WO2002026827A1 - Polypeptides extracellulaires de recpteurs eph b et ligands ephrine b, et molecules d'acide nucleiques correspondantes - Google Patents

Polypeptides extracellulaires de recpteurs eph b et ligands ephrine b, et molecules d'acide nucleiques correspondantes Download PDF

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Publication number
WO2002026827A1
WO2002026827A1 PCT/EP2001/011252 EP0111252W WO0226827A1 WO 2002026827 A1 WO2002026827 A1 WO 2002026827A1 EP 0111252 W EP0111252 W EP 0111252W WO 0226827 A1 WO0226827 A1 WO 0226827A1
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polypeptide
eph
cells
vector
nucleic acid
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PCT/EP2001/011252
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English (en)
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Georg Martiny-Baron
Jeanette Marjorie Wood
Gene Liau
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Novartis Ag
Novartis Pharma Gmbh
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Priority to AU2002212292A priority Critical patent/AU2002212292A1/en
Publication of WO2002026827A1 publication Critical patent/WO2002026827A1/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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • 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
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention relates in particular to isolated extracellular polypeptides of Eph B receptors and ephrin B ligands and the corresponding isolated nucleic acid molecules comprising the sequences coding for such polypeptides, to recombinant vectors, such as viral vectors, that comprise such nucleic acid molecules, to host cells comprising such recombinant vectors, to pharmaceutical compositions comprising such polypeptides, nucleic acid molecules or recombinant vectors, and to the use of such vectors in the production of pharmaceutical compositions for treating a mammalian disease, including a human disease especially tumour diseases and ocular neovascularization, by means of gene therapy.
  • recombinant vectors such as viral vectors, that comprise such nucleic acid molecules
  • host cells comprising such recombinant vectors
  • pharmaceutical compositions comprising such polypeptides, nucleic acid molecules or recombinant vectors, and to the use of such vectors in the production of pharmaceutical compositions for treating a
  • the extracellular domain consists of a N-terminal globular domain, a cystein-rich domain and two fibronectin type III domains [Himanen et al., Nature 396, 486- 491 (1998)].
  • Eph A A1-A8
  • Eph B B1-B6
  • ephrin A ligands (A1 -A5) are inserted in the membrane by a glycosyl phosphatidyl inositol anchor (GPI), and ephrin B ligands (B1-B3) have a transmembrane domain and a cytoplasmatic protein section consisting of 82-88 amino acids [Eph Nomenclature Committee, Cell 90, 403-404 (1997); Beckmann et al., EMBO J. 13, 3757-3762 (1994)].
  • GPI glycosyl phosphatidyl inositol anchor
  • Ephrins of the A class bind predominantly to Eph A receptors and ephrins of the B type to Eph B receptors. Within the classes, however, the ligands have overlapping specifities. Proteins of the Eph/ephrin family play a role in cell and axon orientation. In contrast to chemoa -tractive substances, Eph/ephrin interactions in the neuronal and epidermal tissues lead to the production of repulsive signals between the cell populations, which express the corresponding receptors and ligands.
  • mice whose genes from the Eph/ephrin family have been specifically inactivated, it could be shown that various Eph/ephrins, in particular from the B class, take part in the development of neural crest and in the development of various regions of the brain. Eph/ephrins are thereby made responsible in particular for the complex topography of neuronal systems, which is controlled by direct cell interaction [Brambilla R. and Klein R., Mol. Cell. Biol. 15, 4921-4929 (1995); Henkemeyer et al., Cell 86, 35-46 (1996)].
  • Eph B receptors and ephrin B ligands also take part in the cell communication between endothelial cells and the surrounding mesenchymal cells.
  • Mice which cannot express Eph B2 and Eph B3 die in utero after about 11.5 days.
  • Analysis of the embryos showed that these animals have defects in the reorganisation of the embryonal vascular system, and exhibited disrupted cell contact between the mesenchymal pericytes and the endodermal endothelial cells [Adams et al., Genes and Dev. 13, 295-306 (1999)].
  • Eph B receptors and ephrin B ligands All previous work on the function of Eph B receptors and ephrin B ligands was, however, restricted to the embryonal development of the nervous system and of the embryonal vascular system. Surprisingly, it has now been discovered for the first time that Eph B receptors and ephrin B ligands in an adult mammal organism participate functionally in angiogenesis. In addition, it can be shown for the first time that Eph B receptors and ephrin B ligands also take part in the pathophysiology of malignant diseases and that extracellular polypeptides of Eph B receptors and ephrin B ligands are suitable for treating tumour diseases.
