WO2003004529A2 - Ephrin-tie receptor materials and methods - Google Patents

Ephrin-tie receptor materials and methods Download PDF

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Publication number
WO2003004529A2
WO2003004529A2 PCT/IB2002/002524 IB0202524W WO03004529A2 WO 2003004529 A2 WO2003004529 A2 WO 2003004529A2 IB 0202524 W IB0202524 W IB 0202524W WO 03004529 A2 WO03004529 A2 WO 03004529A2
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ephrin
tie
binding
polypeptide
tie receptor
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PCT/IB2002/002524
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French (fr)
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WO2003004529A3 (en
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Kari Alitalo
Hajime Kubo
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Licentia Ltd.
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Publication of WO2003004529A3 publication Critical patent/WO2003004529A3/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators

Definitions

  • the present invention provides materials and methods relating to cellular and molecular biology and medicine, in particular relating to areas of vasculogenesis, angiogenesis, lymphangiogenesis and the interface between arterial and venous systems and nervous systems.
  • Receptor tyrosine kinases RTK
  • integral membrane proteins for transducing cell signals play important roles in numerous processes in embryonic development, including vascularization. See generally Schlessinger et al., Cell, 103: 211-225 (2000), incorporated herein by reference in its entirety. Establishment of the cardiovascular system is one of the first completed processes of embryogenesis and disruption of this process often leads to embryonic lethality.
  • VEGF/VEGFR pathway including the VEGFR-1,-2, and -3 RTKs , as well as a smaller family of receptor tyrosine kinases which contain Ig and EGF homology domains, Tie-1 (sometimes referred to simply as Tie) and Tie- 2 (also referred to as TEK).
  • Tie-1 sometimes referred to simply as Tie
  • Tie- 2 also referred to as TEK
  • the biology of the Tie receptors is reviewed in Jones, Iljin, Dumont, and Alitalo, "Tie Receptors: New Modulators of Angiogenic and Lymphangiogenic Responses," Nature Reviews, 2: 257-67 (April, 2001), incorporated herein by reference in its entirety.
  • Embryonic vascularogenesis the formation of a primitive network of tubules, proceeds through a very controlled series of steps mediated at least partly by the interaction of vascular endothelial growth factor (VEGF) family ligands with VEGF-Receptors (VEGFR) on the endothelium, setting up what is called the primary capillary plexus. Further development from this plexus - the sprouting, branching, and growth of different types of blood vessels - is referred to as angiogenesis.
  • VEGF vascular endothelial growth factor
  • VEGFR VEGF-Receptors
  • angiogenesis is also mediated at least partly through another family of growth factors, the Angiopoietins (Davis et al, Cell, 87: 1161-69 (1996), Valenzuela et al, Proc. Natl. Acad. Sci. 96: 1904-09 (1999), Suri et al, Cell 87: 1171-80 (1996), Maisonpierre et al, Science. 277: 55-60 (1997).
  • Angiopoietins 1-4 Ang 1-4
  • Ang 1-4 are targeted to the vascular endothelium much like VEGF, and bind to the Tie-2 receptor tyrosine kinase.
  • Tie-1 and Tie-2 differ from the VEGFR and other RTKs in that they contain three EGF-like domains and three frbronectin type-Ill repeats in addition to immunoglobulin-like domains typically found in RTKs.
  • Tie-1 and Tie-2 molecules show approximately 46% homology to each other (Partenan et al, Mol Cell. Biol 12: 1698-1707 (1992), Dumont et al, Oncogene, 7: 1471-80 (1992); Maisonpierre et al, Oncogene, 8: 1631-7 (1993).) Both Tie-2 and Tie-1 appear to be expressed in certain cells of hematopoietic origin in addition to the endothelium.
  • Tie-1 also appears to be variably expressed in adult tissues and may be found in capillary vessels of the lung and kidney, in lymphatic endothelium, in the reproductive tract during ovulation, in areas of wound healing, and in melanoma metastasis where Tie-1 was localized to the neovascularized endothelium (Iwama et al, Biochem. Biophys. Res. Comm.
  • Tie-2 knockout mice die approximately embryonic day 9.5 (E9.5) with loss of vascular integrity and endothelial cell numbers (Dumont et al, Genes Dev,. 8: 1897-1909 (1994).)
  • E9.5 embryonic day 9.5
  • Tie-1 deficient mice survive until E13.5-E15.5, retaining major vessel structure with loss of microvascular integrity (Partanen et al, Development. 122: 3013-21. 1996). This differential expression pattern suggests that each receptor serves a unique function in blood vessel development. Tie-1 is also expressed in endothelial cells in lymphatic vessels.
  • Tie receptors may be involved in pathological angiogenesis as well as normal vascular development and maintenance. For example, interruption of Tie-2 signaling using soluble, dominant negative receptors can inhibit angiogenic growth in tumor-bearing mice. Elevated Tie-1 and Tie-2 expression has been observed in the endothelium of the neovasculature in numerous solid tumors, and certain leukemia cell lines express these receptors. The recently uncovered ligands for the Tie-2 receptor, angiopoietins 1- 4, are small, soluble factors secreted by, e.g., smooth muscle cells and leukemia cells.
  • the angiopoietins have only been shown to interact specifically with the Tie-2 receptor, and unlike most other RTK ligands, the angiopoietins show both agonistic (Angl, Ang4) and antagonistic (Ang 2, Ang3) interactions with Tie-2 (Maisonpierre, 1997).
  • Ang-1 stimulates phosphorylation of the Tie-2 receptor leading to vessel remodeling and angiogenesis (Davis et al., Cell 57:1161-69. 1996).
  • Ang-1 deficient mice show the same phenotype as Tie-2 knockouts.
  • Eph receptors Another related group of RTKs that appear to be involved in vascular development are the Eph receptors. (See generally Wilkinson, “Multiple Roles of Eph Receptors and Ephrins in Neural Development,” Nature Reviews, 2: 155-164 (March, 2001), incorporated herein by reference in its entirety.) Eph receptors, which make up the largest family of RTKs with fourteen members, fall into two categories of receptor (Eph A or B) based on ligand structure and interactions. Ephrins, the ligands for Eph receptors, are unique among RTK ligands in that they are membrane- bound ligands.
  • Ephrin-A subclass of ligands are GPI-linked (Brambilla et al, EMBO J., 14: 3116-26 (1995)) whereas the Ephrin-B subclass of ligands are membrane bound via a transmembrane domain and short cytoplasmic tail (Gale et al, Oncogene 13: 1343-52 (1996)). Binding of the ephrins to their RTK receptors (Eph's) induces clustering of the receptors and subsequent autophosphorylation of residues in the cytoplasmic domain.
  • Ephrins must normally be membrane-attached to activate their receptors; the membrane attachement seems to promote clustering/multimerization that may be necessary for activation. [Gale et ⁇ /., Genes & Development, 13: 1055-66 (1999)].
  • ephrins and Eph receptors show promiscuous binding within a particular subgroup, e.g. any ephrin- A binds to any EphR-A.
  • ephrin- B2 binds primarily to the EphB4 receptor (Wang, 1998).
  • Ephrins and Eph-Receptors were originally identified in the nervous system where they play a significant role in neural patterning and neural cell migration during embryogenesis (Wang et al, Neuron 18: 383-96. 1997, Feldheim et al, Neuron, 21: 1303-13 (1998), Kalcheim et al, Development, 106: 85-93 (1989)), but expression is broadly distributed throughout embryonic tissue.
  • Ephrin-Al shows that it can promote chemotaxis of cultured endothelial cells, induce sprouting in a rat corneal pocket assay, and prmote capilllary-like assembly of human umbilical vein endothelial cells (HUVEC).
  • Ephrin-Bl induces human renal microvascular endothelial cells (HRMEC) to form tubules.
  • Ephrin-B2 specifically in arterial endothelial cells while the EphB4 receptor was reciprocally expressed only on venous cells in the developing vasculature.
  • Ephrin-B2 mutants show defects in both arteries and veins during angiogenesis despite expression of Ephrin-B2 only in developing arterial endothelial cells, suggesting bidirectional signaling as a result of Ephrin/EphR interaction.
  • Blood vessel growth like that found in embryogenesis is also important in the adult for continuous remodeling of the reproductive system, tissue repair, collateral neovascularization in ischemia, and tumor growth. Elucidating additional factors involved in the regulation of neovascularization and angiogenesis, as well as their roles in such processes, would aid in the development of therapies directed toward prevention of vascularization of solid tumors and induction of tumor regression, and induction of vascularization to promote faster, more efficient wound healing after injury, surgery, or tissue transplantation, or to treat ischemia by inducing angiogenesis and srteriogenesis of vessels that nourish the ischemic tissue.
  • modulation of angiogenic processes may be instrumental in treatment or cure of many of the most significant diseases that plague humans in the developed world, such as cerebral infarction/bleeding, acute myocardial infarction and ischemia, and cancers.
  • pro-angiogenic therapies it is highly desirable to induce and build new vessels having a structure comparable to that of normal mature vasculature, which functions as well as normal vessels.
  • endothelial cells and perivascular cells should be optimized.
  • Many pro-angiogenic factors identified to date target only endothelial cells, and their administration may result in disorganized vessels in vivo. Based on data from knockout studies, angiopoietins were hoped to facilitate construction of mature vessels, but recent studies have indicated that these hopes may have been misplaced. Application of just angiopoietins, in fact, induced enlarged vessels uncovered by pericytes.
  • anti-angiogenic therapies For anti-angiogenic therapies, a significant and critical side effect is bleeding. In animal models, positive effects are sometimes over-emphasized and the side effects are often ignored. A need exists for improved anti-angiogenic therapies, through either novel agents or novel combinations of agents, that minimize such side- effects. Also desirable are combinations of agents that minimize total dosages of agents that are necessary to achieve a beneficial effect.
  • the present invention addresses one or more needs in the art relating to modulation of angiogenic processes, including lymphangiogenesis, by identifying a novel receptor-ligand interaction between Ephrin and Tie molecules, which had previously been known to interact with Eph receptors and angiopoietins, respectively, but not with each other.
  • This newly established interaction provides novel materials and methods for modulation of angiogenic processes. It will be apparent that certain diseases and conditions are benef ⁇ tted by promoting angiogenesis, whereas others are benefitted by inhibiting it.
  • the identification herein of new involved proteins and new protein-protein interactions in the angiogenic process yields new tools to achieve these therapeutic end-goals.
  • Ephrin-Tie interactions and their importance to angiogenic processes permits novel screening assays to identify new therapeutic molecules to modulate (up-regulate/activate/stimulate or downregulate/inhibit) Ephrin-Tie interactions.
  • the invention provides a method for identifying a modulator of binding between a Tie receptor tyrosine kinase and an Ephrin ligand, comprising steps of:
  • the method further includes a step (d) of making a modulator composition by formulating a modulator identified according to step (c) in a pharmaceutically acceptable carrier.
  • a modulator so formulated is useful in animal studies and also as a therapeutic for administration to image tissues or treat diseases associated with aberrant Ephrin-Tie biology.
  • the method further includes a step (e) of administering the modulator composition to an animal that comprises cells that express the Tie receptor, and determining physiological effects of the modulator composition in the animal.
  • the animal including humans
  • the modulator improves the animal's state (e.g., by reducing disease symptoms, slowing disease progression, curing the disease, or otherwise improving clinical outcome).
  • Step (a) of the foregoing methods involves contacting a Tie receptor composition with an Ephrin composition in the presence and in the absence of a putative modulator compound.
  • Tie receptor composition any composition that includes a whole Tie receptor, or includes at least the portion of the Tie receptor needed for the particular assay - in this case the portion of the Tie receptor involved in Ephrin binding.
  • exemplary Tie receptor compositions include: (i) a composition comprising a purified polypeptide that comprises an entire Tie protein or that comprises a Tie receptor extracellular domain fragment that binds the Ephrin; (ii) a composition containing phospholipid membranes that contain Tie receptor polypeptides on their surface; (iii) a living cell recombinantly modified to express increased amounts of a Tie receptor on its surface; and (iv) any isolated cell or tissue that naturally expresses the Tie receptor on its surface.
  • Tie receptor composition is intended to include such structures as well.
  • fusion proteins are contemplated.
  • soluble Tie peptides may be preferred.
  • the Tie receptor composition comprises a Tie receptor extracellular domain fragment fused to an immunoglobulin Fc fragment.
  • the Tie receptor chosen is preferably of vertebrate origin, more preferably mammalian, still more preferably primate, and still more preferably human. And, while it will be apparent that the assay will likely give its best results if the functional portion of the chosen Tie receptor is identical in amino acid sequence to the native receptor, it will be apparent that the invention can still be practiced if variations have been introduced in the Tie sequence that do not eliminate its Ephrin binding properties. Use of variant sequences with at least 90%, 95%, 96%, 97%, 98%, or 99% amino acid identity is specifically contemplated.
  • Ephrin molecules like Tie receptors, are membrane bound, and similar considerations apply for the Ephrin composition that is used in the invention.
  • Ephrin composition means any composition that includes a whole Ephrin polypeptide, or includes at least the portion of the Ephrin needed for the particular assay - in this case the portion involved in binding to a Tie receptor.
  • Ephrin compositions include: (i) a composition comprising purified complete Ephrin polypeptide or comprising an Ephrin fragment that binds the Tie receptor chosen for the assay; (ii) a phospholipid membrane containing Ephrin polypeptides; (iii) a cell recombinantly modified to express increased amounts of an Ephrin on its surface; and (iv) any cell that naturally expresses the Ephrin on its surface.
  • it may be desirable to bind the Ephrin molecule of interest e.g., a polypeptide comprising an Ephrin extracellular domain fragment
  • a solid support such as a bead or assay plate well.
  • Ephrin composition is intended to include such structures as well. Likewise, fusion proteins are contemplated. For other assay formats, soluble Ephrin peptides may be preferred, hi one preferred variation, the Ephrin composition comprises an Ephrin fragment fused to an immunoglobulin Fc fragment.
  • Ephrin-B2 binds both Tie-1 and Tie-2, and thus Ephrin-B2 is a highly preferred Ephrin for practice of the invention.
  • Ephrin-Al Ephrin- A2
  • practice of the invention is contemplated with any such Ephrins that bind Tie receptors according to binding assays described herein.
  • practice of the invention is contemplated with other Ephrin family members that are subsequently discovered and that bind Tie receptors.
  • the Ephrin chosen is preferably of vertebrate origin, more preferably mammalian, still more preferably primate, and still more preferably human.
  • the assay will likely give its best results if the functional portion of the chosen Ephrin is identical in amino acid sequence to the corresponding portion of the native Ephrin, it will be apparent that the invention can still be practiced if variations have been introduced in the Ephrin sequence that do not eliminate its Tie receptor binding properties. Use of variant sequences with at least 90%, 95%, 96%, 97%, 98%, or 99% amino acid identity is specifically contemplated.
  • the putative modulator compound that is employed can be any organic or inorganic chemical or biological molecule that one would want to test for ability to modulate Tie-Ephrin interactions. Since the most preferred modulators will be those that can be administered as therapeutics, it will be apparent that molecules with limited toxicity are preferred.
  • Step (b) of the above-described method involves detecting binding between Tie receptor the Ephrin in the presence and absence of the putative modulator. Any technique for detecting intermolecular binding may be employed.
  • Tie/Ephrin may comprise a label, such as a radioisotope, a fluorophore, a fluorescing protein (e.g., natural or synthetic green fluorescent proteins), a dye, an enzyme or substrate, or the like.
  • a label such as a radioisotope, a fluorophore, a fluorescing protein (e.g., natural or synthetic green fluorescent proteins), a dye, an enzyme or substrate, or the like.
  • the Tie receptor composition comprises a cell that expresses Tie naturally or recombinantly on its surface
  • it will often be possible to detect Ephrin binding indirectly e.g., by detecting or measuring an Ephrin binding-induced physiological change in the cell.
  • Such possible changes include phosphorylation of the Tie receptor; cell chemotaxis; cell growth, changes in cellular morphology; adhesion; ionic fluxes, or the like.
  • the same also may be true in the situation where the Ephrin composition comprises a cell naturally or recombinantly expressing an Ephrin on its surface.
  • the detecting step can optionally comprise measuring a Tie binding-induced physiological change in the cell.
  • Step (c) of the above-described method involves identifying a modulator compound (from amongst the putative modulators tested) in view of decreased or increased binding between the Tie receptor and the Ephrin ligand in the presence of the putative modulator, as compared to binding in the absence of the putative modulator.
  • a modulator compound from amongst the putative modulators tested
  • more attractive modulators are those that will activate or inhibit Tie-Ephrin binding at lower concentrations, thereby permitting use of the modulators in a pharmaceutical composition at lower effective doses.
  • Angiopoietin molecules are known to act as ligand for at least the Tie- 2 receptor, and there is intense interest in the medical biology community in modulating Angiopoietin-Tie interactions for therapeutic benefit.
  • the identification herein of an additional class of Tie ligands adds a complexity to the design of therapeutic molecules. In some instances, it will be desirable to have a non-specific modulator that can modulate both Tie- Ang interactions and Tie-Ephrin interactions. In other instances, it will be desirable to modulate Tie-Angiopoietin interactions in vivo while affecting Tie-Ephrin interactions as little as possible, and visa versa.
  • the present invention provides counterscreen assays that identify the selectivity of a modulator for Tie-Angiopoietin binding or Tie-Ephrin binding.
  • the invention provides a method for screening for selectivity of a modulator of binding between a Tie receptor tyrosine kinase and an Angiopoietin, comprising steps of: a) contacting a Tie receptor composition with an Ephrin composition in the presence and in the absence of a compound that modulates binding between the Tie receptor and an angiopoietin ligand of the Tie receptor; b) detecting binding between the Tie receptor composition and the Ephrin in the presence and absence of the modulator compound, and c) identifying the selectivity of the modulator compound in view of decreased or increased binding between the Tie receptor and the Ephrin in the presence as compared to the absence of the modulator, wherein increased selectivity of the modulator for modulating Tie-Angiopoietin binding correlate
  • Step (a) of the foregoing counterscreen method involves contacting a Tie receptor composition with an Ephrin composition in the presence and in the absence of a compound that modulates binding between the Tie receptor and an angiopoietin ligand of the Tie receptor.
  • the Tie receptor composition and the Ephrin composition are essentially as described above.
  • the compound that modulates binding between the Tie receptor and the angiopoietin can be any organic or inorganic chemical or biological molecule that one would want to test for ability to modulate Tie-Ephrin interactions, and that also has the useful property of modulating the binding interaction between a Tie receptor and an Angiopoietin. (Such modulators can be identified using methods similar to the methods described above for identifying modulators of Tie-Ephrin interactions, by substituting an Angiopoietin composition for the Ephrin composition.)
  • Step (c) of the counterscreen involves identifying the selectivity of the modulator compound in view of decreased or increased binding between the Tie receptor and the Ephrin in the presence as compared to the absence of the modulator, wherein increased selectivity of the modulator for modulating Tie-Angiopoietin binding correlates with decreased differences in Ephrin-Tie binding.
  • a monoclonal antibody that specifically recognizes an angiopoietin and blocks Angiopoietin binding to Tie would be expected to be highly selective because it does not bind Ephrin or Tie and therefore would not interfere with Tie-Ephrin binding in the counterscreen.
  • the anti- Ang monoclonal antibody would be scored as a highly selective modulator of Ang- Tie interactions because Tie-Ephrin binding would not be substantially decreased or increased in the presence as compared to the absence of the modulator.
  • polyclonal antisera raised against Tie would be expected to be non-selective, and block both Angiopoietin and Ephrin from binding to Tie by blocking the binding site.
  • the invention provides counterscreens to determine the selectivity of a modulator of Tie-Ephrin binding vis-a-vis angiopoietin ligands of the Tie receptor.
  • the invention provides a method for screening for selectivity of a modulator of binding between a Tie receptor tyrosine kinase and an Ephrin , comprising steps of: a) contacting a Tie receptor composition with an Angiopoietin composition in the presence and in the absence of a compound that modulates binding between the Tie receptor and an Ephrin ligand of the Tie receptor; b) detecting binding between the Tie receptor composition and the
  • Angiopoietin ligand in the presence and absence of the modulator compound and c) identifying the selectivity of the modulator compound in view of decreased or increased binding between the Tie receptor and the Angiopoietin ligand in the presence as compared to the absence of the modulator, wherein increased selectivity of the modulator for Tie-Ephrin binding correlates with decreased differences in Angiopoietin-Tie binding.
  • the Tie-2 receptor is highly preferred for use in the foregoing counterscreens, since known angiopoietins bind Tie-2.
  • Any angiopoietin ligand can be used, including Ang-1, Ang-2, Ang-3, and Ang-4.
  • Vertebrate, and more preferrably mammalian, and more preferably primate, and most preferably human Angiopoietins are employed.
  • Ephrin ligands for Tie receptors provides novel materials and methods for affecting biological processes.
  • the invention provides a method of modulating growth, migration, or proliferation of cells in a mammalian organism, comprising a step of: (a) identifying a mammalian organism having cells that express a Tie receptor tyrosine kinase; and
  • composition comprising an agent selected from the group consisting of:
  • composition a molecule that selectively inhibits Epliin binding to the Tie receptor without inhibiting angiopoietin binding to the Tie receptor; wherein the composition is administered in an amount effective to modulate growth, migration, or proliferation of cells that express Tie in the mammalian organism.
  • the mammalian organism is human.
  • the cells preferably comprise vascular endothelial cells.
  • the organism has a disease characterized by aberrant growth, migration, or proliferation of endothelial cells.
  • the administration of the agent beneficially alters the aberrant growth, migration, or proliferation, e.g., by correcting it, or reducing its severity, or reducing its deleterious symptoms or effects.
  • the animal has a cancer, especially a cancerous tumor characterized by vasculature containing Tie-expressing endothelial cells.
  • a composition is selected that will decrease growth, migration, or proliferation of the cells, and thereby retard the growth of the tumor.
  • agents that inhibit other endothelial growth factor/receptor interactions such as inhibitors of the VEGF-family of ligands; endostatins; inhibitory angiopoietins, or the like.
  • Exemplary inhibitors include antibody substances specific for the growth factors or their ligands.
  • the animal may have a circulatory disorder characterized by inadequate vasculature, such as cerebral infraction, acute myocardial infarction, ischemic (e.g., peripheral ischemic in the legs, atriolitis, arterial thrombosis, Burger's disease).
  • a composition is selected that will increase growth, migration, or proliferation of the endothelial cells.
  • a second agent to the patient for modulating endothelial growth, migration, or proliferation
  • said second agent selected from the group consisting of: a VEGF polypeptide, a VEGF-B polypeptide, a VEGF-C polypeptide, a VEGF-D polypeptide, a VEGF-E polypeptide, a P1GF polypeptide, an FGF-2 polypeptide, an HGF polypeptide, and stimulatory angiopoietin polypeptides.
  • the invention provides a polypeptide comprising a fragment of an Ephrin that binds to a Tie receptor, for use in the manufacture of a medicament for the treatment of diseases characterized by aberrant growth, migration, or proliferation of cells that express a Tie receptor.
  • Preferred polypeptides are soluble, such as Ephrin fragments fused to an immunoglobulin fragment.
  • the invention provides novel combination therapies for vascular disorders.
