccagg gtgacggtgc cgaggatgac 7920

gatgagcgca ttgttagatt tcatacacgg tgcctgactg cgttagcaat ttaactgtga 7980

taaactaccg cattaaagct ccaaacttgg ctgtttcctg tgtgaaattg ttatccgctc 8040

acaattccac acattatacg agccggaagc ataaagtgta aaacctgggg tgcctaatga 8100

gtgagaattc ttgaagacga aagggcctcg tgatacgcct atttttatag gttaatgtca 8160

tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc 8220

ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct 8280

gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg 8340

cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg 8400

tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc 8460

tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca 8520

cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt tgacgccggg caagagcaac 8580

tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa 8640

agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg 8700

ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt 8760

ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg 8820

aagccatacc aaacgacgag cgtgacacca cgatgcctgc agcaatggca acaacgttgc 8880

gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga 8940

tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta 9000

ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc 9060

cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg 9120

atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt 9180

cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa 9240

ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt 9300

cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt 9360

ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt 9420

tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga 9480

taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag 9540

caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata 9600

agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg 9660

gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga 9720

gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca 9780

ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa 9840

acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt 9900

tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac 9960

ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt 10020

ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga 10080

ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc 10140

ttacgcatct gtgcggtatt tcacaccgca tatggtgcac tctcagtaca atctgctctg 10200

atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc 10260

gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc 10320

cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc 10380

atcaccgaaa cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc 10440

acagatgtct gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt 10500

ctggcttctg ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcacttgatg 10560

cctccgtgta agggggaatt tctgttcatg ggggtaatga taccgatgaa acgagagagg 10620

atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacgttgtgagggt 10680

aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag 10740

cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag 10800

atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg 10860

aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt 10920

cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct 10980

agccgggtcc tcaacgacag gagcacgatc atgagcaccc gtggccagga cccaacgctg 11040

cccgagatgc gccgcgtgcg gctgctggag atggcggacg cgatggatat gttctgccaa 11100

gggttggttt gcgcattcac agttctccgc aagaattgat tggctccaat tcttggagtg 11160

gtgaatccgt tagcgaggtg ccgccggctt ccattcaggt cgaggtggcc cggctccatg 11220

caccgcgacg caacgcgggg aggcagacaa ggtatagggc ggcgatgcga tgtacgggcc 11280

agatatacgc gtatctgagg ggactagggt gtgtttaggc gaaaagcggg gcttcggttg 11340

tacgcggtta ggagtcccct taggatatag tagtttcgct tttgcatagg gagggggaaa 11400

tgtagtctta tgcaatactc ttgtagtctt gcaacatggt aacgatgagt tagcaacatg 11460

ccttacaagg agagaaaaag caccgtgcat gccgattggt ggaagtaagg tggtacgatc 11520

gtgccttatt aggaaggcaa cagacgggtc tgacatggat tggacgaacc actgaattcc 11580

gcattgcaga gatattgtat ttaagtgcct agctcgatac aataaac 11627

<210>2

<211>1759

<212>DNA

<213> Chicken

<220>

<221> Gene

<222>(1)..(1759)

<223> Chicken c-SRC cNDA

<220>

<221>CDS

<222>(112)..(1710)