  • polypeptides are also suitable for the treatment of ocular neovascularization. It was also surprisingly established that extracellular polypeptides of Eph B receptors and ephrin B ligands possess pharmacokinetic properties of the type which act not only locally but also systemically and have no evident toxic side effects on the organism of mammals.
  • soluble polypeptide indicates that the respective polypeptide is a non-membrane bound polypeptide which is secreted by mammalian cells which express it. If such a "soluble” polypeptide is expressed for example in cultured mammalian cells, said polypeptide can be found in the cell culture supernatant.
  • High affinity in connection with the binding of ephrin B ligands with Eph B receptors is understood to mean a dissociation constant below 500 nM, preferably in the range of 0.05 nM to 100 nM, more preferably 0.1 nM to 20 nM, the typical range of dissociation constants of ephrin B ligands to Eph B receptors.
  • the term "essentially similar" used in connection with amino acid sequences refers to amino acid sequence identities.
  • the extracellular regions of different Eph B receptors or ephrin B ligands within the same species have amino acid sequence identities of typically 40% and more among one another.
  • the extracellular regions of the same Eph B receptors or ephrin B ligands of different species typically have amino acid sequence identities of 80% and more.
  • a sequence must have at least 40%, preferably at least 75%, most preferably at least 90% amino acid sequence identity to the reference sequence, in order to be regarded as "essentially similar".
  • the invention relates to an isolated soluble polypeptide which comprises an amino acid sequence of an extracellular region of an Eph B receptor or an essentially similar amino acid sequence thereof, said isolated soluble polypeptide being capable of binding with high affinity to a mammalian ephrin B ligand.
  • the invention also relates to an isolated soluble polypeptide which comprises an amino acid sequence of an extracellular region of an ephrin B ligand or an essentially similar amino acid sequence thereof, said isolated soluble polypeptide being capable of binding with high affinity to a mammalian Eph B receptor.
  • the invention further relates especially to an isolated soluble polypeptide which comprises an amino acid sequence of an extracellular region of Eph B4 or an essentially similar amino acid sequence thereof, said isolated soluble polypeptide being capable of binding with high affinity to a mammalian ephrin B ligand.
  • the invention relates especially to an isolated soluble polypeptide which comprises an amino acid sequence of an extracellular region of ephrin B2 or an essentially similar amino acid sequence thereof, said isolated soluble polypeptide being capable of binding with high affinity to a mammalian Eph B receptor.
  • An especially preferred embodiment of the invention relates to an isolated soluble polypeptide comprising SEQ ID NO:2 [amino acids (aa) 1 -539 of SwissProt Database Accession No. P54761] or SEQ ID NO:4 (aa 1-539 of SwissProt Database Accession No. P54760), which stand for the murine and human extracellular region of Eph B4, respectively, or comprising an amino acid sequence which is essentially similar to SEQ ID NO:2 or SEQ ID NO:4, respectively, said isolated soluble polypeptide being capable of binding with high affinity to a mammalian ephrin B ligand.
  • a further especially preferred embodiment of the invention relates to an isolated soluble polypeptide which comprises SEQ ID NO:6 (aa 1-229 of SwissProt Database Accession No. P52800) or SEQ ID NO:8 (aa 1-229 of SwissProt Database Accession No. P52799), which stand for the murine and human extracellular region of ephrin B2, respectively, or comprising an amino acid sequence which is essentially similar to SEQ ID NO:6 or SEQ ID NO:8, respectively, said isolated soluble polypeptide being capable of binding with high affinity to a mammalian Eph B receptor.
  • the invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a polypeptide described in the preceding paragraphs i) - vi), especially such a polypeptide mentioned as being preferred, and to the use of such a polypeptide in particular in the production of a pharmaceutical composition for the treatment of a mammalian, including human disease, especially tumour diseases and ocular neovascularization.
  • Another preferred embodiment of the invention relates also to an isolated nucleic acid molecule comprising a nucleotide sequence that encodes a polypeptide described in the preceding paragraphs i) - vi), especially such a polypeptide mentioned as being preferred, to a pharmaceutical composition comprising such a nucleic acid molecule and to the use of such a nucleic acid molecule in particular in the production of a pharmaceutical composition for the treatment of a mammalian, including human disease, especially tumour diseases and ocular neovascularization.