  • pro-angiogenic therapies it is highly desirable to induce and build new vessels having a structure comparable to that of normal mature vasculature, which functions as well as normal vessels.
  • the novel finding of interaction between Tie receptors and ephrin-B2 offers a means to address this long-felt need.
  • the invention provides a method of promoting growth of vessels in a mammalian organism, comrpising steps of: (a) identifying a mammalian organism in need of neovascularization; and
  • the organism is human. Also in preferred embodiments, the organism has a disease or condition that would be expected to benefit from neovascularization, such as ischemic tissue, an infarction, a new or chronic wound, or a tissue graft or transplant.
  • a disease or condition that would be expected to benefit from neovascularization, such as ischemic tissue, an infarction, a new or chronic wound, or a tissue graft or transplant.
  • a related aspect of the invention comprises gene therapy whereby a gene encoding the protein of interest is administered in a manner to effect expression of the protein of interest in the animal.
  • the gene of interest is attached to a suitable promoter to promote expression of the protein in the target cell of interest, and is delivered in any gene therapy vector capable of delivering the gene to the cell, including adenoviras vectors, adeno-associated virus vectors, liposomes, naked DNA transfer, and others.
  • the invention provides a novel method of modulating Tie recpeptor activity.
  • the invention provides a method of modulating Tie receptor activity in a mammalian subject, comprising steps of:
  • composition comprising an agent selected from the group consisting of:
  • Use of soluble forms of Ephrins that bind Tie receptors is specifically contemplated as a preferred embodiment.
  • Tie-binding fragments of Ephrins are desirably produced as fusions with immunoglobulin peptides or other peptides that promote solubility and increased circulating half-life. Solublization also is improved by pegylation or other covalent modifications to the amino acid sequence.
  • the method can be performed in situations where it is desirable to directly modulate Tie receptor activity, and also in situations where it is desirable to indirectly modulate such activity by modulating Tie-angiopoietin interactions.
  • Administration via any medically suitable route is contemplated. Additional features and variations of the invention will be apparent to those skilled in the art from the entirety of this application, and all such features are intended as aspects of the invention.
  • the invention includes, as an additional aspect, all embodiments of the invention narrower in scope in any way than the variations specifically mentioned above.
  • the applicant(s) invented the full scope of the claims appended hereto, the claims appended hereto are not intended to encompass within their scope the prior art work of others. Therefore, in the event that statutory prior art within the scope of a claim is brought to the attention of the applicants by a Patent Office or other entity or individual, the applicant(s) reserve the right to exercise amendment rights under applicable patent laws to redefine the subject matter of such a claim to specifically exclude such statutory prior art or obvious variations of statutory prior art from the scope of such a claim. Variations of the invention defined by such amended claims also are intended as aspects of the invention.
  • Fig. 1 A is a bar graph depicting binding between a soluble Ephrin-B2-
  • Fc fusion protein and receptors Tie-1, Tie-2, Epl ⁇ B4, and VEGFR-3 control.
  • Fig. IB is a photomicrograph showing Ephrin-B2-Fc binding to Tie-1 or Tie-2 receptors expressed on the surface of cells.
  • Figure 2 is a graph depicting the results of an Ephrin-B2/EphB4 binding inhibition assay. DETAILED DESCRIPTION OF THE INVENTION
  • the present invention involves the fields of cell and molecular biology, and many standard techniques relevant to those fields will be relevant to the practice of the present invention. Many such techniques are described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989), and or Ausubel et al., eds., Current Protocols in Molecular Biology, Green Publishers Inc. and Wiley and Sons, NY (1994-2001), both of which are incorporated by reference in their entirety. A. Gene sequences of interest to the present invention.
  • Tie- 1 At least two Tie receptors have been identified, referred to as Tie (Tie- 1) and Tie-2. At least eight Ephrin polypeptide family members have been identified, divided into A and B subclasses. The DNA and deduced amino acid sequences of all known Ephrins, Tie receptors, and Ephrin receptors of any vertebrate species that have been reported in the literature are hereby incorporated by reference. However, due to their special signficance to the invention, the following table is provided for the convenience of the reader:
  • the Angiopoietin Family Members The Angiopoietins are of special interest to the present invention because, like Ephrin-B2, the Angiopoietins have been found to modulate (stimulate or inhibit) Tie-2.
  • the angiopoietin (Ang 1-4) family of molecules were originally identified by cDNA library screening for ligands to the orphan Tie-2 receptor tyrosine kinase. [Davis et al, Cell, 87: 1161-69 (1996)].
  • Ang-1 the first of the angiopoietin ligands identified, was isolated through secretion-trap expression cloning using cell lines which demonstrated binding of secreted factors to Tie-2-Fc molecules.
  • This novel technique isolated a 498 amino acid, 70 kDa glycoprotein.
  • the N-terminal region of the protein showed hydrophobic sequences characteristic of secretory signal sequences.
  • Residues 100-280 of Ang-1 resemble a coiled-coil structure like that found in myosin, while residues 280-498 show homology to a family of proteins which includes fibrinogen, thus this region is the fibrinogen-like domain.
  • Ang-1 shows a binding affinity to Tie-2 less than 4 nM, and induces phosphorylation and activation of the Tie-2 tyrosine kinase.
  • Ang-2 a 496 amino acid protein (Maisonpierre et al, Science. 277: 55-60 (1997)), shows 85% homology to mouse ang-2 and 60% homology to the Human Ang-1 protein.
  • Ang-2 possesses the N-terminal secretory signal sequence found in Ang-1, and also both the coiled-coil and fibrinogen-like domains.
  • Ang-2 also shares 8 of the 9 cysteine residues found throughout the Ang-1 sequence, believed to be important in disulfide bond formation.
  • Ang-2 activity on the .Tie-2 receptor shows that ang-2 binds to Tie-2 but does not induce phosphorylation of the receptor, implicating Ang-2 as an antagonist to Ang-1 activation of Tie-2.
  • Angiopoietin-3 has been isolated by several groups based on sequence similarity to ang-1 and ang-2. See, e.g., Kim et al, FEBSLett. 443: 353-6 (1999); Nishimura et al, FEBSLett. 448: 254-6 (1999). The groups identified either a 503 or 491 amino acid clone of Ang-3, respectively.
  • a third group independently identified a 460 amino acid Ang-3 clone, (ANGPTL3) from human liver tissue. Conklin et al., Genomics, 62: 477-82 (1999). All three clones possess the characteristic N-terminal secretory signal sequence, coiled-coil motif, and fibrinogen-like domains of the other Ang family members.
  • Human Ang-4 identified by Valenzuela, et al (Proc. Natl Acad. Sci
  • the invention involves combination therapies involving use of of an Ephrin polypeptide (or inhibitor thereof) in combination with one of these polypetpides, or in combination with inhibitors (e.g., antibodies) of one of these polypeptides.
  • inhibitors e.g., antibodies
  • the PDGF/VEGF Family of growth factors includes at least the following members: PDGF-A (see e.g., GenBank Ace. No. X06374), PDGF-B (see e.g., GenBank Ace. No. M12783), VEGF (see e.g., GenBank Ace. No. Q16889 referred to herein for clarity as VEGF-A or by particular isoform), P1GF (see e.g., GenBank Ace. No. X54936 placental growth factor), VEGF-B (see e.g., GenBank Ace. No.
  • VEGF-related factor also known as VEGF-related factor (VRF)
  • VEGF-C see e.g., GenBank Ace. No. X94216; also known as VEGF related protein (V P)
  • V P VEGF related protein
  • VEGF-D also known as c-fos-induced growth factor (FIGF); see e.g., Genbank Ace. No.
  • VEGF-E also known as NZ7 VEGF or OV NZ7; see e.g., GenBank Ace. No. S67522
  • NZ2 VEGF also known as OV NZ2; see e.g., GenBank Ace. No. S67520
  • D1701 VEGF-like protein see e.g., GenBank Ace. No.
  • the PDGF/VEGF family proteins are predominantly secreted glycoproteins that form either disulfide-linked or non-covalently bound homo- or heterodimers whose subunits are arranged in an anti-parallel manner [Stacker and Achen, Growth Factors 17:1-11 (1999); Mullev et al, Structure 5:1325-1338 (1997)].
  • the VEGF subfamily is composed of PDGF/VEGF members which share a VEGF homology domain (VHD) characterized by the sequence: C-X(22-24)- P-[PSR]-C-V-X(3)-R-C-[GSTA]-G-C-C-X(6)-C-X(32-41)-C.
  • VHD VEGF homology domain
  • VEGF-A was originally purified from several sources on the basis of its mitogenic activity toward endothelial cells, and also by its ability to induce microvascular permeability, hence it is also called vascular permeability factor (VPF).
  • VEGF-A has subsequently been shown to induce a number of biological processes including the mobilization of intracellular calcium, the induction of plasminogen activator and plasminogen activator inhibitor- 1 synthesis, promotion of monocyte migration in vitro, induction of antiapoptotic protein expression in human endothelial cells, induction of fenestrations in endothelial cells, promotion of cell adhesion molecule expression in endothelial cells and induction of nitric oxide mediated vasodilation and hypotension [Ferrara, JMolMed 77: 527-543 (1999); Neufeld et al, FASEB J 13: 9-22 (1999); Zachary, Intl JBiochem Cell Bio 30: 1169-1174 (1998)].
  • VEGF-A is a secreted, disulfide-linked homodimeric glycoprotein composed of 23 kD subunits.
  • each isoform differs in biological activity, receptor specificity, and affinity for cell surface- and extracellular matrix-associated heparan- sulfate proteoglycans, which behave as low affinity receptors for VEGF-A.
  • VEGF 121 does not bind to either heparin or heparan-sulfate; VEGF 145 and VEGF 165 (GenBank Ace. No. M32977) are both capable of binding to heparin; and VEGF 189 and VEGF 206 show the strongest affinity for heparin and heparan-sulfates.
  • VEGF 21 , VEGF 145 , and VEGF 16 are secreted in a soluble form, although most of VEGF 165 is confined to cell surface and extracellular matrix proteoglycans, whereas VEGF 189 and VEGF 206 remain associated with extracellular matrix.
  • Both VEGF 189 and VEGF 206 can be released by treatment with heparin or heparinase, indicating that these isoforms are bound to extracellular matrix via proteoglycans.
  • Cell-bound VEGF 189 can also be cleaved by proteases such as plasmin, resulting in release of an active soluble VEGFno.
  • proteases such as plasmin
  • VEGF 145 differs in that it is primarily expressed in cells derived from reproductive organs [Neufeld et al, FASEB J 13:9-22 (1999)].
  • VEGF-A The pattern of VEGF-A expression suggests its involvement in the development and maintenance of the normal vascular system, and in angiogenesis associated with tumor growth and other pathological conditions such as rheumatoid arthritis.
  • VEGF-A is expressed in embryonic tissues associated with the developing vascular system, and is secreted by numerous tumor cell lines. Analysis of mice in which VEGF-A was knocked out by targeted gene disruption indicate that VEGF-A is critical for survival, and that the development of the cardiovascular system is highly sensitive to VEGF-A concentration gradients. Mice lacking a single copy of VEGF-A die between day 11 and 12 of gestation. These embryos show impaired growth and several developmental abnormalities including defects in the developing cardiovasculature.
  • VEGF-A is also required post-natally for growth, organ development, regulation of growth plate morphogenesis and endochondral bone formation. The requirement for VEGF-A decreases with age, especially after the fourth postnatal week. In mature animals, VEGF-A is required primarily for active angiogenesis in processes such as wound healing and the development of the corpus luteum. [Neufeld et al, FASEB J 13:9-22 (1999); Ferrara, JMol Med 77:527-543 (1999)]. VEGF-A expression is influenced primarily by hypoxia and a number of hormones and cytokines including epidermal growth factor (EGF), TGF- ⁇ , and various interleukins. Regulation occurs transcriptionally and also post- transcriptionally such as by increased mRNA stability [Ferrara, JMol Med 77:527- 543 (1999)].
  • EGF epidermal growth factor
  • P1GF a second member of the VEGF subfamily, is generally a poor stimulator of angiogenesis and endothelial cell proliferation in comparison to VEGF- A, and the in vivo role of P1GF is not well understood.
  • Three isoforms of P1GF produced by alternative mRNA splicing have been described [Hauser et al, Growth Factors 9:259-268 (1993); Maglione et al, Oncogene 5:925-931 (1993)].
  • P1GF forms both disulfide-liked homodimers and heterodimers with VEGF-A.
  • P1GF- VEGF-A heterodimers are more effective at inducing endothelial cell proliferation and angiogenesis than P1GF homodimers.
  • P1GF is primarily expressed in the placenta, and is also co-expressed with VEGF-A during early embryogenesis in the trophoblastic giant cells of the parietal yolk sac [Stacker and Achen, Growth Factors 17:1-11 (1999)].
  • VEGF-B described in detail in International Patent Publication No. WO 96/26736 and U.S. Patents 5,840,693 and 5,607,918, incorporated herein by reference, shares approximately 44% amino acid identity with VEGF-A.
  • VEGF-B has been shown to have angiogenic properties, and may also be involved in cell adhesion and migration, and in regulating the degradation of extracellular matrix. It is expressed as two isoforms of 167 and 186 amino acid residues generated by alternative splicing.
  • VEGF-B 167 is associated with the cell surface or extracellular matrix via a heparin-binding domain, whereas VEGF-B 186 is secreted.
  • Both VEGF- B 16 and VEGF-B 186 can form disulfide-linked homodimers or heterodimers with VEGF-A.
  • the association to the cell surface of VEGF 165 -VEGF-B 167 heterodimers appears to be determined by the VEGF-B component, suggesting that heterodimerization may be important for sequestering VEGF-A.
  • VEGF-B is expressed primarily in embryonic and adult cardiac and skeletal muscle tissues [Joukov et al, J Cell Physiol 173:211-215 (1997); Stacker and Achen, Growth Factors 17:1-11 (1999)]. Mice lacking VEGF-B survive but have smaller hearts, dysfunctional coronary vasculature, and exhibit impaired recovery from cardiac ischemia [Bellomo et al, Circ Res 2000;E29-E35].
  • a fourth member of the VEGF subfamily, VEGF-C comprises a VHD that is approximately 30% identical at the amino acid level to VEGF-A.
  • VEGF-C is originally expressed as a larger precursor protein, prepro-VEGF-C, having extensive amino- and carboxy-terminal peptide sequences flanking the VHD, with the C- terminal peptide containing tandemly repeated cysteine residues in a motif typical of Balbiani ring 3 protein.
  • Prepro-VEGF-C undergoes extensive proteolytic maturation involving the successive cleavage of a signal peptide, the C-terminal pro-peptide, and the N-terminal pro-peptide.
  • VEGF-C protein consists of a non-covalently- linked homodimer, in which each monomer contains the VHD.
  • the intermediate forms of VEGF-C produced by partial proteolytic processing show increasing affinity for the VEGFR-3 receptor, and the mature protein is also able to bind to the VEGFR- 2 receptor. [Joukov et al., EMBO J., 16:(13):3898-?>911 (1997)]. It has also been demonstrated that a mutant VEGF-C, in which a single cysteine at position 156 is either substituted by another amino acid or deleted, loses the ability to bind VEGFR-2 but remains capable of binding and activating VEGFR-3 [International Patent Publication No. WO 98/33917].
  • VEGF-C mRNA is expressed primarily in the allantois, jugular area, and the metanephros. [Joukov et al, J Cell Physiol 173:211-215 (1997)]. VEGF-C is involved in the regulation of lymphatic angiogenesis: when VEGF-C was overexpressed in the skin of transgenic mice, a hyperplastic lymphatic vessel network was observed, suggesting that VEGF-C induces lymphatic growth [Jeltsch et al, Science, 276:1423-1425 (1997)]. Continued expression of VEGF-C in the adult also indicates a role in maintenance of differentiated lymphatic endothelium [Ferrara, JMol Med 77:527-543 (1999)].
  • VEGF-C also shows angiogenic properties: it can stimulate migration of bovine capillary endothelial (BCE) cells in collagen and promote growth of human endothelial cells [see, e.g., International Patent Publication No. WO 98/33917, incorporated herein by reference].
  • BCE bovine capillary endothelial
  • VEGF-C [see International Patent Publ. No. WO 98/07832, incorporated herein by reference]. Like VEGF-C, VEGF-D is initially expressed as a prepro-peptide that undergoes N-terminal and C-terminal proteolytic processing, and forms non- covalently linked dimers. VEGF-D stimulates mitogenic responses in endothelial cells in vitro. During embryogenesis, VEGF-D is expressed in a complex temporal and spatial pattern, and its expression persists in the heart, lung, and skeletal muscles in adults. Isolation of a biologically active fragment of VEGF-D designated VEGF- D ⁇ N ⁇ C, is described in International Patent Publication No. WO 98/07832, incorporated herein by reference. VEGF-D ⁇ N ⁇ C consists of amino acid residues 93 to 201 of VEGF-D.
  • VEGF-E and NZ2 VEGF are potent mitogens and permeability enhancing factors. Both show approximately 25% amino acid identity to mammalian VEGF-A, and are expressed as disulfide-liked homodimers. Infection by these viruses is characterized by pustular dermititis which may involve endothelial cell proliferation and vascular permeability induced by these viral VEGF proteins.
  • VEGF-like proteins have also been identified from two additional strains of the orf virus, D1701 [GenBank Ace. No. AF106020; described in Meyer et al, EMBO J 18:363-314 (1999)] andNZIO [described in International Patent Application PCT/US99/25869, incorporated herein by reference]. These viral VEGF-like proteins have been shown to bind VEGFR-2 present on host endothelium, and this binding is important for development of infection and viral induction of angiogenesis [Meyer et al, EMBO J 18:363-314 (1999); International Patent Application PCT/US99/25869] .
  • PDGFR- ⁇ see e.g., GenBank Ace. No. NM006206
  • PDGFR- ⁇ see e.g., GenBank Ace. No. NM002609
  • VEGFR-l/Flt-1 fins-like tyrosine kinase-1; GenBank Ace. No. X51602; De Vries et al, Science 255:989-991 (1992)
  • VEGFR-2/KDR/Flk-l kinase insert domain containing receptor/fetal liver kinase-1; GenBank Ace. Nos.
  • VEGF ⁇ ⁇ , VEGF165, VEGF-B, P1GF-1 and P1GF-2 bind VEGF-R1; VEGF 121 , VEGF 145 , VEGF 165 , VEGF- C, VEGF-D, VEGF-E, and NZ2 VEGF bind VEGF-R2; VEGF-C and VEGF-D bind VEGFR-3; VEGF 165 , P1GF-2, and NZ2 VEGF bind neuropilin-1; and VEGF 165 binds neuropilin-2.
  • the PDGF receptors are protein tyrosine kinase receptors (RTKs) that contain five immunoglobulin-like loops in their extracellular domains.
  • RTKs protein tyrosine kinase receptors
  • VEGFR-1, VEGFR-2, and VEGFR-3 comprise a subgroup of the PDGF subfamily of PTKs, distinguished by the presence of seven Ig domains in their extracellular domain and a split kinase domain in the cytoplasmic region.
  • Both neuropilin-1 and neuropilin-2 are non-RTK VEGF receptors.
  • NP-1 has an extracellular portion includes a MAM domain; regions of homology to coagulation factors V and VIII, MFGPs and the DDR tyrosine kinase; and two CUB-like domains.
  • VEGFR-1 A soluble isoform of VEGFR-1 lacking the seventh Ig-like loop, transmembrane domain, and the cytoplasmic region is expressed in human umbilical vein endothelial cells.
  • This VEGFR-1 isoform binds VEGF-A with high affinity and is capable of preventing VEGF-A-induced mitogenic responses [Ferrara, JMolMed 77:521-543 (1999); Zachary, Intl JBiochem Cell Bio 30:1169-1174 (1998)].
  • a C- terminal truncated from of VEGFR-2 has also been reported [Zachary, Intl JBiochem Cell Bio 30: 1169-1174 (1998)].
  • h humans there are two isoforms of the VEGFR-3 protein which differ in the length of their C-terminal ends. Studies suggest that the longer isoform is responsible for most of the biological properties of VEGFR-3.
  • VEGFR-1 vascular endothelial cells
  • monocytes trophoblast cells
  • renal mesangial cells vascular endothelial cells
  • High levels of VEGFR-1 mRNA are also detected in adult organs, suggesting that VEGFR-1 has a function in quiescent endothelium of mature vessels not related to cell growth.
  • VEGFR-1-/- mice die in utero between day 8.5 and 9.5. Although endothelial cells developed in these animals, the formation of functional blood vessels was severely impaired, suggesting that VEGFR-1 may be involved in cell-cell or cell-matrix interactions associated with cell migration.
  • mice expressing a mutated VEGFR-1 in which only the tyrosine kinase domain was missing show normal angiogenesis and survival, suggesting that the signaling capability of VEGFR-1 is not essential.
  • VEGFR-2 expression is similar to that of VEGFR-1 in that it is broadly expressed in the vascular endothelium, but it is also present in hematopoietic stem cells, megakaryocytes, and retinal progenitor cells [Neufeld et al, FASEB J 13:9-22 (1999)]. Although the expression pattern of VEGFR-1 and VEGFR-2 overlap extensively, evidence suggests that, in most cell types, VEGFR-2 is the major receptor through which most of the VEGFs exert their biological activities. Examination of mouse embryos deficient in VEGFR-2 further indicate that this receptor is required for both endothelial cell differentiation and the development of hematopoietic cells [Joukov et al, J Cell Physiol 173:211-215 (1997)].
  • VEGFR-3 is expressed broadly in endothelial cells during early embryogenesis. During later stages of development, the expression of VEGFR-3 becomes restricted to developing lymphatic vessels [Kaipainen, A., et al, Proc. Natl. Acad. Sci. USA, 92: 3566-3570 (1995)]. In adults, the lymphatic endothelia and some high endothelial venules express VEGFR-3, and increased expression occurs in lymphatic sinuses in metastatic lymph nodes and in lymphangioma. VEGFR-3 is also expressed in a subset of CD34 + hematopoietic cells which may mediate the myelopoietic activity of VEGF-C demonstrated by overexpression studies [WO 98/33917].
  • VEGFR-3 Targeted disruption of the VEGFR-3 gene in mouse embryos leads to failure of the remodeling of the primary vascular network, and death after embryonic day 9.5 [Dumont et al, Science, 282: 946-949 (1998)]. These studies suggest an essential role for VEGFR-3 in the development of the embryonic vasculature, and also during lymphangiogenesis.
  • VEGF receptors Structural analyses of the VEGF receptors indicate that the VEGF-A binding site on VEGFR-1 and VEGFR-2 is located in the second and third Ig-like loops. Similarly, the VEGF-C and VEGF-D binding sites on VEGFR-2 and VEGFR- 3 are also contained within the second Ig-loop [Taipale et al, Curr Top Microbiol Immunol 237:85-96 (1999)]. The second Ig-like loop also confers ligand specificity as shown by domain swapping experiments [Ferrara, JMolMed 77:527-543 (1999)].
  • VEGFR-1 and VEGFR-2 are structurally similar, share common ligands (VEGF ⁇ ⁇ and VEGF ⁇ 65 ), and exhibit similar expression patterns during development. However, the signals mediated through VEGFR-1 and VEGFR-2 by the same ligand appear to be slightly different. VEGFR-2 has been shown to undergo autophosphorylation in response to VEGF-A, but phosphorylation of VEGFR-1 under identical conditions was barely detectable.