<400>2

tctgacaccc atctgtctgt ctgtctgtgt gctgcaggag ctgagctgac tctgctgtgg 60

cctcgcgtac cactgtggcc aggcggtagc tgggacgtgc agcccaccac c atg ggg 117

Met Gly

1

agc agc aag agc aag ccc aag gac ccc agc cag cgc cgg cgc agc ctg 165

Ser Ser Lys Ser Lys Pro Lys Asp Pro Ser Gln Arg Arg Arg Ser Leu

5 10 15

gag cca ccc gac agc acc cac cac ggg gga ttc cca gcc tcg cag acc 213

Glu Pro Pro Asp Ser Thr His His Gly Gly Phe Pro Ala Ser Gln Thr

20 25 30

ccc aac aag aca gca gcc ccc gac acg cac cgc acc ccc agc cgc tcc 251

Pro Asn Lys Thr Ala Ala Pro Asp Thr His Arg Thr Pro Ser Arg Ser

35 40 45 50

ttt ggg acc gtg gcc acc gag ccc aag ctc ttc ggg ggc ttc aac act 309

Phe Gly Thr Val Ala Thr Glu Pro Lys Leu Phe Gly Gly Phe Asn Thr

55 60 65

tct gac acc gtt acg tcg ccg cag cgt gcc ggg gca ctg gct ggc ggc 357

Ser Asp Thr Val Thr Ser Pro Gln Arg Ala Gly Ala Leu Ala Gly Gly

70 75 80

gtc acc act ttc gtg gct ctc tac gac tac gag tcc cgg act gaa acg 405

Val Thr Thr Phe Val Ala Leu Tyr Asp Tyr Glu Ser Arg Thr Glu Thr

85 90 95

gac ttg tcc ttc aag aaa gga gaa cgc ctg cag att gtc aac aac acg 453

Asp Leu Ser Phe Lys Lys Gly Glu Arg Leu Gln Ile Val Asn Asn Thr

100 105 110

gaa ggt gac tgg tgg ctg gct cat tcc ctc act aca gga cag acg ggc 501

Glu Gly Asp Trp Trp Leu Ala His Ser Leu Thr Thr Gly Gln Thr Gly

115 120 125 130

tac atc ccc agt aac tat gtc gcg ccc tca gac tcc atc cag gct gaa 549

Tyr Ile Pro Ser Asn Tyr Val Ala Pro Ser Asp Ser Ile Gln Ala Glu

135 140 145

gag tgg tac ttt ggg aag atc act cgt cgg gag tcc gag cgg ctg ctg 597

Glu Trp Tyr Phe Gly Lys Ile Thr Arg Arg Glu Ser Glu Arg Leu Leu

150 155 160

ctc aac ccc gaa aac ccc cgg gga acc ttc ttg gtc cgg gag agc gag 645

Leu Asn Pro Glu Asn Pro Arg Gly Thr Phe Leu Val Arg Glu Ser Glu

165 170 175

acg aca aaa ggt gcc tat tgc ctc tcc gtt tct gac ttt gac aac gcc 693

Thr Thr Lys Gly Ala Tyr Cys Leu Ser Val Ser Asp Phe Asp Asn Ala

180 185 190

aag ggg ctc aat gtg aag cac tac aag atc cgc aag ctg gac agc ggc 741

Lys Gly Leu Asn Val Lys His Tyr Lys Ile Arg Lys Leu Asp Ser Gly

195 200 205 210

ggc ttc tac atc acc tca cgc aca cag ttc agc agc ctg cag cag ctg 789

Gly Phe Tyr Ile Thr Ser Arg Thr Gln Phe Ser Ser Leu Gln Gln Leu

215 220 225

gtg gcc tac tac tcc aaa cat gct gat ggc ttg tgc cac cgc ctg acc 837

Val Ala Tyr Tyr Ser Lys His Ala Asp Gly Leu Cys His Arg Leu Thr

230 235 240

aac gtc tgc ccc acg tcc aag ccc cag acc cag gga ctc gcc aag gac 885

Asn Val Cys Pro Thr Ser Lys Pro Gln Thr Gln Gly Leu Ala Lys Asp

245 250 255

gcg tgg gaa atc ccc cgg gag tcg ctg cgg ctg gag gtg aag ctg ggg 933

Ala Trp Glu Ile Pro Arg Glu Ser Leu Arg Leu Glu Val Lys Leu Gly

