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WO1997023629A1 - Nouvelle tyrosine kinase de type recepteur et son utilisation - Google Patents

Nouvelle tyrosine kinase de type recepteur et son utilisation Download PDF

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Publication number
WO1997023629A1
WO1997023629A1 PCT/AU1996/000826 AU9600826W WO9723629A1 WO 1997023629 A1 WO1997023629 A1 WO 1997023629A1 AU 9600826 W AU9600826 W AU 9600826W WO 9723629 A1 WO9723629 A1 WO 9723629A1
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Prior art keywords
leu
esk
gly
arg
ser
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PCT/AU1996/000826
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English (en)
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Andrew Wallace Boyd
Jason Lickliter
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Amrad Operations Pty. Ltd.
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Priority to AU10890/97A priority Critical patent/AU1089097A/en
Publication of WO1997023629A1 publication Critical patent/WO1997023629A1/fr

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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
    • 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
    • C07K14/70503—Immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates generally to a novel receptor-type tyrosine kinase, to genetic sequences encoding same and to uses therefor.
  • RTKs receptor tyrosine kinases
  • the RTKs are transmembrane molecules which transduce signals from the extracellular environment into the cytoplasm. They include well-studied regulators of cell proliferation and differentiation such as c-kit and the receptors for epidermal growth factor, platelet-derived growth factor and macrophage colony-stimulating factor (1). Signalling is initiated when a cognate ligand binds to the RTK extracellular domain. This triggers a sequence of events resulting in the activation of an intracellular tyrosine kinase domain. Critical to this process is ligand-mediated receptor dimerization and reciprocal tyrosine phosphorylation by the dimerized molecules (2).
  • Eph was the first-isolated member of a new subfamily of RTKs (3). This group is distinguished by the sequential arrangement of a cysteine-rich region and two fibronectin-type-III repeats in the extracellular domain (4). At least 28 members have now been identified making this the largest subfamily of RTKs. They have been found in diverse species, including zebrafish (5), frogs (6), chickens (4,7,8), mice (7,9,10-12,13), rats (14,15) and humans (3,16,17,18,19). Certain features of the expression pattern of the Eph subfamily suggest key functions during embryonic development.
  • Eph and Erk are over expressed in some epithelial tumor cell lines and carcinomas (3,26), while Hek overexpression occurs sporadically in leukemia (32). Furthermore, artificial overexpression of Hek or Eph in NTH-3T3 cells resulted in a transformed phenotype, as evidenced by the ability to form colonies in agar and tumors in nude mice (27). These molecules may also be involved in tumor progression. In transgenic models of murine mammary cancer, overexpression of the Eph-subfamily members Myk-1 and Myk-2 correlated with the development of poorly- differentiated and invasive tumors (13). Embryonic stem (ES) cells are derived from the inner cell mass of the blastocyst (28). They are undifferentiated and totipotent.
  • LIF leukemia inhibitory factor
  • the inventors considered that RTKs expressed by ES cells and embryoid bodies are likely to be involved in the initial differentiation and organization of embryonic tissues.
  • the inventors used reverse transcriptase (RT) -mediated polymerase chain reaction (PCR) [RT-PCR] to identify Eph subfamily RTKs in ES cells.
  • RT-PCR reverse transcriptase -mediated polymerase chain reaction
  • the inventors have identified a novel RTK member from the Eph-subfamily.
  • one aspect ofthe present invention provides a nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding a novel member of the Eph subfamily of RTKs or a derivative, homologue or chemical analogue thereof.
  • the present invention is directed to a nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding an RTK or a derivative, homologue or chemical analogue thereof having the following characteristics:
  • the novel RTK of the present invention is of animal or mammalian origin.
  • Preferred mammals include but are not limited to humans, primates, livestock animals (e.g. sheep, cows, horses, pigs, donkeys), laboratory test animals (e.g. rabbits, mice, rats, guinea pigs), companion animals (e.g. dogs, cats) or captive wild animals (e.g. foxes, kangaroos, deer).
  • Preferred non-mammalian animals include fish and birds.
  • the present invention is particularly exemplified herein by reference to a novel RTK of murine origin but this is done with the understanding that the present invention extends to all animal and mammalian homologues of the novel murine Eph-subfamily RTK and in particular a human form of the RTK.
  • the novel RTK ofthe present invention is referred to as "Esk” for "embryonic stem cell kinase"
  • the present invention extends to derivatives, homologues and chemical analogues of Esk, which Esk has an amino acid sequence set forth in Figure 2 [SEQ ID NO:2].
  • Derivatives include single or multiple amino acid substitutions, deletions and/or additions to the sequence and encompass mutants, part and fragments thereof.
  • the term "derivative” also encompasses soluble or solubilized or otherwise secreted forms of the Esk molecule.
  • Derivatives also encompass chimeric molecules comprising Esk or a derivative, homologue, or analogue thereof and at least one other molecule such as another receptor or ligand.
  • Homologues include novel Esks of animal or mammalian origin having at least about 79%, more preferably at least about 85%, even more preferably at least about 90% and still more preferably at least about 95% or above sequence similarity to the amino acid sequence set forth in Figure 2 [SEQ ID NO:2]. Preferred homology comparisons are done between coding regions or 3' or 5' regulatory regions or particularly conserved regions.
  • Analogues of Esk contemplated herein include, but are not limited to, modifications to side chains, incorporation of unnatural amino acids and/or their derivatives during peptide synthesis and the use of crosslinkers and other methods which impose conformational constraints on the peptides or their analogues. Such analogues may also provide stability to molecule administered in vivo or for manipulation of molecules in vitro.