  • VEGFR-2 mediated signals cause striking changes in the morphology, actin reorganization, and membrane ruffling of porcine aortic endothelial cells recombinantly overexpressing this receptor.
  • VEGFR-2 also mediated ligand-induced chemotaxis and mitogenicity; whereas VEGFR- 1-transfected cells lacked mitogenic responses to VEGF-A. Mutations in VEGF-A that disrupt binding to VEGFR-2 fail to induce proliferation of endothelial cells, whereas VEGF-A mutants that are deficient in binding VEGFR-1 are still capable of promoting endothelial proliferation.
  • VEGFR-1 vascular endothelial growth factor-1
  • VEGFR-2 is thought to be the main transducer of VEGF-A signals in endothelial cells.
  • mice deficient in VEGFR-1 die as embryos while mice expressing a VEGFR-1 receptor capable of binding VEGF-A but lacking the tyrosine kinase domain survive and do not exhibit abnormal embryonic development or angiogenesis.
  • analyses of VEGF-A mutants that bind only VEGFR-2 show that they retain the ability to induce mitogenic responses in endothelial cells.
  • VEGF- mediated migration of monocytes is dependent on VEGFR-1, indicating that signaling through this receptor is important for at least one biological function.
  • VEGFR-1 may function in cell types other than endothelial cells.
  • Neuropilin-1 was originally cloned as a receptor for the collapsin/semaphorin family of proteins involved in axon guidance [Stacker and Achen, Growth Factors 17:1-11 (1999)]. It is expressed in both endothelia and specific subsets of neurons during embryogenesis, and it thought to be involved in coordinating the developing neuronal and vascular system. Although activation of neuropilin-1 does not appear to elicit biological responses in the absence of the VEGF family tyrosine-kinase receptors, their presence on cells leads to more efficient binding of VEGF 165 and VEGFR-2 mediated responses. [Neufeld et al, FASEB J 13:9-22 (1999)] Mice lacking neuropilin-1 show abnormalities in the developing embryonic cardiovascular system. [Neufeld et al, FASEB J 13:9-22 (1999)]
  • Neuropilin-2 was identified by expression cloning and is a collapsin/semaphorin receptor closely related to neuropilin-1.
  • Neuropilin-2 is an isoform-specif ⁇ c VEGF receptor in that it only binds VEGF 165 .
  • neuropilin-2 is expressed in both endothelia and specific neurons, and is not predicted to function independently due to its relatively short intracellular domain. The function of neuropilin-2 in vascular development is unknown [Neufeld et al, FASEB J 13:9-22 (1999); WO 99/30157].
  • Hepatocyte growth factor (HGF) is thought to be a factor influencing cell growth and cell motility for various epithelial cells.
  • HGF is a heterodimeric molecule composed of the 69 kDa alpha-subunit and the 34 kDa beta-subunit. HGF has no amino acid sequence homology with other known peptide growth factors, but shows 38% homology with plasmin HGF, derived from a single chain precursor of 728 amino acid residues, is proteolytically processed to form a two-chain mature HGF.
  • the 34 kDa beta-subunit and the 69 kDa alpha-subunit of HGF which contains 4 kringle structures.
  • HGF has mitogenic activity for renal tubular epithelial cells, has the potential to promote cell migration for some epithelial cells, including normal human keratinocytes, and may play an important role in wound healing and embryogenesis. See generally U.S. Patent Nos. 6,248,722, 6,214,344, and 5,004,805, incorporated herein by reference.
  • glutamic acid, aspartic acid a basic side chain (arginine, lysine, histidine); a polar amide side chain (glutamine, asparagine); a hydrophobic, aliphatic side chain (leucine, isoleucine, valine, alanine, glycine); an aromatic side chain (phenylalanine, tryptophan, tyrosine); a small side chain (glycine, alanine, serine, threonine, methionine); or an aliphatic hydroxyl side chain (serine, threonine).
  • a basic side chain arginine, lysine, histidine
  • a polar amide side chain glutamine, asparagine
  • a hydrophobic, aliphatic side chain leucine, isoleucine, valine, alanine, glycine
  • an aromatic side chain phenylalanine, tryptophan, tyrosine
  • binding assays and tyrosine phophorylation assays are available to determine whether a particular ligand or ligand variant (a) binds and (b) stimulates or inhibits RTK activity.
  • Two manners for defining genera of polypeptide variants include percent amino acid identity to a native polypeptide (e.g., 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity preferred), or the ability of encoding-polynucleotides to hybridize to each other under specified conditions.
  • One exemplary set of conditions is as follows: hybridization at 42°C in 50% formamide, 5X SSC, 20 mM Na « PO 4 , pH 6.8; and washing in IX SSC at 55°C for 30 minutes.
  • Formula for calculating equivalent hybridization conditions and/or selecting other conditions to achive a desired level of stringency are well known.
  • Any suitable vector may be used to introduce a transgene of interest into an animal.
  • Exemplary vectors that have been described in the literature include replication-deficient retroviral vectors, including but not limited to lentivirus vectors [Kim et al, J. Virol, 72(1): 811-816 (1998); Kingsman & Johnson, Scrip Magazine, October, 1998, pp. 43-46.]; adeno-associated viral vectors [Gnatenko et al, J. Investig. Med, 45: 87-98 (1997)]; adenoviral vectors [See, e.g., U.S. Patent No. 5,792,453; Quantin et al, Proc. Natl Acad. Sci.
  • preferred polynucleotides include a suitable promoter and polyadenylation sequence to promote expression in the target tissue of interest.
  • the Tie promoter U.S. Patent No. 5,877,020, incorporated by reference
  • suitable promoters/enliancers for mammalian cell expression include, e.g., cytomegalovirus promoter/enhancer [Lehner et al, J. Clin. Microbiol, 29:2494- 2502 (1991); Boshart et al, Cell, ⁇ .-521-530 (1985)]; Rous sarcoma virus promoter [Davis et al, Hum. Gene Ther., 4:151 (1993)]; or simian virus 40 promoter.
  • Anti-sense polynucleotides are polynucleotides which recognize and hybridize to polynucleotides encoding a protein of interest and can therefore inhibit transcription or translation of the protein. Full length and fragment anti-sense polynucleotides may be employed. Commercial software is available to optimize antisense sequence selection and also to compare selected sequences to known genomic sequences to help ensure uniqueness/specificity for a chosen gene. Such • uniqueness can be further confirmed by hybridization analyses. Antisense nucleic acids (preferably 10 to 20 base pair oligonucleotides) are introduced into cells (e.g., by a viral vector or colloidal dispersion system such as a liposome).
  • the antisense nucleic acid binds to the target nucleotide sequence in the cell and prevents transcription or translation of the target sequence.
  • Phosphorothioate and methylphosphonate antisense oligonucleotides are specifically contemplated for therapeutic use by the invention.
  • the antisense oligonucleotides may be further modified by poly-L-lysine, transferrin polylysine, or cholesterol moieties at their 5' end.
  • Genetic control can also be achieved through the design of novel transcription factors for modulating expression of the gene of interest in native cells and animals.
  • the Cys 2 -His 2 zinc finger proteins which bind DNA via their zinc finger domains, have been shown to be amenable to structural changes that lead to the recognition of different target sequences.
  • These artificial zinc finger proteins recognize specific target sites with high affinity and low dissociation constants, and are able to act as gene switches to modulate gene expression.
  • Knowledge of the particular target sequence of the present invention facilitates the engineering of zinc finger proteins specific for the target sequence using known methods such as a combination of structure-based modeling and screening of phage display libraries [Segal et al, (1999) Proc Natl Acad Sci USA 96:2158-2163; Liu et al, (1997) Proc Natl Acad Sci USA 94:5525-30; Greisman and Pabo (1997) Science 275:651-61; Choo et al, (1997) JMol Biol 273:525-32]. Each zinc finger domain usually recognizes three or more base pairs.
  • a zinc finger protein consisting of 6 tandem repeats of zinc fingers would be expected to ensure specificity for a particular sequence [Segal et al., (1999) Proc Natl Acad Sci USA 96:2158-2763].
  • the artificial zinc finger repeats designed based on target sequences, are fused to activation or repression domains to promote or suppress gene expression [Liu et al., (1997) Proc Natl Acad Sci USA :5525-30].
  • the zinc finger domains can be fused to the TATA box-binding factor (TBP) with varying lengths of linker region between the zinc finger peptide and the TBP to create either transcriptional activators or repressors [Kim et al, (1997) Proc Natl Acad Sci USA 94:3616-3620].
  • TBP TATA box-binding factor
  • Such proteins, and polynucleotides that encode them, have utility for modulating expression in vivo in both native cells, animals and humans.
  • the novel transcription factor can be delivered to the target cells by transfecting constructs that express the transcription factor (gene therapy), or by introducing the protein.
  • Engineered zinc finger proteins can also be designed to bind RNA sequences for use in therapeutics as alternatives to antisense or catalytic RNA methods [McColl et al., (1999) Proc Natl Acad Sci USA 96:9521-6; Wu et al, (1995) Proc Natl Acad Sci USA 92:344-348].
  • Antibodies are useful for modulating Tie-Ephrin interactions due to the ability to easily generate antibodies with relative specificity, and due to the continued improvements in technologies for adopting antibodies to human therapy.
  • the invention contemplates use of antibodies (e.g., monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies, bifunctional/bispecific antibodies, humanized antibodies, human antibodies, and complementary determining region (CDR)-grafted antibodies, including compounds which include CDR sequences which specifically recognize a polypeptide of the invention) specific for polypeptides of interest to the invention, especially Tie receptors and Ephrins.
  • antibodies e.g., monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies, bifunctional/bispecific antibodies, humanized antibodies, human antibodies, and complementary determining region (CDR)-grafted antibodies, including compounds which include CDR sequences which specifically recognize a polypeptide of the invention
  • Preferred antibodies are human antibodies which are produced and identified according to methods described in WO93/11236, published June 20, 1993, which is incorporated herein by reference in its entirety.
  • Antibody fragments including Fab, Fab', F(ab') 2 , and F v , are also provided by the invention.
  • the term "specific for,” when used to describe antibodies of the invention, indicates that the variable regions of the antibodies of the invention recognize and bind the polypeptide of interest exclusively (i. e. , able to distinguish the polypeptides of interest from other known polypeptides of the same family, by virtue of measurable differences in binding affinity, despite the possible existence of localized sequence identity, homology, or similarity between family members). It will be understood that specific antibodies may also interact with other proteins (for example, S.
  • aureus protein A or other antibodies in ELISA techniques through interactions with sequences outside the variable region of the antibodies, and in particular, in the constant region of the molecule.
  • Screening assays to determine binding specificity of an antibody of the invention are well known and routinely practiced in the art. For a comprehensive discussion of such assays, see Harlow et al (Eds), Antibodies A Laboratorv Manual: Cold Spring Harbor Laboratory; Cold Spring Harbor , NY (1988), Chapter 6.
  • Antibodies of the invention can be produced using any method well known and routinely practiced in the art.
  • Non-human antibodies may be humanized by any methods known in the art.
  • the non-human CDRs are inserted into a human antibody or consensus antibody framework sequence. Further changes can then be introduced into the antibody framework to modulate affinity or immunogenicity.
  • Polypeptides according to the invention may be administered in any suitable manner using an appropriate pharmaceutically-acceptable vehicle, e.g., a pharmaceutically-acceptable diluent, adjuvant, excipient or carrier.
  • a pharmaceutically-acceptable carrier solution such as water, saline, phosphate-buffered saline, glucose, or other carriers conventionally used to deliver therapeutics.
  • the "administering" that is performed according to the present invention may be performed using any medically-accepted means for introducing a therapeutic directly or indirectly into a mammalian subject, including but not limited to injections (e.g., intravenous, intramuscular, subcutaneous, or catheter); oral ingestion; intranasal or topical administration; and the like.
  • injections e.g., intravenous, intramuscular, subcutaneous, or catheter
  • oral ingestion e.g., intranasal or topical administration
  • intranasal or topical administration e.g., intravascular, such as by intravenous, intra-arterial, or intracoronary arterial injection.
  • the therapeutic composition may be delivered to the patient at multiple sites.
  • the multiple administrations may be rendered simultaneously or may be administered over a period of several hours. In certain cases it may be beneficial to provide a continuous flow of the therapeutic composition.
  • Additional therapy may be administered on a period basis, for example, daily, weekly or monthly.
  • Polypeptides for administration may be formulated with uptake or absorption enhancers to increase their efficacy.
  • enhancer include for example, salicylate, glycocholate/linoleate, glycholate, aprotinin, bacitracin, SDS caprate and the like. See, e.g., Fix (J. Pharm. Sci., 85(12) 1282-1285, 1996) and Oliyai and Stella (Ann. Rev. Pharmacol Toxicol, 32:521-544, 1993).
  • the amounts of peptides in a given dosage will vary according to the size of the individual to whom the therapy is being administered as well as the characteristics of the disorder being treated.
  • exemplary treatments it may be necessary to administer about 50mg/day, 75 mg/day, lOOmg/day, 150mg day, 200mg/day, 250 mg/day. These concentrations may be administered as a single dosage form or as multiple doses. Standard dose-response studies, first in animal models and then in clinical testing, reveal optimal dosages for particular disease states and patient populations.
  • kits which comprise compounds or compositions of the invention packaged in a manner which facilitates their use to practice methods of the invention
  • a kit includes a compound or composition described herein as useful for practice of a method of the invention (e.g., polynucleotides or polypeptides for administration to a person), packaged in a container such as a sealed bottle or vessel, with a label affixed to the container or included in the package that describes use of the compound or composition to practice the method of the invention.
  • the compound or composition is packaged in a unit dosage form.
  • the kit may further include a device suitable for administering the composition according to a preferred route of administration.
  • Ephrin-B2 comprises a ligand for Tie receptors.
  • Tie-1 and Tie-2 the following constructs were created and/or purchased from commercial suppliers:
  • a EphB4-Fc construct in which murine EphB4 amino acids 1 to 539 (SEQ ID NO: 32) were fused to the Fc portion of human IgGl;
  • a VEGFR-3 -Fc construct in which an extracellular domain portion of VEGFR-3 comprising the first three immunoglobulin-like domains were fused to the Fc portion of human IgGl (see Karpanen et al, Cancer Res., 61: 1786-1790 (2001);
  • a biotinylated Ephrin-B2-Fc construct in which murine Ephrin-B2 amino acids 1 to 227 (SEQ ID NO: 16) were fused to the Fc portion of human IgGl (R&D Systems);
  • microtiter plates were coated with 1 microgram/ml of either Tie-l-Fc, Tie-2 -Fc or, as controls, EphB4-Fc or VEGFR-3-Fc protein. After blocking the plate with phosphate buffered saline containing 1% bovine serum albumin and Tween detergent (1%BSA/PBS-T), biotinylated ephrin-B2-Fc protein was applied on the microtiter plates overnight at four degrees Centigrade.
  • phosphate buffered saline containing 1% bovine serum albumin and Tween detergent 1%BSA/PBS-T
  • biosensor analysis was performed using a sensor chip labeled with ephrin-B2 and the Tie-1 receptor.
  • Biosensor analysis was carried out with a BIAcore 2000TM (BIAcore AB, Uppsala, Sweden).
  • Ephrin-B2-Fc (10 ⁇ g/ml in 10 mM acetate buffer, pH 5) was coupled to the carboxymethylated dextran layer of a CM5 sensor chip at concentrations of 2600, 1000, 500 and 0 Resonance Units (RU) using standard amine coupling chemistry.
  • RU Resonance Units
  • Tie-1- Fc, EphB4-Fc and control proteins FGFR4-Fc and Fas-Fc were diluted in running buffer (1% BSA/PBS-T).
  • high salt and acidic solution 3M NaCl in 10 mM acetate, pH 4.2
  • a low pH pulse 100 mM HC1 for 30 seconds
  • Evaluation of binding affinities of Tie-1 and EphB4 for ephrin-B2 included both simultaneous and separate k a (association rate constant) and ka (dissociation rate constant) measurements according to the Langmuir binding model.
  • Kinetic parameters were determined using Evaluation software 3.02 (BIAcore AB). Tie-l-Fc (100 nM), EphB4 (100 nM) and control Fc proteins (FGFR4-
  • Fc and Fas-Fc were injected over the sensor chip surface containing immobilized ephrin-B2 (either 2600, 1000, 500, or 0 RU) and the binding of Tie- 1 and EphB4 measured. Analysis of the biosensor binding curves confirmed that ephrin-B2-Fc binds to EphB4 and Tie-1, but not to control Fc proteins.
  • COS1 cells were transiently tranfected (lipofectamine method) with pcDNA3 plasmids containing cDNAs encoding full-length human Tie-1, Tie-2, or VEGFR-3-Fc, or with empty plasmid pcDNA3 (Invitrogen) as a control. After 48 hours of incubation, the transfected COS cells were washed in HBH (Hank's buffer with 0.5%BSA and 20mM HEPES) and were fixed with 4% PFA for 15 minutes. The cells were washed with PBS and blocked with 1%BSA/PBS for 1 hour at 4 degrees C.
  • the cells were then incubated with 15 nM of biotinylated ephrin-B2-Fc in DMEM/FBS overnight at 4 degrees C. After washing with PBS, 1/100 HRP-conjugated anti-Fc antibodies (KPL) was incubated for 1 hour at room temperature. The ephrin-B2 binding to cells was visualized by DAB substrates with C1 2 .
  • Tie-1 transfected cells showed a weak staining, while Tie-2 transfected cells showed a strong staining.
  • Control VEGFR-3-Fc did not bind to the COS cells expressing Tie-1 or Tie-2.
  • ephrin-B2 did not bind to the COS cells expressing VEGFR-3.
  • Eprhin-B2/Tie-1 binding in the cell-free and cell- based assays was noteworthy. Without intending to be limited to a particular theory, one explanation for differential binding may be due to dimerization of Tie-l-Fc in the cell-free assay, creating a dimerized receptor that more efficiently binds ligands.
  • EXAMPLE 2 Binding between Tie receptors and other Ephrin molecules
  • the procedures described in Example 1 are modified by substituting other Ephrin family members for Ephrin-2B, and where appropriate, substituting other Eph receptors as controls. In this way, the ability of Tie-1 and Tie-2 to bind other Ephrin family members is characterized.
  • Ephrin and Tie receptor molecules provide a therapeutic indication for modulators of Ephrin-induced Tie receptor signaling, to modulate Tie-receptor-mediated biological processes.
  • the following examples provide proof of this therapeutic concept.
  • Tie receptors are useful as Ephrin inhibitory molecules due to the ability to block Ephrin-mediated signaling of Tie receptors or Eph receptors expressed on cell surfaces.
  • COS1 cells are transiently transfected with EphB4, e.g., via lipofectamine method, and incubated for 2 days. Then, 1 ⁇ g/ml of ephrin-B2 with 5 times molar of Tie-l-Fc or Tie-2-Fc is applied. After incubation for 1 hour, the cells are lysed with in TKB buffer (l%NP-40, 20mM Tris-HCl [pH7.5], 150mM NaCl, 5mM EDTA, 10% glycerol) supplemented with aprotinin, leupeptin, phenyhnethylsulfonyl fluoride, and sodium vanadate.
  • TKB buffer l%NP-40, 20mM Tris-HCl [pH7.5], 150mM NaCl, 5mM EDTA, 10% glycerol
  • Immunoprecipitation is carried out from equal amounts of cell lysates by adding specific antibodies and protein A-Sepharose or protein G-Sepharose and incubating for 1 hour.
  • the immunoprecipitates are washed with lysis buffer followed by elution with Laemmli buffer and applied in PAGE gels.
  • the proteins are transferred to a nitrocellulose membrane and blotted using anti-phosphotyrosine antibodies.
  • Location of Eph-B4 on the gels in confirmed with anti-Eph-B4 antibodies.
  • Reduced Eph-B4 autophosphorylation is indicative of successful Tie-Fc-mediated inhibition of Ephrin-B2/Eph-B4 binding.
  • Example 2 reveals additional Ephrin/Tie receptor interactions, the foregoing experiments can be repeated using such Ephrin molecules and their known Eph binding partners.
  • EphB4 Eph-recombinantly tranfected COS cells.
  • Use of primary cultures of neuronal cells expressing Eph receptors is specifically contemplated, e.g., cultured cerebellar granule cells derived from embryos.
  • Eph-receptor-specific antibodies can be employed to identify other cells (e.g., cells involved in vasculature, such as human microvascular endothelial cells (EC), human cutaneous fat pad microvascular cells (HUCEC), bovine capillary EC, porcine aortic EG, murine endothelial F-2 cells, or LE-2 cells.)
  • EXAMPLE 4 Use of soluble Tie peptides to modulate Ephrin biological activities.
  • Ephrin molecules are membrane-bound proteins and at least some Eph/Ephrin interactions are therefore thought to be cell-cell interactions.
  • Ephrin-B2 deletion experiments have revealed that ephrin-B2 marked specifically arterial endothelial cells while the EphB4 receptor was reciprocally expressed only on venous cells in the developing vasculature.
  • ephrin-B2 mutants show defects in both arteries and veins during angiogenesis despite expression of ephrin-B2 only in developing arterial endothelial cells, indicating bi-directional signaling as a result of ephrin EphR interaction.
  • Ephrin-expressing cells can be used to demonstrate the ability of soluble Tie receptor molecules to modulate ephrin-mediated cellular processes.
  • HAEC human aortic endothelial cells
  • ephrin-B2 human aortic endothelial cells
  • Ephrin-B2/EphB4 are thought to play a role in migration of cells
  • a cell migration assay using HAEC or other suitable cells can be used to demonstrate inhibitory effects of Tie molecules.
  • polycarbonate filter wells Transwell, Costar, 8 micrometer pore
  • fibronectin Sigma
  • PBS 50 microg/ml fibronectin
  • HAEC passage 4-9, 1 x 10 5 cells
  • HAEC passage 4-9, 1 x 10 5 cells
  • serum-free media supplemented with ephrin-B2 (1 ⁇ g/ml), or ephrin-B2 in the presence of varying concentrations of Tie-l-Fc, Tie-2-Fc, or control VEGFR-3-Fc protein.
  • ephrin-B2 1 ⁇ g/ml
  • ephrin-B2 ephrin-B2 in the presence of varying concentrations of Tie-l-Fc, Tie-2-Fc, or control VEGFR-3-Fc protein.
  • After 5 hours, cells adhering to the top of the transwell are removed with a cotton swab, and the cells that migrate to the underside of the filter are fixed and stained.
  • 6 randomly selected 400x microscope fields are counted per filter.
  • Ephrins cluster before they can signal or transduce signals, and this characteristic provides another parameter for assay.
  • the effects of clustered Ephrin-B2-F c by anti-F c antibodies are tested on microtiter plates.
  • Anti- Ephrin antibodies are used to examine Ephrin-B2 clustering at the cell surface. Differences in clustering behavior are evaluated before and after treatment with soluble Tie-R or a Tie-R-expressing cell.
  • Soluble Ephrin-B2-Fc is used as described above to contact cells that naturally or recombinantly express Tie receptor on their surface.
  • COS cells recombinantly modified to transiently or stably express Tie-1 or Tie-2 can be employed.