260 265 270

cag ggc tgc ttt gga gag gtc tgg atg ggg acc tgg aac ggc acc acc 981

Gln Gly Cys Phe Gly Glu Val Trp Met Gly Thr Trp Asn Gly Thr Thr

275 280 285 290

aga gtg gcc ata aag act ctg aag ccc ggc acc atg tcc ccg gag gcc 1029

Arg Val Ala Ile Lys Thr Leu Lys Pro Gly Thr Met Ser Pro Glu Ala

295 300 305

ttc ctg cag gaa gcc caa gtg atg aag aag ctc cgg cat gag aag ctg 1077

Phe Leu Gln Glu Ala Gln Val Met Lys Lys Leu Arg His Glu Lys Leu

310 315 320

gtt cag ctg tac gca gtg gtg tcg gaa gag ccc atc tac atc gtc act 1125

Val Gln Leu Tyr Ala Val Val Ser Glu Glu Pro Ile Tyr Ile Val Thr

325 330 335

gag tac atg agc aag ggg agc ctc ctg gat ttc ctg aag gga gag atg 1173

Glu Tyr Met Ser Lys Gly Ser Leu Leu Asp Phe Leu Lys Gly Glu Met

340 345 350

ggc aag tac ctg cgg ctg cca cag ctc gtc gat atg gct gct cag att 1221

Gly Lys Tyr Leu Arg Leu Pro Gln Leu Val Asp Met Ala Ala Gln Ile

355 360 365 370

gca tcc ggc atg gcc tat gtg gag agg atg aac tac gtg cac cga gac 1269

Ala Ser Gly Met Ala Tyr Val Glu Arg Met Asn Tyr Val His Arg Asp

375 380 385

ctg cgg gcg gcc aac atc ctg gtg ggg gag aac ctg gtg tgc aag gtg 1317

Leu Arg Ala Ala Asn Ile Leu Val Gly Glu Asn Leu Val Cys Lys Val

390 395 400

gct gac ttt ggg ctg gca cgc ctc atc gag gac aac gag tac aca gca 1365

Ala Asp Phe Gly Leu Ala Arg Leu Ile Glu Asp Asn Glu Tyr Thr Ala

405 410 415

cgg caa ggt gcc aag ttc ccc atc aag tgg aca gcc ccc gag gca gcc 1413

Arg Gln Gly Ala Lys Phe Pro Ile Lys Trp Thr Ala Pro Glu Ala Ala

420 425 430

ctc tat ggc cgg ttc acc atc aag tcg gat gtc tgg tcc ttc ggc atc 1461

Leu Tyr Gly Arg Phe Thr Ile Lys Ser Asp Val Trp Ser Phe Gly Ile

435 440 445 450

ctg ctg act gag ctg acc acc aag ggc cgg gtg cca tac cca ggg atg 1509

Leu Leu Thr Glu Leu Thr Thr Lys Gly Arg Val Pro Tyr Pro Gly Met

455 460 465

gtc aac agg gag gtg ctg gac cag gtg gag agg ggc tac cgc atg ccc 1557

Val Asn Arg Glu Val Leu Asp Gln Val Glu Arg Gly Tyr Arg Met Pro

470 475 480

tgc ccg ccc gag tgc ccc gag tcg ctg cat gac ctc atg tgc cag tgc 1605

Cys Pro Pro Glu Cys Pro Glu Ser Leu His Asp Leu Met Cys Gln Cys

485 490 495

tgg cgg agg gac cct gag gag cgg ccc act ttt gag tac ctg cag gcc 1653

Trp Arg Arg Asp Pro Glu Glu Arg Pro Thr Phe Glu Tyr Leu Gln Ala

500 505 510

ttc ctg gag gac tac ttc acc tcg aca gag ccc cag tac cag cct gga 1701

Phe Leu Glu Asp Tyr Phe Thr Ser Thr Glu Pro Gln Tyr Gln Pro Gly

515 520 525 530

gag aac cta taggcctgga gctcctcctg gaccagaggc ctcgctgtgg ggtacaggg 1759

Glu Asn Leu

<210>3

<211>533

<212>PRT

<213> Chicken

<400>3

Met Gly Ser Ser Lys Ser Lys Pro Lys Asp Pro Ser Gln Arg Arg Arg