  • side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH4; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5'- phosphate followed by reduction with NaBH ⁇
  • the guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.
  • the carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivitisation, for example, to a corresponding amide.
  • Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4-chloromercuribenzoate, 4- chloromercuriphenylsulphonic acid, phenylmercury chloride, 2-chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH.
  • Tryptophan residues may be modified by, for example, oxidation with N-bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides.
  • Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.
  • Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate.
  • Examples of the incorporation of unnatural amino acids and derivatives during polypeptide synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3- hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids.
  • a list of unnatural amino acids contemplated for use in accordance with the present invention is given in Table 1.
  • Non-conventional Code Non-conventional Code amino acid amino acid
  • D-cysteine Dcys L-N-methylnorleucine Nmnle
  • D-glutamine Dgln L-N-methylnorvaline Nmnva
  • D- ⁇ -methylcysteine Dmcys N-(4-aminobutyl)glycine Nglu D- ⁇ -methylglutamine Dmgln N-(2-aminoethyl)glycine Naeg
  • peptides can be confo ⁇ nationally constrained by, for example, incorporation of C ⁇ and N, -methylamino acids, introduction of double bonds between and
  • Particularly useful derivatives contemplated by the present invention are soluble forms of the Esk receptor.
  • a soluble receptor is also referred to herein as a secreted Esk protein and is most preferably in recombinant form.
  • Soluble Esk molecules are useful reagents for ligand isolation, as antagonists of Esk-ligand interaction, as a substrate for antibody production which antibodies are in turn useful diagnostic reagents.
  • another aspect of the present invention provides a secreted recombinant Esk protein or derivative thereof comprising conserved cysteine residues and fibronectin type HI repeats and a portion ofthe amino acid sequence substantially set forth in SEQ ID NO:2 or having at least 79% homology thereto which is not part of the membrane bound region of the corresponding anchored receptor.
  • the amino acid sequence of the secreted recombinant Esk is encoded by a nucleotide sequence substantially as set forth in SEQ LD NO:2 or having at least 82% homology thereto.
  • the nucleic acid molecule comprises a nucleotide sequence (or a complementary form thereof) substantially as set forth in Figure 2 [SEQ LD NO: 1 ] or having at least about 82% similarity to all or part thereof or is capable of hybridising to the sequence set forth in SEQ LD NO: 1 or a complementary form thereof under low stringency conditions.
  • Preferred percentage nucleotide similarities include at least about 84%, more preferably at least about 90% and even more preferably at least about 95% or above.
  • the nucleic acid molecules ofthe present invention include single or multiple nucleotide substitutions, deletions and/or additions to the nucleotide sequence set forth in Figure 2 [SEQ LD NO: 1] and mutants, parts and fragments thereof, which are all encompassed by the term "derivative" of the nucleotide sequence set forth in Figure 2 [SEQ LD NO: 2].
  • Reference herein to a low stringency at 42 P C includes and encompasses from at least about 1% v/v to at least about 15% v/v formamide and from at least about IM to at least about 2M salt for hybridisation, and at least about IM to at least about 2M salt for washing conditions.
  • Alternative stringency conditions may be applied where necessary, such as medium stringency, which includes and encompasses from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5M to at least about 0.9M salt for hybridisation, and at least about 0.5M to at least about 0.9M salt for washing conditions, or high stringency, which includes and encompasses from at least about 31% v/v to at least about 50% v/v formamide and from at least about 0.0 IM to at least about 0.15M salt for hybridisation, and at least about 0.0 IM to at least about 0.15M salt for washing conditions.
  • medium stringency which includes and encompasses from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5M to at least about 0.9M salt for hybridisation, and at least about 0.5M to at least about 0.9M salt for washing conditions
  • high stringency which includes and encompasses from at least about 31% v/v to at least about 50% v/
  • the nucleic acid molecules of the present invention are preferably carried by a vector molecule and more particularly an expression vector.
  • Preferred expression vectors direct expression in mammalian, insect and or bacterial cells.
  • the present invention also extends to cells carrying the recombinant nucleic acid molecules ofthe present invention.
  • Preferred nucleic acid molecules are DNA and more preferably cDNA.
  • the present invention is also directed to a recombinant Esk polypeptide having the following characteristics:
  • (i) is an RTK belonging to the Eph subfamily of RTKs as determined by cysteine residues and fibronectin type II repeats; (ii) comprises protein tyrosine kinase catalytic domain motifs; and 10 (iii) comprises an amino acid sequence substantially as set forth in Figure 2 [SEQ ID NO:
  • LD NO: 2 or having at least about 79% similarity to all or part thereof.
  • the recombinant Esk is preferably in isolated form meaning that a composition comprises at least about 20%, more preferably at least about 30%, still more preferably at least about 40- 15 50%, even more preferably at least about 60-70% and yet even more preferably at least about 80-90% or above of Esk as determined by activity, molecular weight, or immunological reactivity.
  • the preparation may also be sequencably pure or of a purity suitable for use in a pharmaceutical composition.
  • the present invention extends to the ligand(s) of Esk and to agonists and antagonists of Esk- ligand interaction.
  • Agonists and antagonists may be, for example, antibodies or derivatives of the Esk or derivatives of the ligand.
  • Derivatives of Esk or its ligand include soluble or solubilised forms thereof.
  • Reference herein to "Esk” includes both anchored forms (ie. membrane bound forms) ofthe receptor as well as soluble (ie. secreted) forms ofthe receptor.