  • native endothelial cells express Tie receptors and can also be employed, including isolated human umbilical vein endothelial cells (HUVEC), human microvascular endothelial cells (HMVEC), human cutaneous fat pad microvascular cells (HUCEC), bovine capillary EC, porcine aortic EC, murine endothelial F-2 cells, or LE-2 cells.
  • HMVEC human umbilical vein endothelial cells
  • HMVEC human microvascular endothelial cells
  • HUCEC human cutaneous fat pad microvascular cells
  • bovine capillary EC bovine capillary EC
  • porcine aortic EC murine endothelial F-2 cells, or LE-2 cells.
  • Tie receptor phosphorylation is assayed essentially as described in Example 3, except anti-Tie antibodies are used to confirm the location of the Tie peptides on the Western blot.
  • the ability of soluble EphB4-Fc, Tie-l-Fc, or Tie-2-Fc in molar excess to antagonize the effects of Ephrin-2B on the Tie receptors also can be evaluated.
  • Embyronic endothelial cells are cultured in the presence or absence of an Ephrin, such as Ephrin-B2, to assay effects on cell growth using any cell growth or migration assay, such as assays that measure increase in cell number or assays that measure tritiated thymidine incorporation. See, e.g., Thompson et al, Am. J. Physiol Heart Circ. Physiol, 281: H396-403 (2001).
  • the angiopoietins have been shown to act as ligands for Tie-2 receptor, mediating angiogenic effect in vivo.
  • the identification of Ephrin-B2 as a ligand for Tie-2 has therapeutic indications for angiogenesis.
  • the following protocols are used to measure inhibition of Angiopoietin-mediated Tie-2 activity by soluble Ephrin-B2 peptides.
  • Procedures described in preceding examples are modified to assay Ephrin-B2 inhibition of Angiopoietin stimulation of Tie-2.
  • COS cells transfected with a normal human Tie-2 cDNA and transiently expressing Tie-2 are stimulated with either 100 ng/ml Ang2-, or Angl -conditioned media from 293T cells transfected with Signal Pig Plus Angl, as well as several concentrations of Ang3 and Ang4 (Rjantie et al, Mol Cell. Biol, 21: 4647-55 (2001)) in the presence and absence of varying molar ratios of Ephrin-B2-Fc.
  • the cells are processed and Tie-2 phosphorylation is assayed as described above.
  • the protocol is repeated substituting HUVEC cells or other endothelial cells that naturally express Tie-2 for the COS cells.
  • the effects of Ephrin-B2 are assayed by evaluating the effects on cellular migration.
  • the cell migration assay outlined in Example 4 is modified by using Angiopoietins in the presence or absence of Ephrin-B2-Fc, Tie-l-Fc, Tie-2-Fc, or VEGFR-3 -Fc to measure the effects of Ephrin-B2 on the ability of angiopoietins to modulate endothelial cell migration.
  • EXAMPLE 7 Angiogenesis Assays
  • angiogenesis stimulators and inhibitors may work in concert through the same or different receptors, and on different portions of the circulatory system (e.g., arterieries or veins or capillaries; vascular or lymphatic).
  • Angiogenesis assays are employed to measure the effects of ephrin molecules, such as Ephrin-B2, on angiogenic processes, alone or in combination with other angiogenic and anti-angiogenic factors to determine preferred combination therapy involving Ephrins and other modulators. Exemplary procedures include the following.
  • HMVEC cells (passage 5-9) are grown to confluency on collagen coated beads (Pharmacia) for 5-7 days.
  • the beads are plated in a gel matrix containing 5.5 mg/ml fibronectin (Sigma), 2 units/ml thrombin (Sigma), DMEM/2% fetal bovine serum (FBS) and the following test and control proteins: 20 ng/ml VEGF, 20 ng/ml VEGF plus 10 micrograms/ml ephrin-B2-Fc, and several combinations of angiogenic factors and Fc fusion proteins. Serum free media supplemented with test and control proteins is added to the' gel matrix every 2 days and the number of endothelial cell sprouts exceeding bead length are counted and evaluated.
  • transwell migration assay previously described may also be used in conjunction with the sprouting assay to determine factors involved in angiogenesis.
  • the effects of Ephrins are assayed alone or in combination with known angiogenic or anti-angiogenic agents.
  • CAM Chorioallantoic Membrane
  • erythromycin / ophthamic ointment is applied to the eyes. Eyes are examined by a slit-lamp biomicroscope over a course of 3-12 days. Vessel length and clock-hours of circumferential neovascularization are measured. Furthermore, eyes are cut into sections and are immunostained for blood vessel and/or lymphatic markers (LYVE-1 [Prevo et al., J. Biol. Chem., 276: 19420-19430 (2001)], podoplanin
  • the following model is useful for demonstrating effects of materials and method of the invention in the treatment of ischemia or its symptoms or effects.
  • LDPI Laser Doppler perfusion imaging
  • mice are operated on and treated with intramuscular injection of materials of the invention, such as Fc fusion proteins (e.g., Eprhin-Fc fusions, Eph-Fc fusions) or anti- Ephrin antibodies; gene therapy vectors encoding the foregoing; and other materials described herein, either alone or in combination with other angiogenic factors, and compared with control mice.
  • Fc fusion proteins e.g., Eprhin-Fc fusions, Eph-Fc fusions
  • anti- Ephrin antibodies e.g., anti- Ephrin antibodies
  • Injections at various concentrations are made once every other day during the first week, once every 3 days during the second week, and twice during the third and fourth weeks. At predetermined time points, necrosis and hind-limb perfusion are examined. Mice are then sacrificed for histologic analysis.
  • Improved blood flow and/or improved vessel formation is indicative of the efficacy of single or combination agent therapy.
  • Ephrin materials described herein are tested for their effects on atherosclerosis using any suitable model.
  • An exemplary model is set forth below.
  • mice are fed a normal chow diet until 6 weeks of age. Then the mice are switched to a high-fat diet containing 20% fat and 0.3% cholesterol. They show remarkable atherosclerotic lesions after 12 weeks.
  • Ephrin DNAs and Proteins and antibodies and other materials of the invention described herein they are administered in various concentrations and combinations three times from 12 to 18 weeks. Aortic sinuses are compared in each study protocol.
  • Ephrin materials described herein are tested for their ability to reduce restenosis of vessels in any restenosis model. See, e.g., International Patent Publication No. WO/00/24412, incorporated herein by reference, for discussion of models, of formulations of agents for admimstration, and for materials and methods of administration. Treatment of restenosis in a manner described in that application, using Ephrin materials and methods described herein, is specifically contemplated..
  • the treatment performed is performed in the first group of rabbits 3 days after the denudation, and the animals are euthanized 2 or 4 weeks after the administration.
  • the whole aorta is denuded twice with a 4.0F arterial embolectomy catheter (Sorin Biomedical). Three days later, the administration is performed with a 3. OF channeled-balloon local drug delivery catheter (Boston Scientific). Under fluoroscopic control, the catheter is positioned caudal to the left renal artery in a segment free of side branches.
  • a virus titer of 1.15 x 10 10 pfu is used in the final volume of 2 mL in 0.9% saline, and the gene transfer is performed at 6 atm pressure for 10 minutes (0.2 mL/min).
  • the rabbits have only the administration of the agents without a cholesterol diet or balloon denudation, and they are euthanized 2 or 4 weeks after that. The evaluation is done by the histologic analysis.
  • mice Six- to 8-week-old nude (nu/nu) mice (SLC, Shizuoka, Japan) undergo subcutaneous transplantation of C6 rat glioblastoma cells or PC-3 prostate cancer cells in 0.1 mL phosphate-buffered saline (PBS) on the right flank.
  • PBS phosphate-buffered saline
  • the Ephrin- related materials of the invention are administered to the animals at various concentrations and dosing regimens. Tumor size is measured in 2 dimensions, and tumor volume is calculated using the formula, width 2 x length/2. After 14 days, the mice are humanely killed and autopsied to evaluate the quantity and physiology of tumor vasculature.
  • the assay can also be performed using other tumor cell lines implanted in nude mice or other mouse strains.
  • Use of wild type mice implated with LLC lung cancer cells and B16 melanoma cells is specifically contemplated. E Orthotopic tumor implantation
  • Approximately 1 x 10 7 MCF-7 breast cancer cells in PBS are inoculated into the fat pads of the second (axillar) mammary gland of ovarectomized SCID mice or nude mice, carrying s.c. 60-day slow-release pellets containing 0.72 mg of 17 ⁇ -estradiol (Innovative Research of America).
  • the ovarectomy and implantation of the pellets are done 4-8 days before tumor cell inoculation.
  • the Ephrin-related materials of the invention are administered to the animals at various concentrations and dosing regimens. Tumor size is measured in 2 dimensions, and tumor volume is calculated using the formula, width 2 x length/2. After 14 days, the mice are humanely killed and autopsied to evaluate the quantity and physiology of tumor vasculature.
  • Lymphatic metastasis model The following protocol indicates the ability of Ephrin-related materials of the invention for inhibition of lymphatic metastasis.
  • MDA-MB-435 breast cancer cells are injected bilaterally into the second mammary fat pads_of athymic,- female, eight week old nude mice.
  • the cells often metastasize to lymph node by 12 weeks.
  • the Ephrin-related materials of the invention are administered to the animals at various concentrations and dosing regimens.
  • the Ephrin-related materials are administered in combination with other materials for reducing tumor metastasis. See, e.g., International Patent Publication No. WO 00/21560, incorporated herein by reference in its entirety. Mice are killed after 12 weeks and lymph nodes are investigated by histologic analysis.
  • the administration protocol is the same as above.

Abstract

The present invention provides materials and methods relating to the interaction between Tie receptors and Ephrin polypeptides.

Description

EPHRIN-TIE RECEPTOR MATERIALS AND METHODS
FIELD OF THE INVENTION
The present invention provides materials and methods relating to cellular and molecular biology and medicine, in particular relating to areas of vasculogenesis, angiogenesis, lymphangiogenesis and the interface between arterial and venous systems and nervous systems.
BACKGROUND OF THE INVENTION
Receptor tyrosine kinases (RTK), integral membrane proteins for transducing cell signals, play important roles in numerous processes in embryonic development, including vascularization. See generally Schlessinger et al., Cell, 103: 211-225 (2000), incorporated herein by reference in its entirety. Establishment of the cardiovascular system is one of the first completed processes of embryogenesis and disruption of this process often leads to embryonic lethality. Vascular development is mediated primarily through the VEGF/VEGFR pathway, including the VEGFR-1,-2, and -3 RTKs , as well as a smaller family of receptor tyrosine kinases which contain Ig and EGF homology domains, Tie-1 (sometimes referred to simply as Tie) and Tie- 2 (also referred to as TEK). The biology of the Tie receptors is reviewed in Jones, Iljin, Dumont, and Alitalo, "Tie Receptors: New Modulators of Angiogenic and Lymphangiogenic Responses," Nature Reviews, 2: 257-67 (April, 2001), incorporated herein by reference in its entirety.
Embryonic vascularogenesis, the formation of a primitive network of tubules, proceeds through a very controlled series of steps mediated at least partly by the interaction of vascular endothelial growth factor (VEGF) family ligands with VEGF-Receptors (VEGFR) on the endothelium, setting up what is called the primary capillary plexus. Further development from this plexus - the sprouting, branching, and growth of different types of blood vessels - is referred to as angiogenesis. In addition to the requirement for the VEGF/VEGFR interactions, angiogenesis is also mediated at least partly through another family of growth factors, the Angiopoietins (Davis et al, Cell, 87: 1161-69 (1996), Valenzuela et al, Proc. Natl. Acad. Sci. 96: 1904-09 (1999), Suri et al, Cell 87: 1171-80 (1996), Maisonpierre et al, Science. 277: 55-60 (1997). Angiopoietins 1-4 (Ang 1-4) are targeted to the vascular endothelium much like VEGF, and bind to the Tie-2 receptor tyrosine kinase. Tie-1 and Tie-2 differ from the VEGFR and other RTKs in that they contain three EGF-like domains and three frbronectin type-Ill repeats in addition to immunoglobulin-like domains typically found in RTKs. Tie-1 and Tie-2 molecules show approximately 46% homology to each other (Partenan et al, Mol Cell. Biol 12: 1698-1707 (1992), Dumont et al, Oncogene, 7: 1471-80 (1992); Maisonpierre et al, Oncogene, 8: 1631-7 (1993).) Both Tie-2 and Tie-1 appear to be expressed in certain cells of hematopoietic origin in addition to the endothelium.
Expression of both Tie receptors becomes more intense in actively growing structures such as blood vessels. Tie-1 also appears to be variably expressed in adult tissues and may be found in capillary vessels of the lung and kidney, in lymphatic endothelium, in the reproductive tract during ovulation, in areas of wound healing, and in melanoma metastasis where Tie-1 was localized to the neovascularized endothelium (Iwama et al, Biochem. Biophys. Res. Comm. 195: 301- 9 (1993); Batard et al., "The Tie Receptor Tyrosine Kinase is Expressed by Human Hematopoietic Progenitor Cells and by a Subset of Megakaryocytic Cells," Blood,
' 87: 2212-2220 (1996); Korhonen et al, Blood, 80: 2548-55 (1992), Kaipainen et al,
Cancer Res. 5424: 6571-7 (1994)). In genetic deletion experiments, Tie-2 knockout mice die approximately embryonic day 9.5 (E9.5) with loss of vascular integrity and endothelial cell numbers (Dumont et al, Genes Dev,. 8: 1897-1909 (1994).) One of the most prominent defects in Tie-2 -knockout mice embryos is the incomplete development of the heart region. Tie-1 deficient mice survive until E13.5-E15.5, retaining major vessel structure with loss of microvascular integrity (Partanen et al, Development. 122: 3013-21. 1996). This differential expression pattern suggests that each receptor serves a unique function in blood vessel development. Tie-1 is also expressed in endothelial cells in lymphatic vessels.
There is at least some evidence that the Tie receptors may be involved in pathological angiogenesis as well as normal vascular development and maintenance. For example, interruption of Tie-2 signaling using soluble, dominant negative receptors can inhibit angiogenic growth in tumor-bearing mice. Elevated Tie-1 and Tie-2 expression has been observed in the endothelium of the neovasculature in numerous solid tumors, and certain leukemia cell lines express these receptors. The recently uncovered ligands for the Tie-2 receptor, angiopoietins 1- 4, are small, soluble factors secreted by, e.g., smooth muscle cells and leukemia cells. The angiopoietins have only been shown to interact specifically with the Tie-2 receptor, and unlike most other RTK ligands, the angiopoietins show both agonistic (Angl, Ang4) and antagonistic (Ang 2, Ang3) interactions with Tie-2 (Maisonpierre, 1997). Ang-1 stimulates phosphorylation of the Tie-2 receptor leading to vessel remodeling and angiogenesis (Davis et al., Cell 57:1161-69. 1996). Ang-1 deficient mice show the same phenotype as Tie-2 knockouts. However, Ang-1, Tie-1 double knockouts show failure to develop the right-hand side venous system while leaving the left side intact and functional (Loughna and Sato, Mol Cell, 7: 233-39 (2001).) Analysis of embryos lacking Tie-2 or Ang-1 suggests that these molecules may be required for endothelial cells to properly associate with underlying support cells. The persistent expression and phosphorylation of Tie-2 in quiescent adult endothelium is consistent with a role for Tie-2 in transducing a continuing survival stimulus to endothelial cells .
Another related group of RTKs that appear to be involved in vascular development are the Eph receptors. (See generally Wilkinson, "Multiple Roles of Eph Receptors and Ephrins in Neural Development," Nature Reviews, 2: 155-164 (March, 2001), incorporated herein by reference in its entirety.) Eph receptors, which make up the largest family of RTKs with fourteen members, fall into two categories of receptor (Eph A or B) based on ligand structure and interactions. Ephrins, the ligands for Eph receptors, are unique among RTK ligands in that they are membrane- bound ligands. The Ephrin-A subclass of ligands are GPI-linked (Brambilla et al, EMBO J., 14: 3116-26 (1995)) whereas the Ephrin-B subclass of ligands are membrane bound via a transmembrane domain and short cytoplasmic tail (Gale et al, Oncogene 13: 1343-52 (1996)). Binding of the ephrins to their RTK receptors (Eph's) induces clustering of the receptors and subsequent autophosphorylation of residues in the cytoplasmic domain. (Schlessenger, Cell: 103: 211-25 (2000).) The Ephrins must normally be membrane-attached to activate their receptors; the membrane attachement seems to promote clustering/multimerization that may be necessary for activation. [Gale et α/., Genes & Development, 13: 1055-66 (1999)]. In general, ephrins and Eph receptors show promiscuous binding within a particular subgroup, e.g. any ephrin- A binds to any EphR-A. However, it appears that ephrin- B2 binds primarily to the EphB4 receptor (Wang, 1998).
Ephrins and Eph-Receptors were originally identified in the nervous system where they play a significant role in neural patterning and neural cell migration during embryogenesis (Wang et al, Neuron 18: 383-96. 1997, Feldheim et al, Neuron, 21: 1303-13 (1998), Kalcheim et al, Development, 106: 85-93 (1989)), but expression is broadly distributed throughout embryonic tissue.
Recent evidence suggests that ephrin/EphR interactions play an important role in vascular development. Studes of Ephrin-Al indicate that it can promote chemotaxis of cultured endothelial cells, induce sprouting in a rat corneal pocket assay, and prmote capilllary-like assembly of human umbilical vein endothelial cells (HUVEC). Ephrin-Bl induces human renal microvascular endothelial cells (HRMEC) to form tubules.
Deletion of the ephrin-B2 gene (knockout mice) demonstrated a lethal phenotype similar to either a Tie-2 or Angl knockout, which lack vascular remodeling and endothelial cell integrity, and also lack endothelial sprouts, a sign of growth of vessels into avascular areas (Wang et al, Cell 93: 741-53, 1998). A knockout of EphB4, the known ligand for Ephrin-B2, dies at similar stages, and EphB2 and EphB3 knockouts also are lethal and characterized by defective vasculature development. [Gale et α/., Genes & Development, 13: 1055-66 (1999)]. Examination of expression in the circulatory system has identified Ephrin-B2 specifically in arterial endothelial cells while the EphB4 receptor was reciprocally expressed only on venous cells in the developing vasculature. However, Ephrin-B2 mutants show defects in both arteries and veins during angiogenesis despite expression of Ephrin-B2 only in developing arterial endothelial cells, suggesting bidirectional signaling as a result of Ephrin/EphR interaction.
Blood vessel growth like that found in embryogenesis is also important in the adult for continuous remodeling of the reproductive system, tissue repair, collateral neovascularization in ischemia, and tumor growth. Elucidating additional factors involved in the regulation of neovascularization and angiogenesis, as well as their roles in such processes, would aid in the development of therapies directed toward prevention of vascularization of solid tumors and induction of tumor regression, and induction of vascularization to promote faster, more efficient wound healing after injury, surgery, or tissue transplantation, or to treat ischemia by inducing angiogenesis and srteriogenesis of vessels that nourish the ischemic tissue. In fact, modulation of angiogenic processes may be instrumental in treatment or cure of many of the most significant diseases that plague humans in the developed world, such as cerebral infarction/bleeding, acute myocardial infarction and ischemia, and cancers.
For pro-angiogenic therapies, it is highly desirable to induce and build new vessels having a structure comparable to that of normal mature vasculature, which functions as well as normal vessels. To accomplish this, the interaction between endothelial cells and perivascular cells should be optimized. Many pro-angiogenic factors identified to date target only endothelial cells, and their administration may result in disorganized vessels in vivo. Based on data from knockout studies, angiopoietins were hoped to facilitate construction of mature vessels, but recent studies have indicated that these hopes may have been misplaced. Application of just angiopoietins, in fact, induced enlarged vessels uncovered by pericytes. A need exists for improved angiogenic therapies that lead to development of neo vasculature having increased characteristics of normal mature vessels.
For anti-angiogenic therapies, a significant and critical side effect is bleeding. In animal models, positive effects are sometimes over-emphasized and the side effects are often ignored. A need exists for improved anti-angiogenic therapies, through either novel agents or novel combinations of agents, that minimize such side- effects. Also desirable are combinations of agents that minimize total dosages of agents that are necessary to achieve a beneficial effect.
SUMMARY OF THE INVENTION
The present invention addresses one or more needs in the art relating to modulation of angiogenic processes, including lymphangiogenesis, by identifying a novel receptor-ligand interaction between Ephrin and Tie molecules, which had previously been known to interact with Eph receptors and angiopoietins, respectively, but not with each other. This newly established interaction provides novel materials and methods for modulation of angiogenic processes. It will be apparent that certain diseases and conditions are benefϊtted by promoting angiogenesis, whereas others are benefitted by inhibiting it. The identification herein of new involved proteins and new protein-protein interactions in the angiogenic process yields new tools to achieve these therapeutic end-goals.
For example, the discovery of Ephrin-Tie interactions and their importance to angiogenic processes permits novel screening assays to identify new therapeutic molecules to modulate (up-regulate/activate/stimulate or downregulate/inhibit) Ephrin-Tie interactions.
In one embodiment, the invention provides a method for identifying a modulator of binding between a Tie receptor tyrosine kinase and an Ephrin ligand, comprising steps of:
(a) contacting a Tie receptor composition with an Ephrin composition in the presence and in the absence of a putative modulator compound;
(b) detecting binding between Tie receptor and the Ephrin in the presence and absence of the putative modulator; and (c) identifying a modulator compound in view of decreased or increased binding between the Tie receptor and the Ephrin ligand in the presence of the putative modulator, as compared to binding in the absence of the putative modulator. h one variation, the method further includes a step (d) of making a modulator composition by formulating a modulator identified according to step (c) in a pharmaceutically acceptable carrier. A modulator so formulated is useful in animal studies and also as a therapeutic for administration to image tissues or treat diseases associated with aberrant Ephrin-Tie biology. Thus, in still another variation, the method further includes a step (e) of administering the modulator composition to an animal that comprises cells that express the Tie receptor, and determining physiological effects of the modulator composition in the animal. In a preferred variation, the animal (including humans) has a disease or condition characterized by aberrant Ephrin-Tie biology, and the modulator improves the animal's state (e.g., by reducing disease symptoms, slowing disease progression, curing the disease, or otherwise improving clinical outcome). Step (a) of the foregoing methods involves contacting a Tie receptor composition with an Ephrin composition in the presence and in the absence of a putative modulator compound. By "Tie receptor composition" is meant any composition that includes a whole Tie receptor, or includes at least the portion of the Tie receptor needed for the particular assay - in this case the portion of the Tie receptor involved in Ephrin binding. Exemplary Tie receptor compositions include: (i) a composition comprising a purified polypeptide that comprises an entire Tie protein or that comprises a Tie receptor extracellular domain fragment that binds the Ephrin; (ii) a composition containing phospholipid membranes that contain Tie receptor polypeptides on their surface; (iii) a living cell recombinantly modified to express increased amounts of a Tie receptor on its surface; and (iv) any isolated cell or tissue that naturally expresses the Tie receptor on its surface. For certain assay formats, it may be desirable to bind the Tie molecule of interest (e.g., a polypeptide comprising a Tie receptor extracellular domain fragment) to a solid support such as a bead or assay plate well. "Tie receptor composition" is intended to include such structures as well. Likewise, fusion proteins are contemplated. For other assay formats, soluble Tie peptides may be preferred. In one preferred variation, the Tie receptor composition comprises a Tie receptor extracellular domain fragment fused to an immunoglobulin Fc fragment. Although two family members are currently known, Tie-1 and Tie-2, practice of the invention with other Tie receptor family members that are subsequently discovered is contemplated. The Tie receptor chosen is preferably of vertebrate origin, more preferably mammalian, still more preferably primate, and still more preferably human. And, while it will be apparent that the assay will likely give its best results if the functional portion of the chosen Tie receptor is identical in amino acid sequence to the native receptor, it will be apparent that the invention can still be practiced if variations have been introduced in the Tie sequence that do not eliminate its Ephrin binding properties. Use of variant sequences with at least 90%, 95%, 96%, 97%, 98%, or 99% amino acid identity is specifically contemplated.