1 5 10 15

Ser Leu Glu Pro Pro Asp Ser Thr His His Gly Gly Phe Pro Ala Ser

20 25 30

Gln Thr Pro Asn Lys Thr Ala Ala Pro Asp Thr His Arg Thr Pro Ser

35 40 45

Arg Ser Phe Gly Thr Val Ala Thr Glu Pro Lys Leu Phe Gly Gly Phe

50 55 60

Asn Thr Ser Asp Thr Val Thr Ser Pro Gln Arg Ala Gly Ala Leu Ala

65 70 75 80

Gly Gly Val Thr Thr Phe Val Ala Leu Tyr Asp Tyr Glu Ser Arg Thr

85 90 95

Glu Thr Asp Leu Ser Phe Lys Lys Gly Glu Arg Leu Gln Ile Val Asn

100 105 110

Asn Thr Glu Gly Asp Trp Trp Leu Ala His Ser Leu Thr Thr Gly Gln

115 120 125

Thr Gly Tyr Ile Pro Ser Asn Tyr Val Ala Pro Ser Asp Ser Ile Gln

130 135 140

Ala Glu Glu Trp Tyr Phe Gly Lys Ile Thr Arg Arg Glu Ser Glu Arg

145 150 155 160

Leu Leu Leu Asn Pro Glu Asn Pro Arg Gly Thr Phe Leu Val Arg Glu

165 170 175

Ser Glu Thr Thr Lys Gly Ala Tyr Cys Leu Ser Val Ser Asp Phe Asp

180 185 190

Asn Ala Lys Gly Leu Asn Val Lys His Tyr Lys Ile Arg Lys Leu Asp

195 200 205

Ser Gly Gly Phe Tyr Ile Thr Ser Arg Thr Gln Phe Ser Ser Leu Gln

210 215 220

Gln Leu Val Ala Tyr Tyr Ser Lys His Ala Asp Gly Leu Cys His Arg

225 230 235 240

Leu Thr Asn Val Cys Pro Thr Ser Lys Pro Gln Thr Gln Gly Leu Ala

245 250 255

Lys Asp Ala Trp Glu Ile Pro Arg Glu Ser Leu Arg Leu Glu Val Lys

260 265 270

Leu Gly Gln Gly Cys Phe Gly Glu Val Trp Met Gly Thr Trp Asn Gly

275 280 285

Thr Thr Arg Val Ala Ile Lys Thr Leu Lys Pro Gly Thr Met Ser Pro

290 295 300

Glu Ala Phe Leu Gln Glu Ala Gln Val Met Lys Lys Leu Arg His Glu

305 310 315 320

Lys Leu Val Gln Leu Tyr Ala Val Val Ser Glu Glu Pro Ile Tyr Ile

325 330 335

Val Thr Glu Tyr Met Ser Lys Gly Ser Leu Leu Asp Phe Leu Lys Gly

340 345 350

Glu Met Gly Lys Tyr Leu Arg Leu Pro Gln Leu Val Asp Met Ala Ala

355 360 365

Gln Ile Ala Ser Gly Met Ala Tyr Val Glu Arg Met Asn Tyr Val His

370 375 380

Arg Asp Leu Arg Ala Ala Asn Ile Leu Val Gly Glu Asn Leu Val Cys

385 390 395 400

Lys Val Ala Asp Phe Gly Leu Ala Arg Leu Ile Glu Asp Asn Glu Tyr

405 410 415

Thr Ala Arg Gln Gly Ala Lys Phe Pro Ile Lys Trp Thr Ala Pro Glu

420 425 430

Ala Ala Leu Tyr Gly Arg Phe Thr Ile Lys Ser Asp Val Trp Ser Phe

435 440 445

Gly Ile Leu Leu Thr Glu Leu Thr Thr Lys Gly Arg Val Pro Tyr Pro

450 455 460

Gly Met Val Asn Arg Glu Val Leu Asp Gln Val Glu Arg Gly Tyr Arg

465 470 475 480

Met Pro cys Pro Pro Glu Cys Pro Glu Ser Leu His Asp Leu Met Cys

485 490 495

Gln Cys Trp Arg Arg Asp Pro Glu Glu Arg Pro Thr Phe Glu Tyr Leu

500 505 510

Gln Ala Phe Leu Glu Asp Tyr Phe Thr Ser Thr Glu Pro Gln Tyr Gln

515 520 525

Pro Gly Glu Asn Leu

530

<210>4

<211>2187

<212>DNA

<213> human

<220>

<221> Gene

<222>(1)..(2187)