  • Modulating expression of Esk may have important potential in therapeutic regimens for the treatment or prophylaxis of cancers caused or exacerbated by aberations in Esk or aberations in Esk-ligand interaction. This will be particularly important for the treatment of mucositis. This condition remains the major adverse effect of chemotherapy and radiotherapy of
  • vagi itis and vulvitis Sjogrens syndrome and related autoimmune diseases; infection of the lung (eg. shock lung, inhalation of noxious fumes, infection); liver (eg. regeneration after viral illness or toxic damage); pancreatitis; urological disease involving tubules, pelvicalyceal system, ureters, bladder or urethra and salivary glands.
  • lung eg. shock lung, inhalation of noxious fumes, infection
  • liver eg. regeneration after viral illness or toxic damage
  • pancreatitis urological disease involving tubules, pelvicalyceal system, ureters, bladder or urethra and salivary glands.
  • the present invention contemplates a method for modulating Esk-ligand interaction in an animal, said method comprising administering to said animal a modulating effective amount of an agonist or antagonist of Esk-ligand interaction.
  • modulating includes facilitating Esk-ligand interaction or inhibiting, reducing or otherwise interfering with Esk-ligand interaction. Either form of modulation may be required depending on, for example, the type of treatment such as the treatment of cancer or the promotion or inhibition of cell apoptosis.
  • the present invention contemplates a pharmaceutical composition
  • a pharmaceutical composition comprising an Esk-ligand interaction modulating effective amount of an agonist or antagonist of Esk-ligand interaction and one or more pharmaceutically acceptable carriers and/or diluents.
  • compositions are generally known in the art and reference can conveniently be made to Remington's Pharmaceutical Sciences, 17th end., Mack Publishing Co., Easton, Pennsylvania, USA.
  • the active ingredients of a pharmaceutical composition comprising the Esk agonists or antagonists or their derivatives are contemplated herein to exhibit excellent therapeutic activity, for example, in modulating Esk-ligand interaction when administered to an animal in an amount which depends on the particular case. For example, from about 0.5 ⁇ g to about 20 mg per kilogram of body weight per day may be administered. Dosage regimes may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily, weekly or monthly, or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
  • the active compounds may be administered in any convenient manner such as by the oral, intravenous (where water soluble), intramuscular, subcutaneous, intranasal, intradermal or suppository routes or by implanting (eg using slow release molecules), topical administration or following or during surgery or biopsy or other invasive procedure.
  • the active ingredients which comprise the Esk agonists or antagonists or chemical analogues thereof may be required to be coated in a material to protect said ingredients from the action of enzymes, acids and other natural conditions which may inactivate said ingredients.
  • Esk agonists or antagonists In order to administer Esk agonists or antagonists by other than parenteral administration, they will be coated by, or administered with, a material to prevent its inactivation.
  • homologues may be administered in an adjuvant, co-administered with enzyme inhibitors or in liposomes.
  • adjuvants contemplated by the present invention include, but are not limited to, cytokines (e.g. interferons) as well as resorcinols, non-ionic surfactants such as polyoxyethelene oleyl ether and n-hexadecyl polyethylene ether.
  • the active compounds may also be administered parenterally or intraperitoneally.
  • Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of superfactants.
  • the preventions of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thirmerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged abso ⁇ tion of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solven with various of the ingredients enumerated above, as required, followed by filtered sterilisation.
  • dispersions are prepared by incorporating the various sterilised active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • sterile powders for the preparation of sterile injectable solutions the preferred methods of preparation of vacuum drying and the freeze-drying technique which yield a powder ofthe active ingredient plus any additional desired ingredient from previously sterile- filtered solution thereof.
  • the active, compound may be orally administered, for example, with an inert diluent or with a assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tables, or it may be incorporated directly with the food of the diet.
  • the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 1% by weight of active compound.
  • compositions and preparations may, of course, be varied and may conveniently be between 5 to about 80% of the weight of the unit.
  • the amount of active compound in such therapeutically useful compositions in such that a suitable dosage will be obtained.
  • Preferred compositions or preparations according to the present invention are prepared so that a oral dosage unit form contains between about 0.1 ⁇ g and 2000 mg of active compound.
  • the tablets, troches, pills, capsules and the like may also contain the following: A binder such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil of wintergreen, or cherry flavouring.
  • a binder such as gum tragacanth, acacia, corn starch or gelatin
  • excipients such as dicalcium phosphate
  • a disintegrating agent such as corn starch, potato starch, alginic acid and the like
  • a lubricant such as magnesium stearate
  • a sweetening agent such as sucrose, lactose or saccharin
  • a flavouring agent such as peppermint, oil of
  • tablets, pills, or capsules may be coated with shellac, sugar or both.
  • a syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour.
  • any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed.
  • the active compound may be inco ⁇ orated into sustained-release preparations and formulations.
  • a pharmaceutically acceptable carrier and/or diluent indues any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and abso ⁇ tion delaying agents and the like.
  • the use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the acive ingredient, use thereof in the therapeutic compositions is contemplated. Supplementary active ingredients can also be inco ⁇ orated into the compositions.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the mallian subjects to be treated; each unit coating a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the novel dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active material and the particular therapeutic affect to be achieved, and (b) the limiations inherent in the art of compound such a active material for the treatment of disease in living subjects having a diseased condition in which bodily health is impaired as herein disclosed in detail.
  • the principial active ingredient is compounded for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable carrier in dosage unit form as hereinbefore disclosed.