  • the invention relates to a recombinant vector, especially a recombinant viral vector such as a recombinant adenoviral vector, comprising a nucleic acid molecule described in the preceding paragraph viii), especially such a nucleic acid molecule mentioned as being preferred, to a pharmaceutical composition comprising such a recombinant vector and to the use of such a recombinant vector in particular in the production of a pharmaceutical composition for the treatment of a mammalian, including human disease, especially tumour diseases and ocular neovascularization, by means of gene therapy.
  • a recombinant vector especially a recombinant viral vector such as a recombinant adenoviral vector, comprising a nucleic acid molecule described in the preceding paragraph viii), especially such a nucleic acid molecule mentioned as being preferred
  • a pharmaceutical composition comprising such a recombinant vector and to the use of such a recombinant vector in particular in
  • Another embodiment of the invention relates to a host cell comprising a recombinant vector as described in the preceding paragraph ix), especially such a recombinant vector mentioned as being preferred.
  • the present invention also relates to a method for the treatment of a tumour disease in a mammal, including human, comprising: effecting an increase, in a mammal in need of such treatment, of the in vivo concentration of a polypeptide described in the preceding paragraphs i) - vi), especially such a polypeptide mentioned as being preferred, to a tumour inhibiting effective amount, wherein said polypeptide has anti-tumour activity in vivo.
  • the present invention further also relates to a method for the treatment of ocular neovascularization in a mammal, including human, comprising: effecting an increase, in a mammal in need of such treatment, of the in vivo concentration of a polypeptide described in the preceding paragraphs i) - vi), especially such a polypeptide mentioned as being preferred, to an ocular neovascularization inhibiting effective amount, wherein said polypeptide has anti-ocular neovascularization activity in vivo.
  • the invention most preferably relates to the sequences SEQ ID NO:1-8, especially the human sequences SEQ ID NO:3 [nucleotides (nt) 86-1702 of GeneBank Database Accession No. U07695; nucleotide sequence coding for the extracellular domain of human Eph B4], SEQ ID NO:4, SEQ ID NO:7 (nt 8-694 of GeneBank Database Accession No. U81262; nucleotide sequence coding for the extracellular domain of human ephrin B2) and SEQ ID NO:8, and to their use according to the present invention.
  • polypeptides useful in the practice of the present invention i.e., the polypeptides described in paragraphs i) - vi) above; hereinafter also referred to as "polypeptides of the invention" can be delivered to an individual in need of treatment using conventional pharmaceutical formulations.
  • An additional embodiment of the invention relates to the administration of a pharmaceutical composition, in conjunction with a pharmaceutically acceptable carrier, for any of the therapeutic effects discussed above.
  • Such pharmaceutical compositions contain as active ingredients the polypeptides of the invention or anti-idotypic antibodies to these polypeptides or mimetics of these polypeptides, said active ingredients being administered alone or in combination with at least one other agent, such as a stabilizing agent, in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water.
  • the compositions may be administered to a patient alone, or in combination with other agents, drugs or hormones.
  • compositions encompassed by the invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-articular. intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.
  • these pharmaceutical compositions may contain suitable pharmaceutically-acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Further details on techniques for formulation and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton, Pa.).
  • compositions for oral use can be obtained through combination of active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • Suitable excipients are carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; gums including arabic and tragacanth; and proteins such as gelatin and collagen.
  • disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.
  • Dragee cores may be used in conjunction with suitable coatings, such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • suitable coatings such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification orto characterize the quantity of active compound, i.e., dosage.
  • compositions which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol.
  • Push-fit capsules can contain active ingredients mixed with a filler or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate, and, optionally, stabilizers.
  • the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers.
  • compositions suitable for parenteral administration may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiologically buffered saline.
  • Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • suspensions of the active compounds may be prepared as appropriate oily injection suspensions.
  • Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.
  • Non-lipid polycationic amino polymers may also be used for delivery.
  • the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • penetrants appropriate to the particular barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art.
  • compositions of the present invention may be manufactured in a manner that is known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee- making, emulsifying, encapsulating, entrapping, or lyophilizing processes.
  • the pharmaceutical composition may be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc.. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms.
  • the preferred preparation may be a lyophilized powder which may contain any or all of the following: 1-50 mM histidine, 0.1-2% sucrose, and 2-7% mannitol, at a pH range of 4.5 to 5.5, that is combined with buffer prior to use.