<223> human c-SRC-cDNA

<220>

<221>CDS

<222>(134)..(L483)

<400>4

gcgccgcgtc ccgcaggccg tgatgccgcc cgcgcggagg tggcccggac cgcagtgccc 60

caagagagct ctaatggtac caagtgacag gttggcttta ctgtgactcg gggacgccag 120

agctcctgag aag atg tca gca ata cag gcc gcc tgg cca tcc ggt aca 169

Met Ser Ala Ile Gln Ala Ala Trp Pro Ser Gly Thr

1 5 10

gaa tgt att gcc aag tac aac ttc cac ggc act gcc gag cag gac ctg 217

Glu Cys Ile Ala Lys Tyr Asn Phe His Gly Thr Ala Glu Gln Asp Leu

15 20 25

ccc ttc tgc aaa gga gac gtg ctc acc att gtg gcc gtc acc aag gac 265

Pro Phe Cys Lys Gly Asp Val Leu Thr Ile Val Ala Val Thr Lys Asp

30 35 40

ccc aac tgg tac aaa gcc aaa aac aag gtg ggc cgt gag ggc atc atc 313

Pro Asn Trp Tyr Lys Ala Lys Asn Lys Val Gly Arg Glu Gly Ile Ile

45 50 55 60

cca gcc aac tac gtc cag aag cgg gag ggc gtg aag gcg ggt acc aaa 361

Pro Ala Asn Tyr Val Gln Lys Arg Glu Gly Val Lys Ala Gly Thr Lys

65 70 75

ctc agc ctc atg cct tgg ttc cac ggc aag atc aca cgg gag cag gct 409

Leu Ser Leu Met Pro Trp Phe His Gly Lys Ile Thr Arg Glu Gln Ala

80 85 90

gag cgg ctt ctg tac ccg ccg gag aca ggc ctg ttc ctg gtg cgg gag 457

Glu Arg Leu Leu Tyr Pro Pro Glu Thr Gly Leu Phe Leu Val Arg Glu

95 100 105

agc acc aac tac ccc gga gac tac acg ctg tgc gtg agc tgc gac ggc 505

Ser Thr Asn Tyr Pro Gly Asp Tyr Thr Leu Cys Val Ser Cys Asp Gly

110 115 120

aag gtg gag cac tac cgc atc atg tac cat gcc agc aag ctc agc atc 553

Lys Val Glu His Tyr Arg Ile Met Tyr His Ala Ser Lys Leu Ser Ile

125 130 135 140

gac gag gag gtg tac ttt gag aac ctc atg cag ctg gtg gag cac tac 601

Asp Glu Glu Val Tyr Phe Glu Asn Leu Met Gln Leu Val Glu His Tyr

145 150 155

acc tca gac gca gat gga ctc tgt acg cgc ctc att aaa cca aag gtc 649

Thr Ser Asp Ala Asp Gly Leu Cys Thr Arg Leu Ile Lys Pro Lys Val

160 165 170

atg gag ggc aca gtg gcg gcc cag gat gag ttc tac cgc agc ggc tgg 697

Met Glu Gly Thr Val Ala Ala Gln Asp Glu Phe Tyr Arg Ser Gly Trp

175 180 185

gcc ctg aac atg aag gag ctg aag ctg ctg cag acc atc ggg aag ggg 745

Ala Leu Asn Met Lys Glu Leu Lys Leu Leu Gln Thr Ile Gly Lys Gly

190 195 200

gag ttc gga gac gtg atg ctg ggc gat tac cga ggg aac aaa gtc gcc 793

Glu Phe Gly Asp Val Met Leu Gly Asp Tyr Arg Gly Asn Lys Val Ala

205 210 215 220

gtc aag tgc att aag aac gac gcc act gcc cag gcc ttc ctg gct gaa 841

Val Lys Cys Ile Lys Asn Asp Ala Thr Ala Gln Ala Phe Leu Ala Glu

225 230 235

gcc tca gtc atg acg caa ctg cgg cat agc aac ctg gtg cag ctc ctg 889

Ala Ser Val Met Thr Gln Leu Arg His Ser Asn Leu Val Gln Leu Leu

240 245 250