  • a unit dosage form can, for example, contain the principal active compound in amounts ranging from 0.5 ⁇ g to about 2000 mg. Expressed in proportions, the active compound is generally present in from about 0.5 ⁇ g to about 2000 mg/ml of carrier.
  • the dosages are determined by reference to the usual dose and manner of administration of the said ingredients.
  • the animal to be treated is preferably a mammal such as a human, primate, livestock animal, laboratory test animal, companion animal or captive wild animal. Most preferably, the animal is human.
  • the present invention further extends to antibodies to the Esk molecules herein described.
  • the antibodies may be monoclonal or polyclonal.
  • Antibodies to the Esk molecules of the present invention are useful as therapeutic agents in modulating Esk-ligand interaction or as diagnostic agents to assay for Esk molecules or Esk-ligand intemation.
  • Assay techniques are well known in the art and include, for example, sandwhich assays and ELISA.
  • Another aspect of the present invention contemplates a method for assaying for
  • Esk expression on a cell said method comprising contacting a biological sample containing cells putatively expressing Esk with an Esk-binding effective amount of an antibody thereto for a time and under conditions sufficient for said antibody to bind to said Esk and then detecting said Esk-antibody binding.
  • Esk on a cell can be detected using a wide range of immunoassay techniques such as those described in US Patent Nos. 4,016,043, 4,424,279 and 4,018,653. This includes both single-site and two-site, or "sandwhich", assays ofthe non-competitive types, as well as in the traditional competitive binding assays. Sandwhich assays are among the most useful and commonly used assays and are favoured for use in the present invention. A number of variations ofthe sandwhich assay technique exist, and all are intended to be encompassed by the present invention.
  • Esk antibody is immobilised onto a solid substrate to form a first complex and the sample containing cells to be tested b rough into contact with the bound molecule.
  • an antibody labelled with a receptor molecule capable of producing a detectable signal and specific to another antigen to the cell, is then added and incubated, allowing time sufficient for the formation of a tertiary complex of Esk-antibody-labelled antibody. Any unreacted material is washed away, and the presence ofthe first antibody is determined by observation of a signal produced by the reporter molecule on the second antibody.
  • the results may either be qualitative, by simple observation ofthe visible signal or may be quantitated by comparing with a control sample containing known amounts of hapten.
  • Variations of the forward assay include a simultaneous assay, in which both sample and labelled antibody are added simultaneously to the bound antibody, or a reverse assay in which the labelled antibody and sample to be tested are first combined, incubated and then added simultaneously to the bound antibody.
  • a labelled Esk antibody may be added directly to the sample of cells and the reporter molecule defected.
  • the solid substrate is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene polyvinyl chloride or polypropylene.
  • the solid supports may be in the form of tubes, beads, discs or microplates, or any other surface suitable for conducting an immunoassay.
  • the binding processes are well-known in the art and generally consist of cross-linking covalently binding or physically adsorbing the molecule to the insoluble carrier.
  • reporter molecule is meant a molecule which, by its chemical nature, profices an analytical identifiable signal which allows the detection of antigen-bound antibody. Detection may be either qualitative or quantitative.
  • the most commonly used reporter molecule in this type of assay re either enzymes, fluorophores or radionuclide containing molecules (i.e. radioisotopes).
  • an enzyme immunoassay an enzyme is conjugated to the second antibody, generally by means of glutaraldehyde or periodate. As will be readily recognised, however, a wide variety of different conjugation techniques exist which are readily available to one skilled in the art.
  • Commonly used enzymes include horseradish peroxidase, glucose oxidase, ⁇ -galactosidase and alkaline phosphatase, amongst others.
  • the substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable colour change. It is also possible to employ fluorogenic substrates which yield a fluorescent product.
  • fluorescent compounds such as fluorescein and rhodamine
  • fluorescent compounds may be chemically coupled to antibodies without altering their binding capacity.
  • the fluorochrome-labelled antibody When activated by illumination with light of a particular wavelength, the fluorochrome-labelled antibody adsorbs the light energy, inducing a state of excitability in the molecule, followed by emission of the light at a characteristic colour visually detectable with a light microscope.
  • the fluorescent labelled antibody is allowed to bind to the first antibody-hapten complex. After washing off the unbound reagent, the remaining ternary complex is then exposed to the light ofthe appropriate wavelength, the fluorescent observed indicates the presence of the hapten of interest.
  • the present invention further extends to genetic molecules derived from an Esk gene useful as probes, antisense or sense molecules for diagnostic or therapeutic situations.
  • Figure 1 is a diagrammatic representation showing the position of the four degenerate oligonucleotide primers (PI-P4) used for RT-PCR.
  • the relatively-conserved peptide motifs on which the primers were based are shown above a schematic representation of the basic domain structure of Eph-subfamily molecules. The particular amino acid sequences used in the figure are from eph.
  • Pl and P2 were sense primers:
  • P3 and P4 were antisense primers. Their sequences were as follows:
  • TMD denotes the transmembrane domain.
  • Figure 2 is a reprsentation showing the nucleotide and deduced amino acid sequence of Esk.
  • the signal peptide and transmembrance domain are boxed.
  • conserveed cysteine residues in the extracellular domain are circled and two fibronectin type III repeats are in a striped box.
  • arrowheads indicate the highly-conserved Gly-X-Gly-X-X-Gly motif and a dot marks the invariant lysine residue.
  • Two motifs assocaited with substrate specificity for tyrosine are underlined.
  • the stop codon which terminates the coding region is indicated by an asterisk.
  • Figure 3 is a photographic representation of expression of Esk.