  • compositions After pharmaceutical compositions have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition.
  • labeling would include amount, frequency, and method of administration.
  • compositions suitable for use in the invention include compositions where the active ingredients are contained in an effective amount to achieve the intended purpose.
  • the determination of an effective dose is well within the capability of those skilled in the art.
  • a therapeutically effective dose of active agent can be estimated initially either in cell culture assays or in animal models, usually mice, rabbits, dogs, or pigs.
  • the animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
  • a therapeutically effective dose refers to that amount of active ingredient, for example the polypeptides of the invention, which ameliorates the symptoms or condition.
  • Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50.
  • Pharmaceutical compositions which exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use. The dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.
  • the exact dosage will be determined by the practitioner, in light of factors related to the subject that requires treatment. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Factors which may be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long-acting pharmaceutical compositions may be administered every 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate of the particular formulation.
  • Normal dosage amounts may vary from about 0.1 to about 20 mg/kg per day, preferably from 2.5 to 20 mg/kg per day, depending upon the route and method of administration.
  • Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art. Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc..
  • Various biodegradable and biocompatible polymeric matrices, including microcapsules, nanospheres, and implants, are useful in the practice of the present invention.
  • Microspheres are fine spherical particles containing active drugs. They are differentiated from nanospheres primarily by the size of the particle; microspheres have a diameter of less than approximately 1000 ⁇ m, while nanospheres are submicronic ( ⁇ 1 ⁇ m). Microsphere systems contain either homogeneous monolithic microspheres, in which the drug is dissolved or dispersed homogeneously throughout the polymer matrix, or reservoir-type microspheres, in which the drug is surrounded by the polymer matrix membrane shell. Monolithic and reservoir systems can also be combined. For instance, active drug can be dispersed within, or adsorbed onto, the polymer surface in a reservoir-type microsphere.
  • Biodegradable polymers can consist of either natural or synthetic materials that vary in purity.
  • Natural polymers include polypeptides and proteins (e.g., albumin, fibrinogen, gelatin, collagen), polysaccharides (e.g., hyaluronic acid, starch, chitosan), and virus envelopes and living cells (e.g., erythrocytes, fibroblasts, myoblasts).
  • Natural materials require cross-linking in the microencapsulation process, leading to the denaturation of the polymer and the embedded drug. As a result, synthetic polymers are most commonly used.
  • Frequently used synthetic polymers include poly(-hydroxy) acids such as polylactic acid (PLA), polyhydroxybutryic acid, and copoly (lactic/glycolic) acid (PLGA). These compounds are biocompatible, lack immunogenicity, and have physical properties that permit them to be easily shaped (to control the bioerosion rate).
  • PLA polylactic acid
  • PLGA copoly (lactic/glycolic) acid
  • Colloidal particulate carriers can also be used in the methods of the present invention for delivering the polypeptides of the invention.
  • Liposomes are the preferred colloidal vehicle, and are composed of a phospholipid bilayer that may act as a carrier for both hydrophilic and hydrophobic medications. Liposomes can be made from, e.g., neutral lipids, charged phospholipids, and cholesterol. The addition of an amphophilic polymer such as polyethylene glycol (PEG) onto the surface of a liposome can slow the clearance of liposomes.
  • PEG polyethylene glycol
  • PEGs are polymers comprised of repeating ethylene oxide subunits with two terminal hydroxyl groups that can be chemically activated.
  • PEG molecules come in a number of different configurations.
  • PEG chains include linear and branched structures in which one or more PEG chains are joined with linkers such as lysine or triazine.
  • PEGs may be attached, preferably covalently, to a polypeptide of the invention at a single site or at multiple sites.
  • branched-chain PEGs attach at single or fewer sites than do linear PEGs, branched PEGs may be less likely to interfere with the biologic activity of the native molecule than would the attachment of multiple small linear-chain PEGs, and so are preferred.
  • Pharmaceutical formulations suitable for oral administration of proteins are described, e.g., in U.S. Patents 5,008,114; 5,505,962; 5,641 ,515; 5,681 ,811 ; 5,700,486; 5,766,633; 5,792,451 ; 5,853,748; 5,972,387; 5,976,569; and 6,051 ,561.
  • polypeptides of the invention can also be delivered to an individual in need of treatment using gene therapy methods. Any of the methods for gene therapy available in the art can be used according to the present invention. Exemplary methods are described below.