ggc gtg atc gtg gag gag aag ggc ggg ctc tac atc gtc act gag tac 937

Gly Val Ile Val Glu Glu Lys Gly Gly Leu Tyr Ile Val Thr Glu Tyr

255 260 265

atg gcc aag ggg agc ctt gtg gac tac ctg cgg tct agg ggt cgg tca 985

Met Ala Lys Gly Ser Leu Val Asp Tyr Leu Arg Ser Arg Gly Arg Ser

270 275 280

gtg ctg ggc gga gac tgt ctc ctc aag ttc tcg cta gat gtc tgc gag 1033

Val Leu Gly Gly Asp Cys Leu Leu Lys Phe Ser Leu Asp Val Cys Glu

285 290 295 300

gcc atg gaa tac ctg gag ggc aac aat ttc gtg cat cga gac ctg gct 1081

Ala Met Glu Tyr Leu Glu Gly Asn Asn Phe Val His Arg Asp Leu Ala

305 310 315

gcc cgc aat gtg ctg gtg tct gag gac aac gtg gcc aag gtc agc gac 1129

Ala Arg Asn Val Leu Val Ser Glu Asp Asn Val Ala Lys Val Ser Asp

320 325 330

ttt ggt ctc acc aag gag gcg tcc agc acc cag gac acg ggc aag ctg 1177

Phe Gly Leu Thr Lys Glu Ala Ser Ser Thr Gln Asp Thr Gly Lys Leu

335 340 345

cca gtc aag tgg aca gcc cct gag gcc ctg aga gag aag aaa ttc tcc 1225

Pro Val Lys Trp Thr Ala Pro Glu Ala Leu Arg Glu Lys Lys Phe Ser

350 355 360

act aag tct gac gtg tgg agt ttc gga atc ctt ctc tgg gaa atc tac 1273

Thr Lys Ser Asp Val Trp Ser Phe Gly Ile Leu Leu Trp Glu Ile Tyr

365 370 375 380

tcc ttt ggg cga gtg cct tat cca aga att ccc ctg aag gac gtc gtc 1321

Ser Phe Gly Arg Val Pro Tyr Pro Arg Ile Pro Leu Lys Asp Val Val

385 390 395

cct cgg gtg gag aag ggc tac aag atg gat gcc ccc gac ggc tgc ccg 1369

Pro Arg Val Glu Lys Gly Tyr Lys Met Asp Ala Pro Asp Gly Cys Pro

400 405 410

ccc gca gtc tat gaa gtc atg aag aac tgc tgg cac ctg gac gcc gcc 1417

Pro Ala Val Tyr Glu Val Met Lys Asn Cys Trp His Leu Asp Ala Ala

415 420 425

atg cgg ccc tcc ttc cta cag ctc cga gag cag ctt gag cac atc aaa 1465

Met Arg Pro Ser Phe Leu Gln Leu Arg Glu Gln Leu Glu His Ile Lys

430 435 440

acc cac gag ctg cac ctg tgacggctgg cctccgcctg ggtcatgggc 1513

Thr His Glu Leu His Leu

445 450

ctgtggggac tgaacctgga agatcatgga cctggtgccc ctgctcactg ggcccgagcc 1573

tgaactgagc cccagcgggc tggcgggcct ttttcctgcg tcccagcctg cacccctccg 1633

gccccgtctc tcttggaccc acctgtgggg cctggggagc ccactgaggg gccagggagg 1693

aaggaggcca cggagcggga ggcagcgccc caccacgtcg ggcttccctg gcctcccgcc 1753

actcgccttc ttagagtttt attcctttcc ttttttgaga ttttttttcc gtgtgtttat 1813

tttttattat ttttcaagat aaggagaaag aaagtaccca gcaaatgggc attttacaag 1873

aagtacgaat cttatttttc ctgtcctgcc cgtgagggtg ggggggaccg ggcccctctc 1933

tagggacccc tcgccccagc ctcattcccc attctgtgtc ccatgtcccg tgtctcctcg 1993

gtcgccccgt gtttgcgctt gaccatgttg cactgtttgc atgcgcccga ggcagacgtc 2053

tgtcaggggc ttggatttcg tgtgccgctg ccacccgccc acccgccttg tgagatggaa 2113