  • Northern blots of poly(A) + RNA from day- 12 mouse embryo and the adult mouse tissues shown were hybridized to a 32 P-labelled probe derived from clone 35C15. This was then stripped from the filters and hybridization to a GAPDH probe was performed. The positions of RNA size markers are indicated to the left of the blots.
  • Figure 4 is a photographic representation showing Northern blot of ES cell, embyoid body and embryonic firbroblast poly (A) + RNA hybridized to 32 P-labelled probes synthesized from Esk,, Mek4 and Eck cDNA. Probes were stipped from the filter between reprobingS. Hybridisaztion to a GAPDH probe was [erformed last. The sizes of transceipts are indicated to the right of the figure.
  • Figure 5 is a diagrammatic representation of all Eph-subfamily clones isolated from ES cells using RT-PCR aligned with the full-length cDNA clone derived from a ⁇ ZAP library. Numebrs represent nucleotides in the Esk full length sequence. ECD, extracellular domain; ICD, intracellular domain.
  • Figure 6 is a reprsentation showing a plot of grains after scoring approximately 130 Chromosomes 6, showing probable localisation of Esk to bands B1-B2. Grains scored from C57BL and BALB/c mice are represented with solid and open dots respectively.
  • Figure 7 is a photographic representation of an analysis of the relationship between Esk and Eph by Southern hybridization.
  • a mouse genomic Southern blot was prepared using DNA digested with the restriction enzymes shown. This was initially hybridized in 40% formamide to a probe derived from the catalytic domain of human Eph (A). The membrance was then stripped and rehybridized in 50% formamide to a p ⁇ be derived from equivalent sequence in mouse Esk(B). The position of DNA size markers is indicated at left.
  • Figure 9 is a photographic representation showing in situ analysis of Esk expression in whole-mounted embryoid bodies (A) and day 9.5 mouse embryos (B). Embryoid bodies were differentiated in vitro from ES cells by culturing without feeder cells in LIF-deficient maxim, for 7-10 days. Sense and antisense digoxigenin-labelled riboprobes were synthesized from cDNA fragments of Esk and hybridized to the whole-mounts. Bound probe was detected using alkaline phosphate-conjugated anti-digoxigenin Fab fragments and staining to detect enzyme activity. Left panels show results of hybridization with sense-control and right panels with Esk antisense probe.
  • Figure 9 is a photographic representation of Esk expression in sections of embryoid bodies and selected mouse tissues. Results of hybridizations with sense-control probe are shown in the left panels and with Esk antisense probe in the middle and right panels.
  • A Embryoid bodies differentiated in vitro.
  • B Adult thymus.
  • C Adult renal cortex.
  • D Day 18 embryo skin. Abbreviations: LP, low power; HP, high power.
  • Figure 10 is a photographic reprsentation showing binding of ligand to Esk chip in a biosense assay for binding to a potential Erk ligands.
  • (LI to L7) controls a FC, L3-FLAG, L7-FLAG and binding to HEK.
  • Single and three letter abbreviations for amino acid residues are used in the specification and are defmed in Table 2.
  • the murine 129/Sv-derived ES cell line, W9.5 was routinely passaged on underlays of irradiated embryonic fibroblasts in Dulbecco's modified Eagle medium supplemented with 1000 units/ml of LIF (AMRAD Operations Pty Ltd, Melbourne, Victoria, Australia), 10" 4 M 2-mercaptoethanol and 15 % v/v fetal calf serum. Cultures were incubated in a 10% v/v CO 2 atmosphere at 37 ⁇ C. In preparation for the studies described below, ES cells were subcultured into delatinized flasks and four passages without a feeder layer were performed to deplete the embryonic fibroblasts. In some of these cultures, LIF was withdrawn 11 days prior to harvesting the cells, to allow differentiation into embryoid bodies (29). Control cultures of embryonic fibroblasts alone were also performed.
  • RNA extraction Prior to RNA extraction, cultures of undifferentiated ES cells were disrupted with trypsin and washed in phosphate buffered saline. Cell pellets were resuspended in guanidine isothiocyanate denaturing buffer and total RNA was extracted using organic solvents (30). cDNA was then synthesized using 1 ⁇ g of total RNA, an aoligo(dT) primer and AMV reverse transcriptase (Promega). Four degenerate PCR primers were derived from three regions of sequence which are relatively conserved across the Eph subfamily (18)[Figurel ⁇ . Sense primers Plor P2 were used with antisense primer P4 to amplify the Es cell cDNA.
  • Reactions were carried out in a 30 ⁇ l volume containing 50 mM KCl, lOmM Tris. HCl (pH 8.3), 1.25 mM MgCl 2 , 0.2 mM each dNTP, 2.5 units of Taq polymerase (Perkin Elmer), 30 pmol of each promer and 3 ⁇ l of the ES cell cDNA synthesis reaction.
  • mRNA was directly extracted from ES cells with oligo9dT)-coated magnetic beads (Dynal; Oslo, Norway) and cDNA was synthesized from 1 ⁇ g of mRNA using an oligo(dT) primer and Superscript II reverse trasnscriptase (Life Technologies).
  • PCR products were electrophoretically separated, purified with the Geneclean II Kit (Bio 101 Inc) and then subjected to a second round of PCR. Products initially amplified using primers Pl and P4 were reamplified with the same primers, while those amplified with P2 and P4 were reasmplified with P2 and P3.
  • Oligo(dT)-cellulose (Pharmacia) as used to isolate poly(A) + RNA from total derived from
  • ES cells embryoid bodies differentiated in vitro from ES cells. Embryonic fibroblasts, day-
  • Inserts were digested from these three clones with the restriction enzymes EcoRI and Xbal, and used as templates for synthesizing 32 P-lebelled probes witht he Prime-It II Random Primer Labelling Kit (Stratagene).