Ephrin molecules, like Tie receptors, are membrane bound, and similar considerations apply for the Ephrin composition that is used in the invention. Thus, "Ephrin composition" means any composition that includes a whole Ephrin polypeptide, or includes at least the portion of the Ephrin needed for the particular assay - in this case the portion involved in binding to a Tie receptor. Exemplary
Ephrin compositions include: (i) a composition comprising purified complete Ephrin polypeptide or comprising an Ephrin fragment that binds the Tie receptor chosen for the assay; (ii) a phospholipid membrane containing Ephrin polypeptides; (iii) a cell recombinantly modified to express increased amounts of an Ephrin on its surface; and (iv) any cell that naturally expresses the Ephrin on its surface. For certain assay formats, it may be desirable to bind the Ephrin molecule of interest (e.g., a polypeptide comprising an Ephrin extracellular domain fragment) to a solid support such as a bead or assay plate well. "Ephrin composition" is intended to include such structures as well. Likewise, fusion proteins are contemplated. For other assay formats, soluble Ephrin peptides may be preferred, hi one preferred variation, the Ephrin composition comprises an Ephrin fragment fused to an immunoglobulin Fc fragment. The data provided herein establishes that Ephrin-B2 binds both Tie-1 and Tie-2, and thus Ephrin-B2 is a highly preferred Ephrin for practice of the invention. However, at least eight Ephrins have been identified to date (Ephrin-Al, Ephrin- A2, Ephrin- A3, Ephrin- A4, Ephrin- A5, Ephrin-Bl, Ephrin-B2, and Ephrin-B3), and practice of the invention is contemplated with any such Ephrins that bind Tie receptors according to binding assays described herein. Moreover, practice of the invention is contemplated with other Ephrin family members that are subsequently discovered and that bind Tie receptors. The Ephrin chosen is preferably of vertebrate origin, more preferably mammalian, still more preferably primate, and still more preferably human. And, while it will be apparent that the assay will likely give its best results if the functional portion of the chosen Ephrin is identical in amino acid sequence to the corresponding portion of the native Ephrin, it will be apparent that the invention can still be practiced if variations have been introduced in the Ephrin sequence that do not eliminate its Tie receptor binding properties. Use of variant sequences with at least 90%, 95%, 96%, 97%, 98%, or 99% amino acid identity is specifically contemplated. The putative modulator compound that is employed can be any organic or inorganic chemical or biological molecule that one would want to test for ability to modulate Tie-Ephrin interactions. Since the most preferred modulators will be those that can be administered as therapeutics, it will be apparent that molecules with limited toxicity are preferred. However, toxicity can be screen in subsequent assays. Screening of chemical libraries such as those customarily kept by pharmaceutical companies, or combinatorial libraries, peptide libraries, and the like is specifically contemplated. Step (b) of the above-described method involves detecting binding between Tie receptor the Ephrin in the presence and absence of the putative modulator. Any technique for detecting intermolecular binding may be employed. For example, one or both of Tie/Ephrin may comprise a label, such as a radioisotope, a fluorophore, a fluorescing protein (e.g., natural or synthetic green fluorescent proteins), a dye, an enzyme or substrate, or the like. Such labels facilitate detection with standard laboratory machinery and techniques.
When the Tie receptor composition comprises a cell that expresses Tie naturally or recombinantly on its surface, it will often be possible to detect Ephrin binding indirectly, e.g., by detecting or measuring an Ephrin binding-induced physiological change in the cell. Such possible changes include phosphorylation of the Tie receptor; cell chemotaxis; cell growth, changes in cellular morphology; adhesion; ionic fluxes, or the like. The same also may be true in the situation where the Ephrin composition comprises a cell naturally or recombinantly expressing an Ephrin on its surface. The detecting step can optionally comprise measuring a Tie binding-induced physiological change in the cell.
Step (c) of the above-described method involves identifying a modulator compound (from amongst the putative modulators tested) in view of decreased or increased binding between the Tie receptor and the Ephrin ligand in the presence of the putative modulator, as compared to binding in the absence of the putative modulator. Generally, more attractive modulators are those that will activate or inhibit Tie-Ephrin binding at lower concentrations, thereby permitting use of the modulators in a pharmaceutical composition at lower effective doses.
Angiopoietin molecules are known to act as ligand for at least the Tie- 2 receptor, and there is intense interest in the medical biology community in modulating Angiopoietin-Tie interactions for therapeutic benefit. The identification herein of an additional class of Tie ligands adds a complexity to the design of therapeutic molecules. In some instances, it will be desirable to have a non-specific modulator that can modulate both Tie- Ang interactions and Tie-Ephrin interactions. In other instances, it will be desirable to modulate Tie-Angiopoietin interactions in vivo while affecting Tie-Ephrin interactions as little as possible, and visa versa. The present invention provides counterscreen assays that identify the selectivity of a modulator for Tie-Angiopoietin binding or Tie-Ephrin binding. For example, the invention provides a method for screening for selectivity of a modulator of binding between a Tie receptor tyrosine kinase and an Angiopoietin, comprising steps of: a) contacting a Tie receptor composition with an Ephrin composition in the presence and in the absence of a compound that modulates binding between the Tie receptor and an angiopoietin ligand of the Tie receptor; b) detecting binding between the Tie receptor composition and the Ephrin in the presence and absence of the modulator compound, and c) identifying the selectivity of the modulator compound in view of decreased or increased binding between the Tie receptor and the Ephrin in the presence as compared to the absence of the modulator, wherein increased selectivity of the modulator for modulating Tie-Angiopoietin binding correlates with decreased differences in Ephrin-Tie binding.
Step (a) of the foregoing counterscreen method involves contacting a Tie receptor composition with an Ephrin composition in the presence and in the absence of a compound that modulates binding between the Tie receptor and an angiopoietin ligand of the Tie receptor. For the purposes of this method, the Tie receptor composition and the Ephrin composition are essentially as described above. The compound that modulates binding between the Tie receptor and the angiopoietin can be any organic or inorganic chemical or biological molecule that one would want to test for ability to modulate Tie-Ephrin interactions, and that also has the useful property of modulating the binding interaction between a Tie receptor and an Angiopoietin. (Such modulators can be identified using methods similar to the methods described above for identifying modulators of Tie-Ephrin interactions, by substituting an Angiopoietin composition for the Ephrin composition.)
Step (c) of the counterscreen involves identifying the selectivity of the modulator compound in view of decreased or increased binding between the Tie receptor and the Ephrin in the presence as compared to the absence of the modulator, wherein increased selectivity of the modulator for modulating Tie-Angiopoietin binding correlates with decreased differences in Ephrin-Tie binding. This is best illustrated by way of two hypothetical examples. A monoclonal antibody that specifically recognizes an angiopoietin and blocks Angiopoietin binding to Tie would be expected to be highly selective because it does not bind Ephrin or Tie and therefore would not interfere with Tie-Ephrin binding in the counterscreen. h step (c), the anti- Ang monoclonal antibody would be scored as a highly selective modulator of Ang- Tie interactions because Tie-Ephrin binding would not be substantially decreased or increased in the presence as compared to the absence of the modulator.
By way of a contrasting example, polyclonal antisera raised against Tie would be expected to be non-selective, and block both Angiopoietin and Ephrin from binding to Tie by blocking the binding site. In the foregoing assay, one would score the polyclonal antisera as having low selectivity because Tie-Ephrin binding would be greatly decreased in the presence of the polyclonal antisera as compared to in its absence.
In a related embodiment, the invention provides counterscreens to determine the selectivity of a modulator of Tie-Ephrin binding vis-a-vis angiopoietin ligands of the Tie receptor. For example, the invention provides a method for screening for selectivity of a modulator of binding between a Tie receptor tyrosine kinase and an Ephrin , comprising steps of: a) contacting a Tie receptor composition with an Angiopoietin composition in the presence and in the absence of a compound that modulates binding between the Tie receptor and an Ephrin ligand of the Tie receptor; b) detecting binding between the Tie receptor composition and the
Angiopoietin ligand in the presence and absence of the modulator compound, and c) identifying the selectivity of the modulator compound in view of decreased or increased binding between the Tie receptor and the Angiopoietin ligand in the presence as compared to the absence of the modulator, wherein increased selectivity of the modulator for Tie-Ephrin binding correlates with decreased differences in Angiopoietin-Tie binding.
The Tie-2 receptor is highly preferred for use in the foregoing counterscreens, since known angiopoietins bind Tie-2. Any angiopoietin ligand can be used, including Ang-1, Ang-2, Ang-3, and Ang-4. Vertebrate, and more preferrably mammalian, and more preferably primate, and most preferably human Angiopoietins are employed. The existence of Ephrin ligands for Tie receptors provides novel materials and methods for affecting biological processes. For example, the invention provides a method of modulating growth, migration, or proliferation of cells in a mammalian organism, comprising a step of: (a) identifying a mammalian organism having cells that express a Tie receptor tyrosine kinase; and
(b) administering to said mammalian organism a composition, said composition comprising an agent selected from the group consisting of:
(i) a polypeptide comprising an Ephrin that binds to the Tie receptor; (ii) a polypeptide comprising a fragment of the Ephrin, wherein the polypeptide and fragment retain Tie binding characteristics of the Ephrin;
(iii) an antibody that specifically binds the polypeptide of (i) or (ii) in a manner that inhibits the polypeptide from binding the Tie receptor, or a fragment of the antibody that specifically binds the polypeptide of (i) or (ii); (iv) a polypeptide comprising an antigen-binding fragment the (iii) and that inhibits the polypeptide of (i) or (ii) from binding the Tie receptor; and
(v) a molecule that selectively inhibits Epliin binding to the Tie receptor without inhibiting angiopoietin binding to the Tie receptor; wherein the composition is administered in an amount effective to modulate growth, migration, or proliferation of cells that express Tie in the mammalian organism.
Preferably, the mammalian organism is human. Also, the cells preferably comprise vascular endothelial cells. In a highly preferred embodiment, the organism has a disease characterized by aberrant growth, migration, or proliferation of endothelial cells. The administration of the agent beneficially alters the aberrant growth, migration, or proliferation, e.g., by correcting it, or reducing its severity, or reducing its deleterious symptoms or effects.
For example, in one variation, the animal has a cancer, especially a cancerous tumor characterized by vasculature containing Tie-expressing endothelial cells. A composition is selected that will decrease growth, migration, or proliferation of the cells, and thereby retard the growth of the tumor. In such circumstances, one may wish to administer agents that inhibit other endothelial growth factor/receptor interactions, such as inhibitors of the VEGF-family of ligands; endostatins; inhibitory angiopoietins, or the like. Exemplary inhibitors include antibody substances specific for the growth factors or their ligands. By way of another example, the animal may have a circulatory disorder characterized by inadequate vasculature, such as cerebral infraction, acute myocardial infarction, ischemic (e.g., peripheral ischemic in the legs, atriolitis, arterial thrombosis, Burger's disease). A composition is selected that will increase growth, migration, or proliferation of the endothelial cells. In such circumstances, it may be desirable to co-administer a second agent to the patient for modulating endothelial growth, migration, or proliferation, said second agent selected from the group consisting of: a VEGF polypeptide, a VEGF-B polypeptide, a VEGF-C polypeptide, a VEGF-D polypeptide, a VEGF-E polypeptide, a P1GF polypeptide, an FGF-2 polypeptide, an HGF polypeptide, and stimulatory angiopoietin polypeptides. Other conditions to treat include inflammatory diseases (e.g.,
Rheumatoid arthritis, chronic wounds and atherosclerosis)..
Similarly, the invention provides a polypeptide comprising a fragment of an Ephrin that binds to a Tie receptor, for use in the manufacture of a medicament for the treatment of diseases characterized by aberrant growth, migration, or proliferation of cells that express a Tie receptor. Preferred polypeptides are soluble, such as Ephrin fragments fused to an immunoglobulin fragment.
In a related variation, the invention provides novel combination therapies for vascular disorders. As explained above, for pro-angiogenic therapies it is highly desirable to induce and build new vessels having a structure comparable to that of normal mature vasculature, which functions as well as normal vessels. The novel finding of interaction between Tie receptors and ephrin-B2 offers a means to address this long-felt need.
Thus, for example, the invention provides a method of promoting growth of vessels in a mammalian organism, comrpising steps of: (a) identifying a mammalian organism in need of neovascularization; and
(b) administering to said mammalian organism at least two agents to promote neovascularization, wherein the first agent comprises a member selected from the group consisting of:
(i) a polypeptide comprising an Ephrin that binds to the Tie receptor;
(ii) a polypeptide comprising a fragment of the Ephrin, wherein the polypeptide and fragment retain Tie binding characteristics of the Ephrin;
(iii) an antibody that specifically binds the polypeptide of (i) or (ii) in a manner that inhibits the polypeptide from binding the Tie receptor, or a fragment of the antibody that specifically binds the polypeptide of (i) or (ii);
(iv) a polypeptide comprising an antigen-binding fragment the (iii) and that inhibits the polypeptide of (i) or (ii) from binding the Tie receptor; and
(v) a molecule that selectively inhibits Ephin binding to the Tie receptor without inhibiting angiopoietin binding to the Tie receptor; wherein the second agent comprises a member selected from the group consisting of: a VEGF polypeptide, a VEGF-B polypeptide, a VEGF-C polypeptide, a VEGF-D polypeptide, a VEGF-E polypeptide, a P1GF polypeptide, an FGF-2 polypeptide, an Ang-1 polypeptide, an Ang-2 polypeptide, an Ang-3 polypeptide, an Ang-4 polypeptide, an HGF polypeptide; and wherein the agents are administered in amounts effective to induce growth of vessels in the mammalian organism. In a preferred embodiment, the organism is human. Also in preferred embodiments, the organism has a disease or condition that would be expected to benefit from neovascularization, such as ischemic tissue, an infarction, a new or chronic wound, or a tissue graft or transplant.
With respect to aspects of the invention that involve administration of protein agents to mammals, a related aspect of the invention comprises gene therapy whereby a gene encoding the protein of interest is administered in a manner to effect expression of the protein of interest in the animal. For example, the gene of interest is attached to a suitable promoter to promote expression of the protein in the target cell of interest, and is delivered in any gene therapy vector capable of delivering the gene to the cell, including adenoviras vectors, adeno-associated virus vectors, liposomes, naked DNA transfer, and others.. In still another aspect, the invention provides a novel method of modulating Tie recpeptor activity. For example, the invention provides a method of modulating Tie receptor activity in a mammalian subject, comprising steps of:
(a) identifying a mammalian organism having cells that express a Tie receptor tyrosine kinase; and
(b) administering to said mammalian organism a composition, said composition comprising an agent selected from the group consisting of:
(i) a polypeptide comprising an Ephrin that binds to the Tie receptor;
(ii) a polypeptide comprising a fragment of the Ephrin, wherein the polypeptide and fragment retain Tie binding characteristics of the Ephrin;
(iii) an antibody that specifically binds the polypeptide of (i) or (ii) in a manner that inhibits the polypeptide from binding the Tie receptor, or a fragment of the antibody that specifically binds the polypeptide of (i) or (ii);
(iv) a polypeptide comprising an antigen-binding fragment the (iii) and that inhibits the polypeptide of (i) or (ii) from binding the Tie receptor; and
(v) a molecule that selectively inhibits Ephin binding to the Tie receptor without inhibiting angiopoietin binding to the Tie receptor; wherein the composition is administered in an amount effective to modulate Tie receptor activity in cells of the organism. Use of soluble forms of Ephrins that bind Tie receptors is specifically contemplated as a preferred embodiment. Tie-binding fragments of Ephrins are desirably produced as fusions with immunoglobulin peptides or other peptides that promote solubility and increased circulating half-life. Solublization also is improved by pegylation or other covalent modifications to the amino acid sequence. The method can be performed in situations where it is desirable to directly modulate Tie receptor activity, and also in situations where it is desirable to indirectly modulate such activity by modulating Tie-angiopoietin interactions. Administration via any medically suitable route (intravenous, systemic, intraperitoneal injections, oral dosing, transdermal, etc) is contemplated. Additional features and variations of the invention will be apparent to those skilled in the art from the entirety of this application, and all such features are intended as aspects of the invention.
Likewise, features of the invention described herein can be re-combined into additional embodiments that also are intended as aspects of the invention, irrespective of whether the combination of features is specifically mentioned above as an aspect or embodiment of the invention. Also, only such limitations which are described herein as critical to the invention should be viewed as such; variations of the invention lacking limitations which have not been described herein as critical are intended as aspects of the invention.
In addition to the foregoing, the invention includes, as an additional aspect, all embodiments of the invention narrower in scope in any way than the variations specifically mentioned above. Although the applicant(s) invented the full scope of the claims appended hereto, the claims appended hereto are not intended to encompass within their scope the prior art work of others. Therefore, in the event that statutory prior art within the scope of a claim is brought to the attention of the applicants by a Patent Office or other entity or individual, the applicant(s) reserve the right to exercise amendment rights under applicable patent laws to redefine the subject matter of such a claim to specifically exclude such statutory prior art or obvious variations of statutory prior art from the scope of such a claim. Variations of the invention defined by such amended claims also are intended as aspects of the invention.
BRIEF DESCRIPTION OF THE DRAWING
Fig. 1 A is a bar graph depicting binding between a soluble Ephrin-B2-
Fc fusion protein and receptors Tie-1, Tie-2, EplιB4, and VEGFR-3 (control).
Fig. IB is a photomicrograph showing Ephrin-B2-Fc binding to Tie-1 or Tie-2 receptors expressed on the surface of cells. Figure 2 is a graph depicting the results of an Ephrin-B2/EphB4 binding inhibition assay. DETAILED DESCRIPTION OF THE INVENTION
The present invention involves the fields of cell and molecular biology, and many standard techniques relevant to those fields will be relevant to the practice of the present invention. Many such techniques are described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989), and or Ausubel et al., eds., Current Protocols in Molecular Biology, Green Publishers Inc. and Wiley and Sons, NY (1994-2001), both of which are incorporated by reference in their entirety. A. Gene sequences of interest to the present invention.
At least two Tie receptors have been identified, referred to as Tie (Tie- 1) and Tie-2. At least eight Ephrin polypeptide family members have been identified, divided into A and B subclasses. The DNA and deduced amino acid sequences of all known Ephrins, Tie receptors, and Ephrin receptors of any vertebrate species that have been reported in the literature are hereby incorporated by reference. However, due to their special signficance to the invention, the following table is provided for the convenience of the reader:
Polypeptide Genbank Ace Nos. SEQ ID NOs
Human (Hu) Tie (Tie-1) X60957; P35590 1 and 2
Murine (Mu) Tie (Tie-1) NM013690 5 and 6
Hu Tie-2 (TEK) Q02763; L06139 3 and 4
Mu Tie-2 (TEK) NM011587 7 and 8
Hu Ephrin- A 1 NM004428
Mu Ephrin-Al NM010107
Hu Ephrin- A2 XM009246
Mu Ephrin- A2 NM007909
Hu Ephrin- A3 XM001787
Mu Ephrin- A3 U92855
Hu Ephrin- A4 XM001784
Mu Ephrin- A4 NM007910
Hu Ephrin- A5 XM003914
Mu Ephrin- A5 NM010109
Hu Ephrin-Bl XM010388 9 and 10
Mu Ephrin-Bl NM010110 11 and 12
Hu Ephrin-B2 XM007084 13 and 14
Mu Eρhrin-B2 NM010111 15 and 16 Hu Ephrin-B3 ' NM001406 17 and 18
Mu Ephrin-B3 NM007911 19 and 20
Hu EphB2 AF025304 21 and 22
Mu EphB2 X76011 23 and 24 Hu EphB3 NM004443 25 and 26
Mu EphB3 X76012 27 and 28
Hu EphB4 NM004444 29 and 30
Mu EphB4 NM010144 31 and 32
Hu VEGFR-3 NM002020 33 and 34 Hu Ang-1 NM001146 35 and 36
Hu Ang-2 NM001147 37 and 38
Hu Ang-3 AF074332 39 and 40
Hu Ang-4 AF113708 41 and 42
The Angiopoietin Family Members The Angiopoietins are of special interest to the present invention because, like Ephrin-B2, the Angiopoietins have been found to modulate (stimulate or inhibit) Tie-2. The angiopoietin (Ang 1-4) family of molecules were originally identified by cDNA library screening for ligands to the orphan Tie-2 receptor tyrosine kinase. [Davis et al, Cell, 87: 1161-69 (1996)]. Ang-1, the first of the angiopoietin ligands identified, was isolated through secretion-trap expression cloning using cell lines which demonstrated binding of secreted factors to Tie-2-Fc molecules. This novel technique isolated a 498 amino acid, 70 kDa glycoprotein. The N-terminal region of the protein showed hydrophobic sequences characteristic of secretory signal sequences. Residues 100-280 of Ang-1 resemble a coiled-coil structure like that found in myosin, while residues 280-498 show homology to a family of proteins which includes fibrinogen, thus this region is the fibrinogen-like domain. Ang-1 shows a binding affinity to Tie-2 less than 4 nM, and induces phosphorylation and activation of the Tie-2 tyrosine kinase.
The remaining members of the angiopoietin family were isolated using homology searches against the Ang-1 cDNA sequence. Human Ang-2 , a 496 amino acid protein (Maisonpierre et al, Science. 277: 55-60 (1997)), shows 85% homology to mouse ang-2 and 60% homology to the Human Ang-1 protein. Ang-2 possesses the N-terminal secretory signal sequence found in Ang-1, and also both the coiled-coil and fibrinogen-like domains. Ang-2 also shares 8 of the 9 cysteine residues found throughout the Ang-1 sequence, believed to be important in disulfide bond formation. Analysis of Ang-2 activity on the .Tie-2 receptor shows that ang-2 binds to Tie-2 but does not induce phosphorylation of the receptor, implicating Ang-2 as an antagonist to Ang-1 activation of Tie-2. Angiopoietin-3 has been isolated by several groups based on sequence similarity to ang-1 and ang-2. See, e.g., Kim et al, FEBSLett. 443: 353-6 (1999); Nishimura et al, FEBSLett. 448: 254-6 (1999). The groups identified either a 503 or 491 amino acid clone of Ang-3, respectively. Nishimura et al cloned Ang-3 from a human aorta cDNA library, and identified a 503 amiήo acid protein having 45.1% identity with human ang-1 and 44:7% identity to ang-2. A third group independently identified a 460 amino acid Ang-3 clone, (ANGPTL3) from human liver tissue. Conklin et al., Genomics, 62: 477-82 (1999). All three clones possess the characteristic N-terminal secretory signal sequence, coiled-coil motif, and fibrinogen-like domains of the other Ang family members. Human Ang-4, identified by Valenzuela, et al (Proc. Natl Acad. Sci
USA. 96:1904-09. 1999), using sequence homology to a mouse genomic library, is a 503 amino acid protein having the leader signal sequence, coiled-coil, and fibrinogen-like sequences indicative of an angiopoietin family member. Both Ang-3 and Ang-4 show conservation of 8 of the 9 cysteines present in Ang-1. Both Ang-3 and Ang-4 show binding to the Tie-2 receptor and not Tie-1, Ang-3 acting as an antagonist while Ang-4 activates Tie-2 as an agonist. hi addition to the foregoing, the invention involves several other polypeptide factors involved in promoting or inhibiting aspects of the angiogenic process. For example, the invention involves combination therapies involving use of of an Ephrin polypeptide (or inhibitor thereof) in combination with one of these polypetpides, or in combination with inhibitors (e.g., antibodies) of one of these polypeptides. The following description will therefore be useful in the practice of the invention.