ttgtaataaa ccacgccatg aggacaccgc cgcccgcctc ggcgcttcct ccaccgaaaa 2173

aaaaaaaaaa aaaa 2187

<210>5

<211>450

<212>PRT

<213> human

<400>5

Met Ser Ala Ile Gln Ala Ala Trp Pro Ser Gly Thr Glu Cys Ile Ala

1 5 10 15

Lys Tyr Asn Phe His Gly Thr Ala Glu Gln Asp Leu Pro Phe Cys Lys

20 25 30

Gly Asp Val Leu Thr Ile Val Ala Val Thr Lys Asp Pro Asn Trp Tyr

35 40 45

Lys Ala Lys Asn Lys Val Gly Arg Glu Gly Ile Ile Pro Ala Asn Tyr

50 55 60

Val Gln Lys Arg Glu Gly Val Lys Ala Gly Thr Lys Leu Ser Leu Met

65 70 75 80

Pro Trp Phe His Gly Lys Ile Thr Arg Glu Gln Ala Glu Arg Leu Leu

85 90 95

Tyr Pro Pro Glu Thr Gly Leu Phe Leu Val Arg Glu Ser Thr Asn Tyr

100 105 110

Pro Gly Asp Tyr Thr Leu Cys Val Ser Cys Asp Gly Lys Val Glu His

115 120 125

Tyr Arg Ile Met Tyr His Ala Ser Lys Leu Ser Ile Asp Glu Glu Val

130 135 140

Tyr Phe Glu Asn Leu Met Gln Leu Val Glu His Tyr Thr Ser Asp Ala

145 150 155 160

Asp Gly Leu Cys Thr Arg Leu Ile Lys Pro Lys Val Met Glu Gly Thr

165 170 175

Val Ala Ala Gln Asp Glu Phe Tyr Arg Ser Gly Trp Ala Leu Asn Met

180 185 190

Lys Glu Leu Lys Leu Leu Gln Thr Ile Gly Lys Gly Glu Phe Gly Asp

195 200 205

Val Met Leu Gly Asp Tyr Arg Gly Asn Lys Val Ala Val Lys Cys Ile

210 215 220

Lys Asn Asp Ala Thr Ala Gln Ala Phe Leu Ala Glu Ala Ser Val Met

225 230 235 240

Thr Gln Leu Arg His Ser Asn Leu Val Gln Leu Leu Gly Val Ile Val

245 250 255

Glu Glu Lys Gly Gly Leu Tyr Ile Val Thr Glu Tyr Met Ala Lys Gly

260 265 270

Ser Leu Val Asp Tyr Leu Arg Ser Arg Gly Arg Ser Val Leu Gly Gly

275 280 285

Asp Cys Leu Leu Lys Phe Ser Leu Asp Val Cys Glu Ala Met Glu Tyr

290 295 300

Leu Glu Gly Asn Asn Phe Val His Arg Asp Leu Ala Ala Arg Asn Val

305 310 315 320

Leu Val Ser Glu Asp Asn Val Ala Lys Val Ser Asp Phe Gly Leu Thr

325 330 335

Lys Glu Ala Ser Ser Thr Gln Asp Thr Gly Lys Leu Pro Val Lys Trp

340 345 350

Thr Ala Pro Glu Ala Leu Arg Glu Lys Lys Phe Ser Thr Lys Ser Asp

355 360 365

Val Trp Ser Phe Gly Ile Leu Leu Trp Glu Ile Tyr Ser Phe Gly Arg

370 375 380

Val Pro Tyr Pro Arg Ile Pro Leu Lys Asp Val Val Pro Arg Val Glu

385 390 395 400

Lys Gly Tyr Lys Met Asp Ala Pro Asp Gly Cys Pro Pro Ala Val Tyr

405 410 415

Glu Val Met Lys Asn Cys Trp His Leu Asp Ala Ala Met Arg Pro Ser

420 425 430

Phe Leu Gln Leu Arg Glu Gln Leu Glu His Ile Lys Thr His Glu Leu

435 440 445

His Leu

450

<210>6

<211>14

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: 9E10-myc epitope tag

<400>6

Val Asp Met Glu Gln Lys Leu Ile Ala Glu Glu Asp Leu Asn

1 5 10

Claims (19)