  • a labelled propbe was also made from glyceraldehyde-3- phosphate dehydrogenase (GAPDH) cDNA.
  • GPDH glyceraldehyde-3- phosphate dehydrogenase
  • hybridized probe was stripped from the membrane by pouring on boiling 0.1 % w/v SDS and cooling to room temperature. Exposure to X-ray film overnight confirmed effective removal of the probe. Hybridisation to the GAPDH probe was performed last. In all cases, hybridisations took place in 50% v/v formamide at 42° C and washses were performed under stringent conditions, with the final wash at 65°C in 0.1 x SSC and 0.1 % w/v SDS (SSC is 0.15 M NaCl and 0.015 m Na citrate, pH 7.6). Autoradiograps were exposed at -70° C.
  • a 32 P-labelled Esk probe was synthesised from a PCR fragment amplified from Esk cDNA using degenerate kinase-domain primers described in Example 2. The fragment included sequence from bases 2098-2437 of Esk. Probe was hybridized to a geomic Southern blot made using standard techniques with DNA extractex from the murine embryonic stem cell line W9.5 and digested with the panel of enzymes showin in Figure 7.
  • Hybridisation was performed in 50% formamide, lOx Denhardt's solutioN (35), 50mM Tris HCl (pH7.5), 1.0 ⁇ NaCl, 2.24 mM tetrasodium pyrophosphate, 1% w/v SDS, 10% w/v dextran sulfate and 0.1 mg/ml sheared heat-denatured herring sperm DNA.
  • the final wash was in O.lxSSC/0.1 % w/v SDS at 65°C (SSC is 0.15 M sodium chloride, 0.015 M sodium ctrate, pH 7.6).
  • SSC 0.15 M sodium chloride, 0.015 M sodium ctrate, pH 7.6
  • a 32 P-labelled Eph probe was synthesised from a cloned cDNA fragment isolated by RT- PCR from human bone marrow cDNA, using the same degenerate kinase-domain primers that generated the Esk probe.
  • the cloned cDNA included sequence from bases 2212-2553 of human Eph. Probe was hybridized to the stripped Southern blot, with hybridization conditions and washes identifical to those used with the Esk probe. An autoradiograph was exposed at -70° C for seven days.
  • ES cells from the W9.5 line were induced to undergo differentiation by passaging them in bacterial-grade Petri dishes without an embryonic fibroblast feeder layer and in the absence of LLF. Embryoid bodies were harvested after 7-10 days of development (39).
  • the Esk cDNA was used to screen a mouse liver cDNA library and identified twenty-four positive clones.
  • One clone containing the largest insert size was analysed in detail and found to include the complete coding region of Esk ( Figure 2).
  • This clone contained a single open reading frame that encoded 977 amino acids, without an initiation codon occuring in a context consistent with the Kozac rules for translation start sites (42).
  • Amino acids 1-26 conformed to the predicted sequence for a signal peptide (43).
  • a sequence of predominantly hydrophobic residues at positions 549-569 were consistent with a transmembrane domain, potentially dividing the mature Esk protein into extracellular and intracellular portions.
  • a Northern blot containing ES cell and embryoid body RNA was sequentially hybridized to Esk, Mek4 and Eck probes.
  • Embryonic fibroblast RNA was included as a control on this blot, because some fibroblast contamination of the ES cell and embryoid body samples could not be excluded.
  • the Esk probe hybridized to all three samples, approximately in proportion to the amount of RNA present. This result indicates significant levels of Esk expression in ES cells and embryoid bodies, and could not be accounted for by fibroblast contamination alone.
  • Mek4 was barely detectable in the ES cell cultures.
  • a 9.5 kb Mek4 transcript was expressed by the embryonic fibroblasts, however, suggesting that the faint bands seen in the ES cell lane may be due to contaminating fibroblast RNA.
  • the Eck probe hybridized to all three samples, but expression was relatively greater in the undifferentiated ES cells. Transcripts were of slightly different sizes in the different lanes.
  • Esk The expression of Esk was investigated with in situ hybridization in embryoid bodies differentiated in vitro, mouse embryos and selected adult mouse tissues. These experiments were performed using two pairs of antisense and sense-control riboprobes derived from non- overlapping regions of Esk sequence. Both antisense probes gave similar results. No specific staining was detected in any of the tissues hybridized to the sense-control probes with the exception of small intestine, where high levels of endogenous alkaline phosphatase activity resulted in false-positive staining of the epithelium. This occured despite the inclusion of levamisole in the post-hybridization washes.
  • Esk was expressed by most embryoid-body cells, an amo ⁇ hous layer visible near the surface at high magnification delineated an outer rind of cells that did not stain.
  • the latter cells have been shown by others to correspond to primitive endoderm, whereas the cells within the interior are primitive ectoderm in type (45; 46). These populations of cells are separated by a layer of basement membrane-like material secreted by the endodermal cells.
  • Example 13 Northern analysis (Example 13) indicated that Esk is expressed in a variety of non-neural adult tissues. To defme the specific cells containing Esk mRNA, in situ hybridisation experiments were performed on sections of some of these tissues. In thymus, Esk expression was observed in aggregates of large cells with abundant cytoplasm, located predominantly in the medulla ( Figure 9B). Mo ⁇ hologically, these cells were consistent with thymic epithelia. No staining of thymocytes was observed. In kidney, Esk expression was localized to the epithelium of proximal and distal convoluted tubules (Figure 9C). Glomeruli and the interstium did not stain. Esk was also expressed in the epithelial cells of the adrenal cortex.