The PDGF/VEGF Family The PDGF/VEGF family of growth factors includes at least the following members: PDGF-A (see e.g., GenBank Ace. No. X06374), PDGF-B (see e.g., GenBank Ace. No. M12783), VEGF (see e.g., GenBank Ace. No. Q16889 referred to herein for clarity as VEGF-A or by particular isoform), P1GF (see e.g., GenBank Ace. No. X54936 placental growth factor), VEGF-B (see e.g., GenBank Ace. No. U48801; also known as VEGF-related factor (VRF)), VEGF-C (see e.g., GenBank Ace. No. X94216; also known as VEGF related protein (V P)), VEGF-D (also known as c-fos-induced growth factor (FIGF); see e.g., Genbank Ace. No.
AJ000185), VEGF-E (also known as NZ7 VEGF or OV NZ7; see e.g., GenBank Ace. No. S67522), NZ2 VEGF (also known as OV NZ2; see e.g., GenBank Ace. No. S67520), D1701 VEGF-like protein (see e.g., GenBank Ace. No. AF106020; Meyer et al, EMBO J 75:363-374), and NZ10 VEGF-like protein (described in International Patent Application PCT/US99/25869) [Stacker and Achen, Growth Factors 17:1-11 (1999); Neufeld et al, FASEB J 13:9-22 (1999); Fenara, JMolMed 77:527-543 (1999)]. The PDGF/VEGF family proteins are predominantly secreted glycoproteins that form either disulfide-linked or non-covalently bound homo- or heterodimers whose subunits are arranged in an anti-parallel manner [Stacker and Achen, Growth Factors 17:1-11 (1999); Mullev et al, Structure 5:1325-1338 (1997)].
The VEGF subfamily is composed of PDGF/VEGF members which share a VEGF homology domain (VHD) characterized by the sequence: C-X(22-24)- P-[PSR]-C-V-X(3)-R-C-[GSTA]-G-C-C-X(6)-C-X(32-41)-C.
VEGF-A was originally purified from several sources on the basis of its mitogenic activity toward endothelial cells, and also by its ability to induce microvascular permeability, hence it is also called vascular permeability factor (VPF). VEGF-A has subsequently been shown to induce a number of biological processes including the mobilization of intracellular calcium, the induction of plasminogen activator and plasminogen activator inhibitor- 1 synthesis, promotion of monocyte migration in vitro, induction of antiapoptotic protein expression in human endothelial cells, induction of fenestrations in endothelial cells, promotion of cell adhesion molecule expression in endothelial cells and induction of nitric oxide mediated vasodilation and hypotension [Ferrara, JMolMed 77: 527-543 (1999); Neufeld et al, FASEB J 13: 9-22 (1999); Zachary, Intl JBiochem Cell Bio 30: 1169-1174 (1998)]. VEGF-A is a secreted, disulfide-linked homodimeric glycoprotein composed of 23 kD subunits. Five human VEGF-A isoforms of 121, 145, 165, 189 or 206 amino acids in length (VEGF121-2o<s), encoded by distinct mRNA splice variants, have been described, all of which are capable of stimulating mitogenesis in endothelial cells. However, each isoform differs in biological activity, receptor specificity, and affinity for cell surface- and extracellular matrix-associated heparan- sulfate proteoglycans, which behave as low affinity receptors for VEGF-A. VEGF121 does not bind to either heparin or heparan-sulfate; VEGF145 and VEGF165 (GenBank Ace. No. M32977) are both capable of binding to heparin; and VEGF189 and VEGF206 show the strongest affinity for heparin and heparan-sulfates. VEGF 21, VEGF145, and VEGF16 are secreted in a soluble form, although most of VEGF165 is confined to cell surface and extracellular matrix proteoglycans, whereas VEGF189 and VEGF206 remain associated with extracellular matrix. Both VEGF189 and VEGF206 can be released by treatment with heparin or heparinase, indicating that these isoforms are bound to extracellular matrix via proteoglycans. Cell-bound VEGF189 can also be cleaved by proteases such as plasmin, resulting in release of an active soluble VEGFno. Most tissues that express VEGF are observed to express several VEGF isoforms simultaneously, although VEGF121 and VEGF165 are the predominant forms, whereas VEGF206 is rarely detected [Ferrara, JMol Med 77:527-543 (1999)] .
VEGF145 differs in that it is primarily expressed in cells derived from reproductive organs [Neufeld et al, FASEB J 13:9-22 (1999)].
The pattern of VEGF-A expression suggests its involvement in the development and maintenance of the normal vascular system, and in angiogenesis associated with tumor growth and other pathological conditions such as rheumatoid arthritis. VEGF-A is expressed in embryonic tissues associated with the developing vascular system, and is secreted by numerous tumor cell lines. Analysis of mice in which VEGF-A was knocked out by targeted gene disruption indicate that VEGF-A is critical for survival, and that the development of the cardiovascular system is highly sensitive to VEGF-A concentration gradients. Mice lacking a single copy of VEGF-A die between day 11 and 12 of gestation. These embryos show impaired growth and several developmental abnormalities including defects in the developing cardiovasculature. VEGF-A is also required post-natally for growth, organ development, regulation of growth plate morphogenesis and endochondral bone formation. The requirement for VEGF-A decreases with age, especially after the fourth postnatal week. In mature animals, VEGF-A is required primarily for active angiogenesis in processes such as wound healing and the development of the corpus luteum. [Neufeld et al, FASEB J 13:9-22 (1999); Ferrara, JMol Med 77:527-543 (1999)]. VEGF-A expression is influenced primarily by hypoxia and a number of hormones and cytokines including epidermal growth factor (EGF), TGF-β, and various interleukins. Regulation occurs transcriptionally and also post- transcriptionally such as by increased mRNA stability [Ferrara, JMol Med 77:527- 543 (1999)].
P1GF, a second member of the VEGF subfamily, is generally a poor stimulator of angiogenesis and endothelial cell proliferation in comparison to VEGF- A, and the in vivo role of P1GF is not well understood. Three isoforms of P1GF produced by alternative mRNA splicing have been described [Hauser et al, Growth Factors 9:259-268 (1993); Maglione et al, Oncogene 5:925-931 (1993)]. P1GF forms both disulfide-liked homodimers and heterodimers with VEGF-A. The P1GF- VEGF-A heterodimers are more effective at inducing endothelial cell proliferation and angiogenesis than P1GF homodimers. P1GF is primarily expressed in the placenta, and is also co-expressed with VEGF-A during early embryogenesis in the trophoblastic giant cells of the parietal yolk sac [Stacker and Achen, Growth Factors 17:1-11 (1999)].
VEGF-B, described in detail in International Patent Publication No. WO 96/26736 and U.S. Patents 5,840,693 and 5,607,918, incorporated herein by reference, shares approximately 44% amino acid identity with VEGF-A. Although the biological functions of VEGF-B in vivo remain incompletely understood, it has been shown to have angiogenic properties, and may also be involved in cell adhesion and migration, and in regulating the degradation of extracellular matrix. It is expressed as two isoforms of 167 and 186 amino acid residues generated by alternative splicing. VEGF-B167 is associated with the cell surface or extracellular matrix via a heparin-binding domain, whereas VEGF-B186 is secreted. Both VEGF- B16 and VEGF-B186 can form disulfide-linked homodimers or heterodimers with VEGF-A. The association to the cell surface of VEGF165-VEGF-B167 heterodimers appears to be determined by the VEGF-B component, suggesting that heterodimerization may be important for sequestering VEGF-A. VEGF-B is expressed primarily in embryonic and adult cardiac and skeletal muscle tissues [Joukov et al, J Cell Physiol 173:211-215 (1997); Stacker and Achen, Growth Factors 17:1-11 (1999)]. Mice lacking VEGF-B survive but have smaller hearts, dysfunctional coronary vasculature, and exhibit impaired recovery from cardiac ischemia [Bellomo et al, Circ Res 2000;E29-E35].
A fourth member of the VEGF subfamily, VEGF-C, comprises a VHD that is approximately 30% identical at the amino acid level to VEGF-A. VEGF-C is originally expressed as a larger precursor protein, prepro-VEGF-C, having extensive amino- and carboxy-terminal peptide sequences flanking the VHD, with the C- terminal peptide containing tandemly repeated cysteine residues in a motif typical of Balbiani ring 3 protein. Prepro-VEGF-C undergoes extensive proteolytic maturation involving the successive cleavage of a signal peptide, the C-terminal pro-peptide, and the N-terminal pro-peptide. Secreted VEGF-C protein consists of a non-covalently- linked homodimer, in which each monomer contains the VHD. The intermediate forms of VEGF-C produced by partial proteolytic processing show increasing affinity for the VEGFR-3 receptor, and the mature protein is also able to bind to the VEGFR- 2 receptor. [Joukov et al., EMBO J., 16:(13):3898-?>911 (1997)]. It has also been demonstrated that a mutant VEGF-C, in which a single cysteine at position 156 is either substituted by another amino acid or deleted, loses the ability to bind VEGFR-2 but remains capable of binding and activating VEGFR-3 [International Patent Publication No. WO 98/33917]. In mouse embryos, VEGF-C mRNA is expressed primarily in the allantois, jugular area, and the metanephros. [Joukov et al, J Cell Physiol 173:211-215 (1997)]. VEGF-C is involved in the regulation of lymphatic angiogenesis: when VEGF-C was overexpressed in the skin of transgenic mice, a hyperplastic lymphatic vessel network was observed, suggesting that VEGF-C induces lymphatic growth [Jeltsch et al, Science, 276:1423-1425 (1997)]. Continued expression of VEGF-C in the adult also indicates a role in maintenance of differentiated lymphatic endothelium [Ferrara, JMol Med 77:527-543 (1999)]. VEGF-C also shows angiogenic properties: it can stimulate migration of bovine capillary endothelial (BCE) cells in collagen and promote growth of human endothelial cells [see, e.g., International Patent Publication No. WO 98/33917, incorporated herein by reference]. VEGF-D is structurally and functionally most closely related to
VEGF-C [see International Patent Publ. No. WO 98/07832, incorporated herein by reference]. Like VEGF-C, VEGF-D is initially expressed as a prepro-peptide that undergoes N-terminal and C-terminal proteolytic processing, and forms non- covalently linked dimers. VEGF-D stimulates mitogenic responses in endothelial cells in vitro. During embryogenesis, VEGF-D is expressed in a complex temporal and spatial pattern, and its expression persists in the heart, lung, and skeletal muscles in adults. Isolation of a biologically active fragment of VEGF-D designated VEGF- DΔNΔC, is described in International Patent Publication No. WO 98/07832, incorporated herein by reference. VEGF-D ΔNΔC consists of amino acid residues 93 to 201 of VEGF-D.
Four additional members of the VEGF subfamily have been identified in poxviruses, which infect humans, sheep and goats. The orf virus-encoded VEGF-E and NZ2 VEGF are potent mitogens and permeability enhancing factors. Both show approximately 25% amino acid identity to mammalian VEGF-A, and are expressed as disulfide-liked homodimers. Infection by these viruses is characterized by pustular dermititis which may involve endothelial cell proliferation and vascular permeability induced by these viral VEGF proteins. [Ferrara, JMol Med 77:527-543 (1999); Stacker and Achen, Growth Factors 17:1-11 (1999)]. VEGF-like proteins have also been identified from two additional strains of the orf virus, D1701 [GenBank Ace. No. AF106020; described in Meyer et al, EMBO J 18:363-314 (1999)] andNZIO [described in International Patent Application PCT/US99/25869, incorporated herein by reference]. These viral VEGF-like proteins have been shown to bind VEGFR-2 present on host endothelium, and this binding is important for development of infection and viral induction of angiogenesis [Meyer et al, EMBO J 18:363-314 (1999); International Patent Application PCT/US99/25869] .
PDGF/VEGF Receptors
Seven cell surface receptors that interact with PDGF/VEGF family members have been identified. These include PDGFR-α (see e.g., GenBank Ace. No. NM006206) , PDGFR-β (see e.g., GenBank Ace. No. NM002609), VEGFR-l/Flt-1 ( fins-like tyrosine kinase-1; GenBank Ace. No. X51602; De Vries et al, Science 255:989-991 (1992)); VEGFR-2/KDR/Flk-l (kinase insert domain containing receptor/fetal liver kinase-1; GenBank Ace. Nos. X59397 (Flk-1) and L04947 (KDR); Terman et al, Biochem Biophys Res Comm 187:1519-1586 (1992); Matthews et al, ProcNatl Acad Sci USA 88:9026-9030 (1991)); VEGFR-3/Flt4 (fms-liko tyrosine kinase 4; U.S. Patent No. 5,776,755 and GenBank Ace. No. X68203 and S66407; Pajusola et al, Oncogene 9:3545-3555 (1994)), neuroρilin-1 (Gen Bank Ace. No. NM003873), and neuropilin-2 (Gen Bank Ace. No. NM003872). The two PDGF receptors mediate signaling of PDGFs as described above. VEGFι ι, VEGF165, VEGF-B, P1GF-1 and P1GF-2 bind VEGF-R1; VEGF121, VEGF145, VEGF165, VEGF- C, VEGF-D, VEGF-E, and NZ2 VEGF bind VEGF-R2; VEGF-C and VEGF-D bind VEGFR-3; VEGF165, P1GF-2, and NZ2 VEGF bind neuropilin-1; and VEGF165 binds neuropilin-2. [Neufeld et al, FASEB J 13:9-22 (1999); Stacker and Achen, Growth Factors 17:1-11 (1999); Ortega et al, Fron Biosci 4:141-152 (1999); Zachary, Intl J Biochem Cell Bio 30:1169-1174 (1998); Petrova et al, Exp Cell Res 253:117-130 (1999)].
The PDGF receptors are protein tyrosine kinase receptors (RTKs) that contain five immunoglobulin-like loops in their extracellular domains. VEGFR-1, VEGFR-2, and VEGFR-3 comprise a subgroup of the PDGF subfamily of PTKs, distinguished by the presence of seven Ig domains in their extracellular domain and a split kinase domain in the cytoplasmic region. Both neuropilin-1 and neuropilin-2 are non-RTK VEGF receptors. NP-1 has an extracellular portion includes a MAM domain; regions of homology to coagulation factors V and VIII, MFGPs and the DDR tyrosine kinase; and two CUB-like domains.
Several of the VEGF receptors are expressed as more than one isoform. A soluble isoform of VEGFR-1 lacking the seventh Ig-like loop, transmembrane domain, and the cytoplasmic region is expressed in human umbilical vein endothelial cells. This VEGFR-1 isoform binds VEGF-A with high affinity and is capable of preventing VEGF-A-induced mitogenic responses [Ferrara, JMolMed 77:521-543 (1999); Zachary, Intl JBiochem Cell Bio 30:1169-1174 (1998)]. A C- terminal truncated from of VEGFR-2 has also been reported [Zachary, Intl JBiochem Cell Bio 30: 1169-1174 (1998)]. h humans, there are two isoforms of the VEGFR-3 protein which differ in the length of their C-terminal ends. Studies suggest that the longer isoform is responsible for most of the biological properties of VEGFR-3.
The expression of VEGFR-1 occurs mainly in vascular endothelial cells, although some may be present on monocytes, trophoblast cells, and renal mesangial cells [Neufeld et al, FASEB J 13:9-22 (1999)]. High levels of VEGFR-1 mRNA are also detected in adult organs, suggesting that VEGFR-1 has a function in quiescent endothelium of mature vessels not related to cell growth. VEGFR-1-/- mice die in utero between day 8.5 and 9.5. Although endothelial cells developed in these animals, the formation of functional blood vessels was severely impaired, suggesting that VEGFR-1 may be involved in cell-cell or cell-matrix interactions associated with cell migration. Recently, it has been demonstrated that mice expressing a mutated VEGFR-1 in which only the tyrosine kinase domain was missing show normal angiogenesis and survival, suggesting that the signaling capability of VEGFR-1 is not essential. [Neufeld et al, FASEB J 13:9-22 (1999); Ferrara, JMol Med 77:521-543 (1999)].
VEGFR-2 expression is similar to that of VEGFR-1 in that it is broadly expressed in the vascular endothelium, but it is also present in hematopoietic stem cells, megakaryocytes, and retinal progenitor cells [Neufeld et al, FASEB J 13:9-22 (1999)]. Although the expression pattern of VEGFR-1 and VEGFR-2 overlap extensively, evidence suggests that, in most cell types, VEGFR-2 is the major receptor through which most of the VEGFs exert their biological activities. Examination of mouse embryos deficient in VEGFR-2 further indicate that this receptor is required for both endothelial cell differentiation and the development of hematopoietic cells [Joukov et al, J Cell Physiol 173:211-215 (1997)].
VEGFR-3 is expressed broadly in endothelial cells during early embryogenesis. During later stages of development, the expression of VEGFR-3 becomes restricted to developing lymphatic vessels [Kaipainen, A., et al, Proc. Natl. Acad. Sci. USA, 92: 3566-3570 (1995)]. In adults, the lymphatic endothelia and some high endothelial venules express VEGFR-3, and increased expression occurs in lymphatic sinuses in metastatic lymph nodes and in lymphangioma. VEGFR-3 is also expressed in a subset of CD34+ hematopoietic cells which may mediate the myelopoietic activity of VEGF-C demonstrated by overexpression studies [WO 98/33917]. Targeted disruption of the VEGFR-3 gene in mouse embryos leads to failure of the remodeling of the primary vascular network, and death after embryonic day 9.5 [Dumont et al, Science, 282: 946-949 (1998)]. These studies suggest an essential role for VEGFR-3 in the development of the embryonic vasculature, and also during lymphangiogenesis.
Structural analyses of the VEGF receptors indicate that the VEGF-A binding site on VEGFR-1 and VEGFR-2 is located in the second and third Ig-like loops. Similarly, the VEGF-C and VEGF-D binding sites on VEGFR-2 and VEGFR- 3 are also contained within the second Ig-loop [Taipale et al, Curr Top Microbiol Immunol 237:85-96 (1999)]. The second Ig-like loop also confers ligand specificity as shown by domain swapping experiments [Ferrara, JMolMed 77:527-543 (1999)]. Receptor-ligand studies indicate that dimers formed by the VEGF family proteins are capable of binding two VEGF receptor molecules, thereby dimerizing VEGF receptors. The fourth Ig-like loop on VEGFR-1, and also possibly on VEGFR-2, acts as the receptor dimerization domain that links two receptor molecules upon binding of the receptors to a ligand dimer [Ferrara, JMol Med 77:527-543 (1999)]. Although the regions of VEGF-A that bind VEGFR-1 and VEGFR-2 overlap to a large extent, studies have revealed two separate domains within VEGF-A that interact with either VEGFR-1 or VEGFR-2, as well as specific amino acid residues within these domains that are critical for ligand-receptor interactions. Mutations within either VEGF receptor-specific domain that specifically prevent binding to one particular VEGF receptor have also been recovered [Neufeld et al, FASEB J 13:9-22 (1999)]. VEGFR-1 and VEGFR-2 are structurally similar, share common ligands (VEGFι ι and VEGFι65), and exhibit similar expression patterns during development. However, the signals mediated through VEGFR-1 and VEGFR-2 by the same ligand appear to be slightly different. VEGFR-2 has been shown to undergo autophosphorylation in response to VEGF-A, but phosphorylation of VEGFR-1 under identical conditions was barely detectable. VEGFR-2 mediated signals cause striking changes in the morphology, actin reorganization, and membrane ruffling of porcine aortic endothelial cells recombinantly overexpressing this receptor. In these cells, VEGFR-2 also mediated ligand-induced chemotaxis and mitogenicity; whereas VEGFR- 1-transfected cells lacked mitogenic responses to VEGF-A. Mutations in VEGF-A that disrupt binding to VEGFR-2 fail to induce proliferation of endothelial cells, whereas VEGF-A mutants that are deficient in binding VEGFR-1 are still capable of promoting endothelial proliferation. Similarly, VEGF stimulation of cells expressing only VEGFR-2 leads to a mitogenic response whereas comparable stimulation of cells expressing only VEGFR-1 also results in cell migration, but does not induce cell proliferation. In addition, phosphoproteins co-precipitating with VEGFR-1 and VEGFR-2 are distinct, suggesting that different signaling molecules interact with receptor-specific intracellular sequences. The emerging hypothesis is that the primary function of VEGFR-1 in angiogenesis may be to negatively regulate the activity of VEGF-A by binding it and thus preventing its interaction with VEGFR-2, whereas VEGFR-2 is thought to be the main transducer of VEGF-A signals in endothelial cells. In support of this hypothesis, mice deficient in VEGFR-1 die as embryos while mice expressing a VEGFR-1 receptor capable of binding VEGF-A but lacking the tyrosine kinase domain survive and do not exhibit abnormal embryonic development or angiogenesis. In addition, analyses of VEGF-A mutants that bind only VEGFR-2 show that they retain the ability to induce mitogenic responses in endothelial cells. However, VEGF- mediated migration of monocytes is dependent on VEGFR-1, indicating that signaling through this receptor is important for at least one biological function. In addition, the ability of VEGF-A to prevent the maturation of dendritic cells is also associated with VEGFR-1 signaling, suggesting that VEGFR-1 may function in cell types other than endothelial cells. [Ferrara, JMol Med 77:521-543 (1999); Zachary, Intl JBiochem Cell Bio 30:1169-1114 (1998)].