1. An article of manufacture comprising a packaging material and a pharmaceutical composition contained within the packaging material, wherein the pharmaceutical composition is capable of modulating angiogenesis in a tissue associated with a disease condition; wherein said packaging material carries a label indicating that said pharmaceutical composition is useful for treating a disease by modulating angiogenesis; and wherein said pharmaceutical composition comprises a Src protein or a nucleic acid having a nucleotide sequence capable of expressing said Src protein, wherein said Src protein is SrcA, Src251 or SrcK 295M.

2. The article of manufacture of claim 1, wherein said pharmaceutical composition further comprises a liposome.

3. The article of manufacture of claim 1, wherein said pharmaceutical composition comprises a viral expression vector capable of expressing said nucleotide sequence.

4. The article of manufacture of claim 1, wherein said pharmaceutical composition comprises a non-viral expression vector capable of expressing said nucleotide sequence.

Use of Src protein, or a nucleic acid having a nucleotide sequence capable of expressing said Src protein, wherein said Src protein is SrcA, Src251 or SrcK295M, in the manufacture of a pharmaceutical composition for modulating angiogenesis in a tissue associated with a disease condition.

6. The use of claim 5, wherein the tissue has poor circulation and the protein is SrcA.

7. The use of claim 5 wherein said tissue is inflammatory tissue and said disease is arthritis or rheumatoid arthritis and said Src protein is Src251 or Src 295M.

8. The use of claim 5 wherein said tissue is a solid tumor or a solid metastatic tumor and said Src protein is Src251 or Src 295M.

9. The use of claim 5 wherein said tissue is retinal tissue and said disease is retinopathy, diabetic retinopathy or macular degeneration and said Src protein is Src251 or Src 295M.

10. The use of claim 5 wherein said tissue is at the site of coronary angioplasty and said tissue is at risk of restenosis, and said Src protein is Src251 or Src 295M.

11. The use of claim 5, wherein said pharmaceutical composition further comprises liposomes.

12. The use of claim 5, wherein said pharmaceutical composition comprises a retroviral expression vector capable of expressing said nucleotide sequence.

13. The use of claim 5, wherein said pharmaceutical composition comprises a non-viral expression vector capable of expressing said nucleotide sequence.

14. A pharmaceutical composition for stimulating angiogenesis in a target mammalian tissue comprising a viral gene transfer vector comprising a nucleic acid and a pharmaceutically acceptable carrier or excipient; the nucleic acid has a nucleic acid segment encoding a Src protein, which is Src a.

15. A pharmaceutical composition for stimulating angiogenesis in a target mammalian tissue comprising a non-viral gene transfer vector comprising a nucleic acid and a pharmaceutically acceptable carrier or excipient; the nucleic acid has a nucleic acid segment encoding a Src protein, which is Src a.

16. A pharmaceutical composition for inhibiting angiogenesis in a target mammalian tissue comprising a viral gene transfer vector comprising a nucleic acid and a pharmaceutically acceptable carrier or excipient; the nucleic acid has a nucleic acid segment encoding a Src protein, wherein the Src protein is Src251 or Src 295M.

17. A pharmaceutical composition for inhibiting angiogenesis in a target mammalian tissue comprising a non-viral gene transfer vector comprising a nucleic acid and a pharmaceutically acceptable carrier or excipient; the nucleic acid has a nucleic acid segment encoding a Src protein that is Src251 or Src 295M.

18. A pharmaceutical composition for stimulating angiogenesis in a target mammalian tissue comprising a therapeutic amount of Src protein in a pharmaceutically acceptable carrier or excipient; the Src protein is SrcA.

19. A pharmaceutical composition for inhibiting angiogenesis in a target mammalian tissue comprising Src protein in a pharmaceutically acceptable carrier or excipient; the Src protein is Src251 or SrcK 295M.