  • a tritiated probe was made from the 1.6 kb cloned PCR fragment and hybridized in situ to preparations of mouse metaphase chromosomes. This resulted in a significant accumulation of silver grains over bands Bl and B2 of mouse Chromosome 6. Out of an initial score of 345 grains for all chromosomes, 36.8% of grains were over the proximal half of Chromosome 6. Background grains were distributed over the other chromosomes with 5 grains over distal Chromosome 15 to tallest secondary peak observed. This compared to 59 grains over band 6B2 and 28 over 6B1.
  • COS cells and CHO cells were propagated in RPMI containing 10% v/v heat-inactivated FCS in a humidified 10% v/v CO 2 atmosphere at 37°C.
  • the COS cells were stably transfected with the polyoma large T antigen, permitting replication of vectors containing an SV40 origin of replication.
  • a concentration of 600 ⁇ g/ml of G418 (Gibco BRL) in RPMI 1-% v/v FCS was used.
  • cDNA sequence was amplified from Esk using a 5' primer derived from sequence beginning five bases upstream of the translation start site and a 3' primer from sequence immediately upstream of the transmembrane domain. Custom Bglll sites were included in both primers and a sphce donor sequence was added to the 3 ' primer. To minimize the possibility of PCR- induced errors, amplifications were performed using recombinant (Stratagene).
  • the reaction was carried out in a 50 ⁇ l volume containing lx cloned Pfu buffer, 0.2 mM each dNTP, 2.5 units cloned Pfu polymerase, 0.2 ⁇ M each primer and approximately 250 ng of pBluescript plasmid containing the full-length Esk clone, and was performed in a Perkin Elmer GeneAmp 2400 PCR machine.
  • PCR amplification conditions were: 30 seconds at 95 ⁇ C, 30 seconds at 55 ⁇ C and 2 minutes and 30 seconds at 72 ⁇ C for 5 cycles; 30 seconds at 95 ⁇ C and 3 minutes at 72°C for 15 cycles.
  • Amplification products were digested with BgHI and ligated into the unique BamKL cloning site of the plg-BOS expression vector.
  • This plasmid is a pEF-BOS derivative which includes a genomic fragment of human IgGI (containing exons encoding the hinge, CH2 and CH3 domains) downstream ofthe cloning site. Expression of this construct was expected to produce a polypeptide of approximately 90 kD in mass, which should become a divalent molecule of approximately 180 kD after the formation of cystine bonds between the hinge regions.
  • the constructs were cloned in DH10 B Kcoli cells and clones containing correctly oriented inserts were identified and DNA prepared and checked by DNA sequencing.
  • a peptide was synthesized from the first 31 amino acids of the predicted mature protein encoded in the Esk sequence. This was coupled to keyhole limpet hemocyanin (KLH) and 200 ⁇ g of the conjugate was emulsified in complete Freund's adjuvant and injected into a New Zealand white rabbit via multiple subcutaneous sites. A second dose of antigen was administered in incomplete Freund's adjuvant after four weeks and serum was collected two weeks later. The serum was used at a dilution of 1 : 100 to probe Western blots.
  • KLH keyhole limpet hemocyanin
  • CHO cells permanently transfected with an Esk and control CHO cells were grown in chamber slides, fixed in PBS containing l%v/v paraformaldehyde for 30 minutes and permeabilized with 100% v/v methanol. After blocking in PBS containing 2% v/v FCS and 2% v/v goat serum, the cells were incubated with either a 1 : 100 dilution ofthe rabbit anti-Esk antiserum described in the previous section or with preimmune serum as a control. In a second set of controls, 40 ⁇ g of Esk peptide coupled to KLH was added with the antiserum. A donkey anti- rabbit lg antibody conjugated to HRP was used as the secondary antibody in all cases and bound antibody was detected with diaminobenzidine (Dako).
  • Dako diaminobenzidine
  • the following method was used for protein A-Sepharose affinity chromatography.
  • the column was pre-eluted with pH 3 0.1 M acetate buffer and re-equilibrated with PBS.
  • the sample was applied and then the column washed to baseline with PBS.
  • the sample was eluted with acetate buffer and peak fractions collected in tubes containing 1/10 vol of IM tris HCl pH8.
  • the column was re-equilibrated with PBS/azide for storage.
  • the purified ESK Fc protein (100 ⁇ g) was homogenised in complete Freund's adjuvant and administered subcutaneously at multiple sites. Animals were immunised every 14 days and bled 10 days after each injection. To purify the specific component the antiserum was exhaustively absorbed on a human immunoglobulin affinity column and then applied to an ESK Fc affinity column, the specific antibody was eluted with 0.2 M glycine HCl pH 2.3, quickly neutralised and concentrated to 0.5-1.0 mg/ml. 2.
  • a polyclonal serum raised against an Esk peptide was initially tested by probing a Western blot containing recombinant Esk and Hek.
  • the antibody recognized Esk specifically, with bands ofthe expected size appearing in both the full-length and truncated Esk samples and no specific bands in the Hek samples.
  • the anti-Esk serum was used for immunochemistry, the cytoplasm of CHO cells transfected with the Esk construct stained strongly. The specificity of this staining for Esk was confirmed by several controls. These included incubations of Esk-transfected cells with preimmune serum and of control CHO cells with the antiserum. Only background staining was observed in either case. In addition, Esk peptide as observed to inhibit staining of the transfectants by the antiserum.