Neuropilin-1 was originally cloned as a receptor for the collapsin/semaphorin family of proteins involved in axon guidance [Stacker and Achen, Growth Factors 17:1-11 (1999)]. It is expressed in both endothelia and specific subsets of neurons during embryogenesis, and it thought to be involved in coordinating the developing neuronal and vascular system. Although activation of neuropilin-1 does not appear to elicit biological responses in the absence of the VEGF family tyrosine-kinase receptors, their presence on cells leads to more efficient binding of VEGF165 and VEGFR-2 mediated responses. [Neufeld et al, FASEB J 13:9-22 (1999)] Mice lacking neuropilin-1 show abnormalities in the developing embryonic cardiovascular system. [Neufeld et al, FASEB J 13:9-22 (1999)]
Neuropilin-2 was identified by expression cloning and is a collapsin/semaphorin receptor closely related to neuropilin-1. Neuropilin-2 is an isoform-specifϊc VEGF receptor in that it only binds VEGF165. Like neuropilin-1, neuropilin-2 is expressed in both endothelia and specific neurons, and is not predicted to function independently due to its relatively short intracellular domain. The function of neuropilin-2 in vascular development is unknown [Neufeld et al, FASEB J 13:9-22 (1999); WO 99/30157]. Hepatocyte growth factor (HGF) is thought to be a factor influencing cell growth and cell motility for various epithelial cells. Matsumoto et al purified HGF from rat platelets and cloned both human and rat HGF cDNA (Gastroenterol Hepatol. 6:509-19. 1991). HGF is a heterodimeric molecule composed of the 69 kDa alpha-subunit and the 34 kDa beta-subunit. HGF has no amino acid sequence homology with other known peptide growth factors, but shows 38% homology with plasmin HGF, derived from a single chain precursor of 728 amino acid residues, is proteolytically processed to form a two-chain mature HGF. The 34 kDa beta-subunit and the 69 kDa alpha-subunit of HGF, which contains 4 kringle structures. HGF has mitogenic activity for renal tubular epithelial cells, has the potential to promote cell migration for some epithelial cells, including normal human keratinocytes, and may play an important role in wound healing and embryogenesis. See generally U.S. Patent Nos. 6,248,722, 6,214,344, and 5,004,805, incorporated herein by reference.
With respect to the Ephrins or other polypeptides used to practice the invention, it will be understood that native sequences will usually be most preferred, but that modifications can be made to most protein sequences without destroying the activity of interest of the protein, especially conservative amino acid substitions. By "conservative amino acid substitution" is meant substitution of an amino acid with an amino acid having a side chain of a similar chemical character. Similar amino acids for making conservative substitutions include those having an acidic side chain
(glutamic acid, aspartic acid); a basic side chain (arginine, lysine, histidine); a polar amide side chain (glutamine, asparagine); a hydrophobic, aliphatic side chain (leucine, isoleucine, valine, alanine, glycine); an aromatic side chain (phenylalanine, tryptophan, tyrosine); a small side chain (glycine, alanine, serine, threonine, methionine); or an aliphatic hydroxyl side chain (serine, threonine).
Moreover, deletion and addition of amino acids is often possible without destroying a desired activity. With respect to the present invention, where binding activity is of particular interest and the ability of molecules to activate or inhibit receptor tyrosine kinases upon binding is of special interest, binding assays and tyrosine phophorylation assays are available to determine whether a particular ligand or ligand variant (a) binds and (b) stimulates or inhibits RTK activity.
Two manners for defining genera of polypeptide variants include percent amino acid identity to a native polypeptide (e.g., 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity preferred), or the ability of encoding-polynucleotides to hybridize to each other under specified conditions. One exemplary set of conditions is as follows: hybridization at 42°C in 50% formamide, 5X SSC, 20 mM Na«PO4, pH 6.8; and washing in IX SSC at 55°C for 30 minutes. Formula for calculating equivalent hybridization conditions and/or selecting other conditions to achive a desired level of stringency are well known. It is understood in the art that conditions of equivalent stringency can be achieved through variation of temperature and buffer, or salt concentration as described Ausubel, et al. (Eds.), Protocols in Molecular Biology, John Wiley & Sons (1994), pp. 6.0.3 to 6.4.10. Modifications in hybridization conditions can be empirically determined or precisely calculated based on the length and the percentage of guanosine/cytosine (GC) base pairing of the probe. The hybridization conditions can be calculated as described in Sambrook, et al, (Eds.), Molecular Cloning: A Laboratorv Manual, Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York (1989), pp. 9.47 to 9.51.
B. Gene Therapy
While much of the application, including the examples, are written in the context of protein-protein interactions and protein administration, it should be clear that genetic manipulations to achieve modulation of protein expression or activity is specifically contemplated. For example, where administration of proteins is contemplated, administration of a gene therapy vector to cause the protein of interest to be produced in vivo also is contemplated. Where inhibition of proteins is contemplated (e.g., though use of antibodies or small molecule inhibitors), inhibition of protein expression in vivo by genetic techniques, such as knock-out techniques or anti-sense therapy, is contemplated.
Any suitable vector may be used to introduce a transgene of interest into an animal. Exemplary vectors that have been described in the literature include replication-deficient retroviral vectors, including but not limited to lentivirus vectors [Kim et al, J. Virol, 72(1): 811-816 (1998); Kingsman & Johnson, Scrip Magazine, October, 1998, pp. 43-46.]; adeno-associated viral vectors [Gnatenko et al, J. Investig. Med, 45: 87-98 (1997)]; adenoviral vectors [See, e.g., U.S. Patent No. 5,792,453; Quantin et al, Proc. Natl Acad. Sci. USA, 89: 2581-2584 (1992); Srratford-Perricadet et al, J. Clin. Invest., 90: 626-630 (1992); and Rosenfeld et al, Cell, 68: 143-155 (1992)]; Lipofectin-mediated gene transfer (BRL); liposomal vectors [See, e.g., U.S. Patent No. 5,631,237 (Liposomes comprising Sendai virus proteins)] ; and combinations thereof. All of the foregoing documents are incorporated herein by reference in the entirety. Replication-deficient adenoviral vectors and adeno-associated viral vectors constitute preferred embodiments.
In embodiments employing a viral vector, preferred polynucleotides include a suitable promoter and polyadenylation sequence to promote expression in the target tissue of interest. For many applications of the present invention, the Tie promoter (U.S. Patent No. 5,877,020, incorporated by reference) will be especially suitable. Other suitable promoters/enliancers for mammalian cell expression include, e.g., cytomegalovirus promoter/enhancer [Lehner et al, J. Clin. Microbiol, 29:2494- 2502 (1991); Boshart et al, Cell, ^.-521-530 (1985)]; Rous sarcoma virus promoter [Davis et al, Hum. Gene Ther., 4:151 (1993)]; or simian virus 40 promoter.
Anti-sense polynucleotides are polynucleotides which recognize and hybridize to polynucleotides encoding a protein of interest and can therefore inhibit transcription or translation of the protein. Full length and fragment anti-sense polynucleotides may be employed. Commercial software is available to optimize antisense sequence selection and also to compare selected sequences to known genomic sequences to help ensure uniqueness/specificity for a chosen gene. Such • uniqueness can be further confirmed by hybridization analyses. Antisense nucleic acids (preferably 10 to 20 base pair oligonucleotides) are introduced into cells (e.g., by a viral vector or colloidal dispersion system such as a liposome). The antisense nucleic acid binds to the target nucleotide sequence in the cell and prevents transcription or translation of the target sequence. Phosphorothioate and methylphosphonate antisense oligonucleotides are specifically contemplated for therapeutic use by the invention. The antisense oligonucleotides may be further modified by poly-L-lysine, transferrin polylysine, or cholesterol moieties at their 5' end.
Genetic control can also be achieved through the design of novel transcription factors for modulating expression of the gene of interest in native cells and animals. For example, the Cys2-His2 zinc finger proteins, which bind DNA via their zinc finger domains, have been shown to be amenable to structural changes that lead to the recognition of different target sequences. These artificial zinc finger proteins recognize specific target sites with high affinity and low dissociation constants, and are able to act as gene switches to modulate gene expression. Knowledge of the particular target sequence of the present invention facilitates the engineering of zinc finger proteins specific for the target sequence using known methods such as a combination of structure-based modeling and screening of phage display libraries [Segal et al, (1999) Proc Natl Acad Sci USA 96:2158-2163; Liu et al, (1997) Proc Natl Acad Sci USA 94:5525-30; Greisman and Pabo (1997) Science 275:651-61; Choo et al, (1997) JMol Biol 273:525-32]. Each zinc finger domain usually recognizes three or more base pairs. Since a recognition sequence of 18 base pairs is generally sufficient in length to render it unique in any known genome, a zinc finger protein consisting of 6 tandem repeats of zinc fingers would be expected to ensure specificity for a particular sequence [Segal et al., (1999) Proc Natl Acad Sci USA 96:2158-2763]. The artificial zinc finger repeats, designed based on target sequences, are fused to activation or repression domains to promote or suppress gene expression [Liu et al., (1997) Proc Natl Acad Sci USA :5525-30]. Alternatively, the zinc finger domains can be fused to the TATA box-binding factor (TBP) with varying lengths of linker region between the zinc finger peptide and the TBP to create either transcriptional activators or repressors [Kim et al, (1997) Proc Natl Acad Sci USA 94:3616-3620]. Such proteins, and polynucleotides that encode them, have utility for modulating expression in vivo in both native cells, animals and humans. The novel transcription factor can be delivered to the target cells by transfecting constructs that express the transcription factor (gene therapy), or by introducing the protein. Engineered zinc finger proteins can also be designed to bind RNA sequences for use in therapeutics as alternatives to antisense or catalytic RNA methods [McColl et al., (1999) Proc Natl Acad Sci USA 96:9521-6; Wu et al, (1995) Proc Natl Acad Sci USA 92:344-348].
C. Antibodies
Antibodies are useful for modulating Tie-Ephrin interactions due to the ability to easily generate antibodies with relative specificity, and due to the continued improvements in technologies for adopting antibodies to human therapy. Thus, the invention contemplates use of antibodies (e.g., monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies, bifunctional/bispecific antibodies, humanized antibodies, human antibodies, and complementary determining region (CDR)-grafted antibodies, including compounds which include CDR sequences which specifically recognize a polypeptide of the invention) specific for polypeptides of interest to the invention, especially Tie receptors and Ephrins. Preferred antibodies are human antibodies which are produced and identified according to methods described in WO93/11236, published June 20, 1993, which is incorporated herein by reference in its entirety. Antibody fragments, including Fab, Fab', F(ab')2, and Fv, are also provided by the invention. The term "specific for," when used to describe antibodies of the invention, indicates that the variable regions of the antibodies of the invention recognize and bind the polypeptide of interest exclusively (i. e. , able to distinguish the polypeptides of interest from other known polypeptides of the same family, by virtue of measurable differences in binding affinity, despite the possible existence of localized sequence identity, homology, or similarity between family members). It will be understood that specific antibodies may also interact with other proteins (for example, S. aureus protein A or other antibodies in ELISA techniques) through interactions with sequences outside the variable region of the antibodies, and in particular, in the constant region of the molecule. Screening assays to determine binding specificity of an antibody of the invention are well known and routinely practiced in the art. For a comprehensive discussion of such assays, see Harlow et al (Eds), Antibodies A Laboratorv Manual: Cold Spring Harbor Laboratory; Cold Spring Harbor , NY (1988), Chapter 6. Antibodies of the invention can be produced using any method well known and routinely practiced in the art.
Non-human antibodies may be humanized by any methods known in the art. In one method, the non-human CDRs are inserted into a human antibody or consensus antibody framework sequence. Further changes can then be introduced into the antibody framework to modulate affinity or immunogenicity.
D. Dosing
Polypeptides according to the invention may be administered in any suitable manner using an appropriate pharmaceutically-acceptable vehicle, e.g., a pharmaceutically-acceptable diluent, adjuvant, excipient or carrier. The composition to be administered according to methods of the invention preferably comprises (in addition to the polynucleotide or vector) a pharmaceutically-acceptable carrier solution such as water, saline, phosphate-buffered saline, glucose, or other carriers conventionally used to deliver therapeutics.
The "administering" that is performed according to the present invention may be performed using any medically-accepted means for introducing a therapeutic directly or indirectly into a mammalian subject, including but not limited to injections (e.g., intravenous, intramuscular, subcutaneous, or catheter); oral ingestion; intranasal or topical administration; and the like. For some cardiovascular disesases a preferred route of administration is intravascular, such as by intravenous, intra-arterial, or intracoronary arterial injection. The therapeutic composition may be delivered to the patient at multiple sites. The multiple administrations may be rendered simultaneously or may be administered over a period of several hours. In certain cases it may be beneficial to provide a continuous flow of the therapeutic composition. Additional therapy may be administered on a period basis, for example, daily, weekly or monthly. Polypeptides for administration may be formulated with uptake or absorption enhancers to increase their efficacy. Such enhancer include for example, salicylate, glycocholate/linoleate, glycholate, aprotinin, bacitracin, SDS caprate and the like. See, e.g., Fix (J. Pharm. Sci., 85(12) 1282-1285, 1996) and Oliyai and Stella (Ann. Rev. Pharmacol Toxicol, 32:521-544, 1993). The amounts of peptides in a given dosage will vary according to the size of the individual to whom the therapy is being administered as well as the characteristics of the disorder being treated. In exemplary treatments, it may be necessary to administer about 50mg/day, 75 mg/day, lOOmg/day, 150mg day, 200mg/day, 250 mg/day. These concentrations may be administered as a single dosage form or as multiple doses. Standard dose-response studies, first in animal models and then in clinical testing, reveal optimal dosages for particular disease states and patient populations.
It will also be apparent that dosing should be modified if traditional therapeutics are administered in combination with therapuetics of the invention. For example, treatment of cancer using traditional chemotherapeutic agents or radiation, in combination with methods of the invention, is contemplated. E. Kits
As an additional aspect, the invention includes kits which comprise compounds or compositions of the invention packaged in a manner which facilitates their use to practice methods of the invention, h a simplest embodiment, such a kit includes a compound or composition described herein as useful for practice of a method of the invention (e.g., polynucleotides or polypeptides for administration to a person), packaged in a container such as a sealed bottle or vessel, with a label affixed to the container or included in the package that describes use of the compound or composition to practice the method of the invention. Preferably, the compound or composition is packaged in a unit dosage form. The kit may further include a device suitable for administering the composition according to a preferred route of administration.
Additional aspects and details of the invention will be apparent from the following examples, which are intended to be illustrative rather than limiting.
EXAMPLE 1 Ephrin-B2 Interacts With Tie Receptors
The following experiments demonstrated that Ephrin-B2 comprises a ligand for Tie receptors.
A. Materials
To investigate the interaction between Ephrin-B2 and the Tie receptors
Tie-1 and Tie-2, the following constructs were created and/or purchased from commercial suppliers:
(a) a Tie- 1 -Fc construct, in which the extracellular domain of human Tie- 1 (amino acids 1 to 760 of SEQ ID NO: 2) were fused to the Fc portion of human IgGl (see Kubo et al., Blood, 96: 546-553 (2000); also available from R&D Systems, Minneapolis, MN, USA);
(b) a Tie-2-Fc construct, in which the extracellular domain portion of human Tie-2 (amino acids 1 to 745 of SEQ ID NO: 4) were fused to the Fc portion of human IgGl (see Kubo et al., Blood, 96: 546-553
(2000); also available from R&D Systems, Minneapolis, MN, USA);
(c) a EphB4-Fc construct, in which murine EphB4 amino acids 1 to 539 (SEQ ID NO: 32) were fused to the Fc portion of human IgGl; (d) a VEGFR-3 -Fc construct, in which an extracellular domain portion of VEGFR-3 comprising the first three immunoglobulin-like domains were fused to the Fc portion of human IgGl (see Karpanen et al, Cancer Res., 61: 1786-1790 (2001); (e) a biotinylated Ephrin-B2-Fc construct, in which murine Ephrin-B2 amino acids 1 to 227 (SEQ ID NO: 16) were fused to the Fc portion of human IgGl (R&D Systems); and
(f) control Fc constructs FGFR-4-Fc and Fas-Fc.
B. Cell-free binding assays.
B.1 Microtiter plate assay
The binding of ephrin-B2 to Tie-1 and Tie-2 first was investigated on microtiter plates. Microtiter plates were coated with 1 microgram/ml of either Tie-l-Fc, Tie-2 -Fc or, as controls, EphB4-Fc or VEGFR-3-Fc protein. After blocking the plate with phosphate buffered saline containing 1% bovine serum albumin and Tween detergent (1%BSA/PBS-T), biotinylated ephrin-B2-Fc protein was applied on the microtiter plates overnight at four degrees Centigrade. Thereafter, the plates were washed with PBS-T, and 1:1000 of avidin-HRP (horseradish peroxidase) was added. The bound ephrin-B2-Fc protein was detected by addition of the ABTS substrate (KPL, Gaithersburg, MD). As shown in Figure 1 A, Tie-l-Fc and Tie-2-Fc were shown to bind to ephrin-B2, at a level comparable to that observed with EphB4-Fc. VEGFR3-Fc could not bind to ephrin-B2.
B.2 Biosensor Analysis
To further investigate the interactions of ephrin-B2 and the Tie-1 receptor, biosensor analysis was performed using a sensor chip labeled with ephrin-
B2-Fc, and the binding affinity of Tie-1/Ephrin-B2 complexes assessed.
Biosensor analysis was carried out with a BIAcore 2000TM (BIAcore AB, Uppsala, Sweden). Ephrin-B2-Fc (10 μg/ml in 10 mM acetate buffer, pH 5) was coupled to the carboxymethylated dextran layer of a CM5 sensor chip at concentrations of 2600, 1000, 500 and 0 Resonance Units (RU) using standard amine coupling chemistry. Following immobilization, residual activation groups were blocked by washing with 1M ethanolamine hydrochloride (pH 8.5) followed by washing with 10 mM diethylamine to remove non-covalently bound material. Tie-1- Fc, EphB4-Fc and control proteins FGFR4-Fc and Fas-Fc were diluted in running buffer (1% BSA/PBS-T). To regenerate the sensor surface between analyses, high salt and acidic solution (3M NaCl in 10 mM acetate, pH 4.2) was used for EphB4, while a low pH pulse (100 mM HC1 for 30 seconds) was used in addition for a complete regeneration of Tie-1. Evaluation of binding affinities of Tie-1 and EphB4 for ephrin-B2 included both simultaneous and separate ka (association rate constant) and ka (dissociation rate constant) measurements according to the Langmuir binding model. Kinetic parameters were determined using Evaluation software 3.02 (BIAcore AB). Tie-l-Fc (100 nM), EphB4 (100 nM) and control Fc proteins (FGFR4-
Fc and Fas-Fc) were injected over the sensor chip surface containing immobilized ephrin-B2 (either 2600, 1000, 500, or 0 RU) and the binding of Tie- 1 and EphB4 measured. Analysis of the biosensor binding curves confirmed that ephrin-B2-Fc binds to EphB4 and Tie-1, but not to control Fc proteins. The binding affinity (K[ =kd/ka) of Tie-1 and EphB4 was determined over three RU levels of ephrin-B2. The affinity of Tie-1 binding to ephrin-B2 ( D =l-5 nM) was 4.4 times lower than the affinity of ephrin-B2/EphB4 binding (KD=340 pM) [values represent the mean ± SEM of the three different levels of ephrin-B2]. A long contact time (10 min) and high concentration (100 nM) of Tie-l-Fc were necessary to detect the interaction between Tie-1 and ephrin-B2, indicating the difference in Tie-1 and EphB4 binding kinetics was likely caused by a lower on rate for Tie-1. The off rate of the interaction, however, was comparable to that of EphB4/ephrin-B2, demonstrating that Tie- l/ephrin-B2 complexes were stable. Analysis of the binding kinetics of Fc proteins over a range of concentrations (up to 500 nM) to immobilized ephrin-B2-Fc (1000 RU) demonstrated that protein binding occurs in a concentration dependent manner.
C. Cell-based binding assay
A confirmatory cell-based binding experiment was conducted. COS1 cells were transiently tranfected (lipofectamine method) with pcDNA3 plasmids containing cDNAs encoding full-length human Tie-1, Tie-2, or VEGFR-3-Fc, or with empty plasmid pcDNA3 (Invitrogen) as a control. After 48 hours of incubation, the transfected COS cells were washed in HBH (Hank's buffer with 0.5%BSA and 20mM HEPES) and were fixed with 4% PFA for 15 minutes. The cells were washed with PBS and blocked with 1%BSA/PBS for 1 hour at 4 degrees C. The cells were then incubated with 15 nM of biotinylated ephrin-B2-Fc in DMEM/FBS overnight at 4 degrees C. After washing with PBS, 1/100 HRP-conjugated anti-Fc antibodies (KPL) was incubated for 1 hour at room temperature. The ephrin-B2 binding to cells was visualized by DAB substrates with C12.
As shown in Figure IB, Tie-1 transfected cells showed a weak staining, while Tie-2 transfected cells showed a strong staining. Expression studies confirmed that the differential binding was not due to weak expression of Tie-1 in the COS cells. Control VEGFR-3-Fc did not bind to the COS cells expressing Tie-1 or Tie-2. Furthermore, ephrin-B2 did not bind to the COS cells expressing VEGFR-3.
The difference in Eprhin-B2/Tie-1 binding in the cell-free and cell- based assays was noteworthy. Without intending to be limited to a particular theory, one explanation for differential binding may be due to dimerization of Tie-l-Fc in the cell-free assay, creating a dimerized receptor that more efficiently binds ligands.
EXAMPLE 2 Binding between Tie receptors and other Ephrin molecules The procedures described in Example 1 are modified by substituting other Ephrin family members for Ephrin-2B, and where appropriate, substituting other Eph receptors as controls. In this way, the ability of Tie-1 and Tie-2 to bind other Ephrin family members is characterized.
EXAMPLE 3
Ephrin Inhibition with soluble Tie peptides
The binding affinity between Ephrin and Tie receptor molecules provides a therapeutic indication for modulators of Ephrin-induced Tie receptor signaling, to modulate Tie-receptor-mediated biological processes. The following examples provide proof of this therapeutic concept.
A. In vitro cell-free assay
To demonstrate the inhibitory effects of Tie-l-Fc and Tie-2 -Fc against ephrin-B2 stimulation, 20ng/ml of the biotinylated ephrin-B2-Fc was first incubated with Tie-l-Fc, Tie-2-Fc, EρhB4-Fc or VEGFR-3-Fc at various molar ratios, and then applied on microtiter plates coated with 1 microgram/ml of EphB4-Fc. The bound biotinylated ephrin-B2-Fc was detected as described in Example 1. The results of the binding inhibition assay are summarized in Figure 2. Five times molar ratio of Tie-1 - Fc fusion proteins substantially inhibited the ephrin-B2-Fc binding to EphB4. This inhibition was at least as effective as inhibition with EplιB4 in parallel studies. VEGFR-3-Fc had no effects.
B. hi vitro cell-based assay with Tie-l-Fc, Tie-2-Fc This assay demonstrates that soluble extracellular domain fragments of
Tie receptors are useful as Ephrin inhibitory molecules due to the ability to block Ephrin-mediated signaling of Tie receptors or Eph receptors expressed on cell surfaces.
COS1 cells are transiently transfected with EphB4, e.g., via lipofectamine method, and incubated for 2 days. Then, 1 μg/ml of ephrin-B2 with 5 times molar of Tie-l-Fc or Tie-2-Fc is applied. After incubation for 1 hour, the cells are lysed with in TKB buffer (l%NP-40, 20mM Tris-HCl [pH7.5], 150mM NaCl, 5mM EDTA, 10% glycerol) supplemented with aprotinin, leupeptin, phenyhnethylsulfonyl fluoride, and sodium vanadate. Immunoprecipitation is carried out from equal amounts of cell lysates by adding specific antibodies and protein A-Sepharose or protein G-Sepharose and incubating for 1 hour. The immunoprecipitates are washed with lysis buffer followed by elution with Laemmli buffer and applied in PAGE gels. The proteins are transferred to a nitrocellulose membrane and blotted using anti-phosphotyrosine antibodies. Location of Eph-B4 on the gels in confirmed with anti-Eph-B4 antibodies. Reduced Eph-B4 autophosphorylation (as detected by the anti-phosphotyrosine antibodies) is indicative of successful Tie-Fc-mediated inhibition of Ephrin-B2/Eph-B4 binding.