  • High level expressing clones were expanded to 1 litre cultures in RPMI 1640 supplemented with 2.5% v/v FCS which had been absorbed on protein A-Sepharose beads to remove all protein A-binding bovine immunoglobulin.
  • the cultures were allowed to grow to confluence and the conditioned medium removed, centrifuged to remove cellular material and concentrated 10-fold.
  • the medium was applied to a protein A Sepharose column as above. Purified protein was analysed by SDS-PAGE to confirm purity. In each case a single band was obtained on SDS-PAGE under reducing conditions. N terminal sequencing ofthe ESK- Fc protein band gave a single un-equivocal sequence:
  • Sek (11), Nuk (15), Eck (21) and Mek (8) are murine Eph-subfamily molecules; Eph (3) is a human molecule.

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Abstract

La présente invention se rapporte de manière générale à une nouvelle tyrosine kinase de type récepteur, à des séquences génétiques codant cette tyrosine kinase et à ses utilisations. Plus particulièrement, la présente invention se rapporte à une tyrosine kinase de type récepteur ayant les caractéristiques suivantes: (i) elle appartient à la sous-famille Eph des tyrosines kinases réceptrices (RTK) définies par des résidus de cystéine préservés et des répétitions de type III de fibronectine, (ii) elle comporte des motifs de domaines catalytiques de tyrosine kinase de protéine et (iii) elle comporte une séquence d'acides aminés sensiblement identique à celle présentée dans SEQ ID NO:2 ou présentant au moins 79 % de similarité avec cette séquence.
PCT/AU1996/000826 1995-12-22 1996-12-20 Nouvelle tyrosine kinase de type recepteur et son utilisation WO1997023629A1 (fr)

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7381410B2 (en) 2003-03-12 2008-06-03 Vasgene Therapeutics, Inc. Polypeptide compounds for inhibiting angiogenesis and tumor growth
US7595044B2 (en) 1998-04-13 2009-09-29 California Institute Of Technology Artery-and vein-specific proteins and uses therefor
US7700297B2 (en) 1998-04-13 2010-04-20 California Institute Of Technology Artery- and vein-specific proteins and uses therefor
US7862816B2 (en) 2003-03-12 2011-01-04 Vasgene Therapeutics, Inc. Polypeptide compounds for inhibiting angiogenesis and tumor growth
US7977463B2 (en) 2004-03-12 2011-07-12 Vasgene Therapeutics, Inc. Polypeptide compounds for inhibiting angiogenesis and tumor growth
US8975377B2 (en) 2007-08-13 2015-03-10 Vasgene Therapeutics, Inc Cancer treatment using humanized antibodies that bind to EphB4
US8981062B2 (en) 2004-03-12 2015-03-17 Vasgene Theapeutics, Inc Polypeptide compounds for inhibiting angiogenesis and tumor growth
US9533026B2 (en) 2004-09-23 2017-01-03 Vasgene Therapeutics, Inc Polypeptide compounds for inhibiting angiogenesis and tumor growth

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
EMBO JOURNAL, Vol. 14(13), (1995), BRAMBILLA et al., "Membrane-bound LERK 2 Ligand can Signal Through Three Different Eph-related Receptor Tyrosine Kinases", pages 3116-3126. *
PROC. NATL. ACAD. SCI. U.S.A., Vol. 93, (January 1996), LICKLITER et al., "Embryonic Stem Cells Express Multiple Eph-subfamily Receptor Tyrosine Kinases", pages 145-150. *
SCIENCE, Vol. 238, (1987), HIRAI et al., "A Novel Putative Tyrosine Kinase Receptor Encoded by the Eph Gene", pages 1717-1720. *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7595044B2 (en) 1998-04-13 2009-09-29 California Institute Of Technology Artery-and vein-specific proteins and uses therefor
US7700297B2 (en) 1998-04-13 2010-04-20 California Institute Of Technology Artery- and vein-specific proteins and uses therefor
US7741272B2 (en) 1998-04-13 2010-06-22 California Institute Of Technology Artery- and vein-specific proteins and uses therefor
US7939071B2 (en) 1998-04-13 2011-05-10 California Institute Of Technology Artery- and vein-specific proteins and uses therefor
US7381410B2 (en) 2003-03-12 2008-06-03 Vasgene Therapeutics, Inc. Polypeptide compounds for inhibiting angiogenesis and tumor growth
US7862816B2 (en) 2003-03-12 2011-01-04 Vasgene Therapeutics, Inc. Polypeptide compounds for inhibiting angiogenesis and tumor growth
US8063183B2 (en) 2003-03-12 2011-11-22 Vasgene Therapeutics, Inc. Polypeptide compounds for inhibiting angiogenesis and tumor growth
US8273858B2 (en) 2003-03-12 2012-09-25 Vasgene Therapeutics, Inc. Polypeptide compounds for inhibiting angiogenesis and tumor growth
US7977463B2 (en) 2004-03-12 2011-07-12 Vasgene Therapeutics, Inc. Polypeptide compounds for inhibiting angiogenesis and tumor growth
US8981062B2 (en) 2004-03-12 2015-03-17 Vasgene Theapeutics, Inc Polypeptide compounds for inhibiting angiogenesis and tumor growth
US9533026B2 (en) 2004-09-23 2017-01-03 Vasgene Therapeutics, Inc Polypeptide compounds for inhibiting angiogenesis and tumor growth
US8975377B2 (en) 2007-08-13 2015-03-10 Vasgene Therapeutics, Inc Cancer treatment using humanized antibodies that bind to EphB4

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