It will be apparent that, to the extent Example 2 reveals additional Ephrin/Tie receptor interactions, the foregoing experiments can be repeated using such Ephrin molecules and their known Eph binding partners.
C. Cell-based assay using cells that naturally express Eph receptors.
The preceding experiment can be modified by substituting cells that naturally express an Eph receptor (especially EphB4) for the Eph-recombinantly tranfected COS cells. Use of primary cultures of neuronal cells expressing Eph receptors is specifically contemplated, e.g., cultured cerebellar granule cells derived from embryos. Additionally, Eph-receptor-specific antibodies can be employed to identify other cells (e.g., cells involved in vasculature, such as human microvascular endothelial cells (EC), human cutaneous fat pad microvascular cells (HUCEC), bovine capillary EC, porcine aortic EG, murine endothelial F-2 cells, or LE-2 cells.)
EXAMPLE 4 Use of soluble Tie peptides to modulate Ephrin biological activities.
A number of Ephrin molecules are membrane-bound proteins and at least some Eph/Ephrin interactions are therefore thought to be cell-cell interactions. Ephrin-B2 deletion experiments have revealed that ephrin-B2 marked specifically arterial endothelial cells while the EphB4 receptor was reciprocally expressed only on venous cells in the developing vasculature. However, ephrin-B2 mutants show defects in both arteries and veins during angiogenesis despite expression of ephrin-B2 only in developing arterial endothelial cells, indicating bi-directional signaling as a result of ephrin EphR interaction. Thus, Ephrin-expressing cells can be used to demonstrate the ability of soluble Tie receptor molecules to modulate ephrin-mediated cellular processes.
A. Cell migration assay
For example, human aortic endothelial cells (HAEC) are known to express ephrin-B2, and such cells can be used to investigate the effect of soluble Tie-l-Fc and Tie-2 -Fc on such cells. Since Ephrin-B2/EphB4 are thought to play a role in migration of cells, a cell migration assay using HAEC or other suitable cells can be used to demonstrate inhibitory effects of Tie molecules.
Using a modified Boyden chamber assay, polycarbonate filter wells (Transwell, Costar, 8 micrometer pore) are coated with 50 microg/ml fibronectin (Sigma), 0.1% gelatin in PBS for 30 minutes at room temperature, followed by equilibration into DMEM/0.1% BSA at 37 degrees C for 1 hour. HAEC (passage 4-9, 1 x 105 cells) are plated in the upper chamber of the filter well and allowed to migrate to the undersides of the filters, toward the bottom chamber of the well, which contains serum-free media supplemented with ephrin-B2 (1 μg/ml), or ephrin-B2 in the presence of varying concentrations of Tie-l-Fc, Tie-2-Fc, or control VEGFR-3-Fc protein. After 5 hours, cells adhering to the top of the transwell are removed with a cotton swab, and the cells that migrate to the underside of the filter are fixed and stained. For quantification of cell numbers, 6 randomly selected 400x microscope fields are counted per filter.
B. Clustering assay
Ephrins cluster before they can signal or transduce signals, and this characteristic provides another parameter for assay. For example, the effects of clustered Ephrin-B2-Fc by anti-Fc antibodies are tested on microtiter plates. Anti- Ephrin antibodies are used to examine Ephrin-B2 clustering at the cell surface. Differences in clustering behavior are evaluated before and after treatment with soluble Tie-R or a Tie-R-expressing cell.
EXAMPLE 5
Cell-based assay to characterize Ephrin-Tie interactions
The procedures described in Examples 3 and 4 are modified to assay the effects of Ephrin molecules on cells expressing Tie receptor on their surface. A. Phosphorylation assay.
Soluble Ephrin-B2-Fc is used as described above to contact cells that naturally or recombinantly express Tie receptor on their surface. By way of example, COS cells recombinantly modified to transiently or stably express Tie-1 or Tie-2 can be employed. Several native endothelial cells express Tie receptors and can also be employed, including isolated human umbilical vein endothelial cells (HUVEC), human microvascular endothelial cells (HMVEC), human cutaneous fat pad microvascular cells (HUCEC), bovine capillary EC, porcine aortic EC, murine endothelial F-2 cells, or LE-2 cells. Tie receptor phosphorylation is assayed essentially as described in Example 3, except anti-Tie antibodies are used to confirm the location of the Tie peptides on the Western blot. The ability of soluble EphB4-Fc, Tie-l-Fc, or Tie-2-Fc in molar excess to antagonize the effects of Ephrin-2B on the Tie receptors also can be evaluated. B. Mitogen assay
Embyronic endothelial cells are cultured in the presence or absence of an Ephrin, such as Ephrin-B2, to assay effects on cell growth using any cell growth or migration assay, such as assays that measure increase in cell number or assays that measure tritiated thymidine incorporation. See, e.g., Thompson et al, Am. J. Physiol Heart Circ. Physiol, 281: H396-403 (2001).
EXAMPLE 6 Modulating Angiopoietin/Tie-2 interactions.
The angiopoietins have been shown to act as ligands for Tie-2 receptor, mediating angiogenic effect in vivo. The identification of Ephrin-B2 as a ligand for Tie-2 has therapeutic indications for angiogenesis. The following protocols are used to measure inhibition of Angiopoietin-mediated Tie-2 activity by soluble Ephrin-B2 peptides.
Procedures described in preceding examples are modified to assay Ephrin-B2 inhibition of Angiopoietin stimulation of Tie-2. COS cells transfected with a normal human Tie-2 cDNA and transiently expressing Tie-2 are stimulated with either 100 ng/ml Ang2-, or Angl -conditioned media from 293T cells transfected with Signal Pig Plus Angl, as well as several concentrations of Ang3 and Ang4 (Rjantie et al, Mol Cell. Biol, 21: 4647-55 (2001)) in the presence and absence of varying molar ratios of Ephrin-B2-Fc. The cells are processed and Tie-2 phosphorylation is assayed as described above. The protocol is repeated substituting HUVEC cells or other endothelial cells that naturally express Tie-2 for the COS cells. In addition to measuring Tie-2 phosphorylation, the effects of Ephrin-B2 are assayed by evaluating the effects on cellular migration. The cell migration assay outlined in Example 4 is modified by using Angiopoietins in the presence or absence of Ephrin-B2-Fc, Tie-l-Fc, Tie-2-Fc, or VEGFR-3 -Fc to measure the effects of Ephrin-B2 on the ability of angiopoietins to modulate endothelial cell migration. EXAMPLE 7 Angiogenesis Assays
There continues to be a long-felt need for additional agents that can stimulate angiogenesis, e.g., to promote wound healing, or to promote successful tissue grafting and transplantation, as well as agents to inhibit angiogenesis (e.g., to inhibit growth of tumors). Moreover, various angiogenesis stimulators and inhibitors may work in concert through the same or different receptors, and on different portions of the circulatory system (e.g., arterieries or veins or capillaries; vascular or lymphatic). Angiogenesis assays are employed to measure the effects of ephrin molecules, such as Ephrin-B2, on angiogenic processes, alone or in combination with other angiogenic and anti-angiogenic factors to determine preferred combination therapy involving Ephrins and other modulators. Exemplary procedures include the following. A. In vitro assays for angiogenesis
1. Sprouting assay
HMVEC cells (passage 5-9) are grown to confluency on collagen coated beads (Pharmacia) for 5-7 days. The beads are plated in a gel matrix containing 5.5 mg/ml fibronectin (Sigma), 2 units/ml thrombin (Sigma), DMEM/2% fetal bovine serum (FBS) and the following test and control proteins: 20 ng/ml VEGF, 20 ng/ml VEGF plus 10 micrograms/ml ephrin-B2-Fc, and several combinations of angiogenic factors and Fc fusion proteins. Serum free media supplemented with test and control proteins is added to the' gel matrix every 2 days and the number of endothelial cell sprouts exceeding bead length are counted and evaluated.
2. Migration assay
The transwell migration assay previously described may also be used in conjunction with the sprouting assay to determine factors involved in angiogenesis. The effects of Ephrins are assayed alone or in combination with known angiogenic or anti-angiogenic agents. B. In vivo assays for angiogenesis
1. Chorioallantoic Membrane (CAM) assay Three-day old fertilized white Leghorn eggs are cracked, and chicken embryos with intact yolks are carefully placed in 20x100 mm plastic Petri dishes. After six days of incubation in 3% CO2 at 37 degrees C, a disk of methylcellulose containing single or various combinations of factors and fusion proteins dried on a nylon mesh (3x3mm) is implanted on the CAM of individual embryos. The nylon mesh disks are made by desiccation of 10 micro liters of 0.45% methylcellulose (in H2O). After 4-5 days of incubation, embryos and CAMs are examined for the formation of new blood vessels in the field of the implanted disks by a stereoscope. Disks of methylcellulose containing PBS are used as negative controls. Antibodies that recognize vessel cell surface molecules are used to further characterize the vessels.
2. Corneal assay
Comeal micropockets are created with a modified von Graefe cataract . knife in both eyes of male 5- to 6-week-old C57BL6/J mice. A micropellet (0.35 x 0.35 mm) of sucrose aluminum sulfate (Bukh Meditec, Copenhagen, Denmark) coated with hydron polymer type NCC (IFN Science, New Brunswick, NJ) containing various concentrations of Ephrin molecules (especially Ephrin-B2) alone or in combination with other factors that modulate vessel growth (e.g., 160 ng of VEGF-C or VEGF, or 80 ng of FGF-2 or Angiopoietins) is implanted into each pocket. The pellet is positioned 0.6-0.8 mm from the limbus. After implantation, erythromycin / ophthamic ointment is applied to the eyes. Eyes are examined by a slit-lamp biomicroscope over a course of 3-12 days. Vessel length and clock-hours of circumferential neovascularization are measured. Furthermore, eyes are cut into sections and are immunostained for blood vessel and/or lymphatic markers (LYVE-1 [Prevo et al., J. Biol. Chem., 276: 19420-19430 (2001)], podoplanin
[Breiteneder-Geleff et al., Am. J. Pathol., 154: 385-94 (1999).] and VEGFR-3) to further characterize affected vessels. EXAMPLE 8 In Vivo Assays
A. Hind-limb Ischemia Model
The following model is useful for demonstrating effects of materials and method of the invention in the treatment of ischemia or its symptoms or effects.
Unilateral hind-limb ischemia is created in 2-year-old C57BL/6J mice by ligating and excising the femoral artery of one hindlimb. Laser Doppler perfusion imaging (LDPI) is employed to document the consequent reduction in hindlimb blood flow, which typically persisted for up to 7 days. In previous reports (Couffinhal et al, Am. J. Pathol 152: 1661-1619 (1998)), serial in vivo examinations by LDPI disclosed that hindlimb blood flow was progressively augmented over the course of 14 days, ultimately reaching a plateau between 21 and 28 days. Morphometric analysis of capillary density performed at the same time points selected for in vivo analysis of blood flow by LDPI confirmed that the histological sequence of neovascularization corresponded temporally to blood flow recovery detected in vivo. In this study, the mice are operated on and treated with intramuscular injection of materials of the invention, such as Fc fusion proteins (e.g., Eprhin-Fc fusions, Eph-Fc fusions) or anti- Ephrin antibodies; gene therapy vectors encoding the foregoing; and other materials described herein, either alone or in combination with other angiogenic factors, and compared with control mice. Injections at various concentrations are made once every other day during the first week, once every 3 days during the second week, and twice during the third and fourth weeks. At predetermined time points, necrosis and hind-limb perfusion are examined. Mice are then sacrificed for histologic analysis.
Improved blood flow and/or improved vessel formation (as assessed by vessel number and/or vessel physiology and maturity) is indicative of the efficacy of single or combination agent therapy.
B. Atherosclerosis model
Ephrin materials described herein are tested for their effects on atherosclerosis using any suitable model. An exemplary model is set forth below.
Apo-E-deficient mice are fed a normal chow diet until 6 weeks of age. Then the mice are switched to a high-fat diet containing 20% fat and 0.3% cholesterol. They show remarkable atherosclerotic lesions after 12 weeks. To investigate the effects of Ephrin DNAs and Proteins and antibodies and other materials of the invention described herein, they are administered in various concentrations and combinations three times from 12 to 18 weeks. Aortic sinuses are compared in each study protocol. C. Restenosis model
Ephrin materials described herein are tested for their ability to reduce restenosis of vessels in any restenosis model. See, e.g., International Patent Publication No. WO/00/24412, incorporated herein by reference, for discussion of models, of formulations of agents for admimstration, and for materials and methods of administration. Treatment of restenosis in a manner described in that application, using Ephrin materials and methods described herein, is specifically contemplated..
For example, sixty New Zealand White rabbits are divided into 2 major groups, the first having a 0.25% cholesterol diet for 2 weeks and balloon denudation before the administration of several agents as described above, and the second having only the administration of the agents. For restenosis, gene therapy Ephrin agents, such as adenoviral Ephrin gene therapy vectors, is specifically envisioned.
The treatment performed is performed in the first group of rabbits 3 days after the denudation, and the animals are euthanized 2 or 4 weeks after the administration. The whole aorta is denuded twice with a 4.0F arterial embolectomy catheter (Sorin Biomedical). Three days later, the administration is performed with a 3. OF channeled-balloon local drug delivery catheter (Boston Scientific). Under fluoroscopic control, the catheter is positioned caudal to the left renal artery in a segment free of side branches. For example, a virus titer of 1.15 x 1010 pfu is used in the final volume of 2 mL in 0.9% saline, and the gene transfer is performed at 6 atm pressure for 10 minutes (0.2 mL/min). In the second study group, the rabbits have only the administration of the agents without a cholesterol diet or balloon denudation, and they are euthanized 2 or 4 weeks after that. The evaluation is done by the histologic analysis. D. Ectopic Tumor Implantation
Six- to 8-week-old nude (nu/nu) mice (SLC, Shizuoka, Japan) undergo subcutaneous transplantation of C6 rat glioblastoma cells or PC-3 prostate cancer cells in 0.1 mL phosphate-buffered saline (PBS) on the right flank. The Ephrin- related materials of the invention are administered to the animals at various concentrations and dosing regimens. Tumor size is measured in 2 dimensions, and tumor volume is calculated using the formula, width2 x length/2. After 14 days, the mice are humanely killed and autopsied to evaluate the quantity and physiology of tumor vasculature.
It will be apparent that the assay can also be performed using other tumor cell lines implanted in nude mice or other mouse strains. Use of wild type mice implated with LLC lung cancer cells and B16 melanoma cells is specifically contemplated. E Orthotopic tumor implantation
Approximately 1 x 107 MCF-7 breast cancer cells in PBS are inoculated into the fat pads of the second (axillar) mammary gland of ovarectomized SCID mice or nude mice, carrying s.c. 60-day slow-release pellets containing 0.72 mg of 17β-estradiol (Innovative Research of America). The ovarectomy and implantation of the pellets are done 4-8 days before tumor cell inoculation. The Ephrin-related materials of the invention are administered to the animals at various concentrations and dosing regimens. Tumor size is measured in 2 dimensions, and tumor volume is calculated using the formula, width2 x length/2. After 14 days, the mice are humanely killed and autopsied to evaluate the quantity and physiology of tumor vasculature.
A similar protocol is employed wherein PC-3 cells are implarntated into the prostate of male mice.
F. Lymphatic metastasis model The following protocol indicates the ability of Ephrin-related materials of the invention for inhibition of lymphatic metastasis.
MDA-MB-435 breast cancer cells are injected bilaterally into the second mammary fat pads_of athymic,- female, eight week old nude mice. The cells often metastasize to lymph node by 12 weeks. The Ephrin-related materials of the invention are administered to the animals at various concentrations and dosing regimens. Moreover, the Ephrin-related materials are administered in combination with other materials for reducing tumor metastasis. See, e.g., International Patent Publication No. WO 00/21560, incorporated herein by reference in its entirety. Mice are killed after 12 weeks and lymph nodes are investigated by histologic analysis. The administration protocol is the same as above.
The foregoing describes and exemplifies the invention but is not intended to limit the invention defined by the claims which follow. Variations to the examples given above will be apparent and are considered aspects of the invention within the claims.

Claims

CLAIMSWhat is claimed is:
1. A method for identifying a modulator of binding between a Tie receptor tyrosine kinase and an Ephrin ligand, comprising steps of:
(a) contacting a Tie receptor composition with an Ephrin composition in the presence and in the absence of a putative modulator compound;
(b) detecting binding between Tie receptor and the Ephrin in the presence and absence of the putative modulator; and
(c) identifying a modulator compound in view of decreased or increased binding between the Tie receptor and the Ephrin in the presence of the putative modulator, as compared to binding in the absence of the putative modulator.
2. A method according to claim 1, further comprising a step of:
(d) making a modulator composition by formulating a modulator identified according to step (c) in a pharmaceutically acceptable carrier.
3. A method according to claim 2, further comprising a step of:
(e) administering the modulator composition to an animal that comprises cells that express the Tie receptor, and determining physiological effects of the modulator composition in the animal.
4. A method according to any one of claims 1-3, wherein the Tie receptor composition comprises a member selected from the group consisting of:
(a) a purified polypeptide comprising a Tie receptor extracellular domain fragment that binds the Ephrin;
(b) a phospholipid membrane containing Tie receptor polypeptides; and
(c) a cell recombinantly modified to express increased amounts of a Tie receptor on its surface.
5. A method according to any one of claims 1-3, wherein the Tie receptor composition comprises a Tie receptor extracellular domain fragment bound to a solid support.
.
6. A method according to any one of claims 1-3, wherein the Tie receptor composition comprises a Tie receptor extracellular domain fragment fused to an immunoglobulin Fc fragment.
7. A method according to any one of claims 1-6, wherein the Tie receptor is selected from the group consisting of a mammalian Tie-1 and a mammalian Tie-2.
8. A method according to claim 7, wherein the Ephrin is Ephrin-B2.
9. A method according to any one of claims 1-8, wherein the Tie receptor is human.
10. A method according to any one of claims 1-9, wherein the Ephrin composition comprises a member selected from the group consisting of:
(a) a purified polypeptide comprising an Ephrin fragment that binds the Tie receptor;
(b) a phospholipid membrane containing Ephrin polypeptides; and
(c) a cell recombinantly modified to express increased amounts of an Ephrin on its surface.
11. A method according to any one of claims 1-9, wherein the Ephrin composition comprises an Ephrin extracellular domain fragment bound to a solid support.
12. A method according to any one of claims 1-9, wherein the Ephrin composition comprises an Ephrin extracellular domain fragment fused to an immunoglobulin Fc fragment.
13. A method according to any one of claims 1-7 or 9-12, wherein the Ephrin comprises at least one mammalian Ephrin selected from the group consisting of Ephrin-Al, Ephrin-A2, Ephrin-A3, Eplιrin-A4, Ephrin-A5, Ephrin-Bl, Ephrin-B2, and Ephrin-B3.
14. A method according to any one of claims 1-13, wherein the Ephrin is human.
15. A method according to any one of claims 1-3, wherein the Tie receptor composition comprises a cell recombinantly modified to express increased amounts of a Tie receptor on its surface, and wherein the detecting step comprises measuring an Ephrin binding-induced physiological change in the cell.
16. A method according to any one of claims 1-3, wherein the Ephrin composition comprises a cell recombinantly modified to express increased amounts of an Ephrin on its surface, and wherein the detecting step comprises measuring a Tie binding-induced physiological change in the cell.
17. A method for screening for selectivity of a modulator of binding between a Tie receptor tyrosine kinase and an Angiopoietin ligand, comprising steps of: a) contacting a Tie receptor composition with an Ephrin ligand composition in the presence and in the absence of a compound that modulates binding between the Tie receptor and an angiopoietin ligand of the Tie receptor; and b) detecting binding between the Tie receptor composition and the Ephrin ligand in the presence and absence of the modulator compound, c) identifying the selectivity of the modulator compound in view of decreased or increased binding between the Tie receptor and the Ephrin ligand in the presence as compared to the absence of the modulator, wherein increased selectivity of the modulator for modulating Tie-Angiopoietin binding correlates with decreased differences in Ephrin-Tie binding.
18. A method for screening for selectivity of a modulator of binding between a Tie receptor tyrosine kinase and an Ephrin ligand, comprising steps of: a) contacting a Tie receptor composition with an Angiopoietin ligand composition in the presence and in the absence of a compound that modulates binding between the Tie receptor and an Ephrin ligand of the Tie receptor; and b) detecting binding between the Tie receptor composition and the Angiopoietin ligand in the presence and absence of the modulator compound, c) identifying the selectivity of the modulator compound in view of decreased or increased binding between the Tie receptor and the Angiopoietin ligand in the presence as compared to the absence of the modulator, wherein increased selectivity of the modulator for Tie-Ephrin binding correlates with decreased differences in Angiopoietin-Tie binding.
19. A method of modulating growth, migration, or proliferation of cells in a mammalian organism, comprising a step of:
(a) identifying a mammalian organism having cells that express a Tie receptor tyrosine kinase; and
(b) administering to said mammalian organism a composition, said composition comprising an agent selected from the group consisting of:
(i) a polypeptide comprising an Ephrin that binds to the Tie receptor;
(ii) a polypeptide comprising a fragment of the Ephrin, wherein the polypeptide and fragment retain Tie binding characteristics of the Ephrin;
(iii) an antibody that specifically binds the polypeptide of (i) or (ii) in a manner that inhibits the polypeptide from binding the Tie receptor, or a fragment of the antibody that specifically binds the polypeptide of (i) or (ii);
(iv) a polypeptide comprising an antigen-binding fragment the (iii) and that inhibits the polypeptide of (i) or (ii) from binding the Tie receptor; and (v) a molecule that selectively inhibits Ephin binding to the Tie receptor without inhibiting angiopoietin binding to the Tie receptor; wherein the composition is administered in an amount effective to modulate growth, migration, or proliferation of cells that express Tie in the mammalian organism.
20. A method according to claim 19, wherein the mammalian organism is human.
21. A method according to claim 19 or 20, wherein the cells comprise vascular endothelial cells.
22. A method according to any one of claims 19-21, wherein the organism has a disease characterized by aberrant growth, migration, or proliferation of endothelial cells.
23. A method according to claim 22, wherein the disease comprises a cancer.
24. A method according to claim 19, further comprising administering a second agent to the patient for modulating endothelial growth, migration, or proliferation, said second agent selected from the group consisting of: a VEGF polypeptide, a VEGF-B polypeptide, a VEGF-C polypeptide, a VEGF-D polypeptide, a VEGF-E polypeptide, a P1GF polypeptide, an FGF-2 polypeptide, an Ang-1 polypeptide, an Ang-2 polypeptide, an Ang-3 polypeptide, an Ang-4 polypeptide, an endostatin polpeptide, an HGF polypeptide, an antibody that specifically binds with any of the foregoing polypeptides, an antibody that specifically binds with a receptor for any of the foregoing polypeptides, or a polypeptide comprising an antigen binding fragment of such antibodies.
25. A polypeptide comprising a fragment of an Ephrin that binds to a Tie receptor, for use in the manufacture of a medicament for the treatment of diseases characterized by aberrant growth, migration, or proliferation of cells that express a Tie receptor.
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