  • the therapeutic comprises a nucleic acid encoding a polypeptide of the invention that is part of an expression vector that expresses a polypeptide of the invention or a chimeric protein thereof in a suitable host.
  • a nucleic acid has a promoter operably linked to the polypeptide coding region, the promoter being inducible or constitutive, and, optionally, tissue-specific.
  • a nucleic acid molecule is used in which the polypeptide coding sequences and any other desired sequences are flanked by regions that promote homologous recombination at a desired site in the genome, thus providing for intrachromosomal expression of the desired nucleic acid.
  • nucleic acid encoding a polypeptide of the invention may be either direct, in which case the patient is directly exposed to the nucleic acid or nucleic acid-carrying vector, or indirect, in which case, cells are first transformed with the nucleic acid in vitro, then transplanted into the patient.
  • direct in which case the patient is directly exposed to the nucleic acid or nucleic acid-carrying vector
  • indirect in which case, cells are first transformed with the nucleic acid in vitro, then transplanted into the patient.
  • a nucleic acid encoding a polypeptide of the invention is directly administered in vivo, where it is expressed to produce the encoded product.
  • This can be accomplished by any of numerous methods known in the art, e.g., by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g., by infection using a defective or attenuated retroviral or other viral vector (see, e.g., U.S. Pat. No.
  • Patents 5,166,320; 5,728,399; 5,874,297; and 6,030,954) (which can be used to target cell types specifically expressing the receptors), etc..
  • a nucleic acid-ligand complex can be formed in which the ligand comprises a f usogenic viral peptide to disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation.
  • the nucleic acid can be targeted in v/Vofor cell specific uptake and expression, by targeting a specific receptor (see, e.g., PCT Publications WO 92/06180; WO 92/22635; WO9220316; WO93/14188; and WO 93/20221).
  • the nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination (see, e.g., U.S. Patents 5,413,923; 5,416,260; and 5,574,205).
  • a viral vector that contains a nucleic acid encoding a polypeptide of the invention is used.
  • a retroviral vector can be used (see, e.g., U.S. Patents 5,219,740; 5,604,090; and 5,834,182). These retroviral vectors have been modified to delete retroviral sequences that are not necessary for packaging of the viral genome and integration into host cell DNA.
  • a nucleic acid encoding a polypeptide of the invention to be used in gene therapy is cloned into the vector, which facilitates delivery of the gene into a patient.
  • Adenoviruses are another type of viral vector that can be used in gene therapy.
  • Adenovirus genomes are linear, double-stranded DNA molecules of approximately 36 kilobase pairs. Each extremity of the viral genome has a short sequence known as the inverted terminal repeat (or ITR), which is necessary for viral replication.
  • ITR inverted terminal repeat
  • the well-characterized molecular genetics of adenovirus render it an advantageous vector for gene transfer. Portions of the viral genome can be substituted with DNA of foreign origin.
  • recombinant adenoviruses are structurally stable and no rearranged viruses are observed after extensive amplification.
  • Adenoviruses are especially attractive vehicles for delivering genes to respiratory epithelia. Adenoviruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are liver cells, the central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Methods for conducting adenovirus-based gene therapy are described in e.g., U.S.
  • the nucleic acid to be introduced for purposes of gene therapy using an adenoviral vector comprises an inducible promoter operably linked to the coding region, such that expression of the nucleic acid is controllable by controlling the presence or absence of the appropriate inducer of transcription.
  • genomic elements may provide for enhanced expression of the DNA sequence encoding a polypeptide of the invention.
  • an adenoviral vector including at least one DNA sequence encoding a polypeptide of the invention, and at least one genomic element affecting the expression of such DNA sequence.
  • genomic element is used as previously defined. Such genomic elements include, but are not limited to, introns, the 5' untranslated region, and the 3' untranslated region, and portions of the introns and 3' and 5' untranslated regions.
  • the adenoviral vector may be as herein described. Promoters which control the DNA sequence may be selected from those described herein and from those known in the art.
  • the vector consisting of infectious, but replication-defective, viral particles, which contain at least one DNA sequence encoding a polypeptide of the invention, is administered in vivo to a host in an amount effective to treat neovascularization or a tumour disease in the host.
  • the host may be a mammalian host, including human and non-human primate hosts.
  • a viral vector can be administered in an amount of from about 10 8 plaque forming units (pfu) to about 10 14 plaque forming units, preferably from about 10 8 plaque forming units to about 10 11 plaque forming units, more preferably from about 10 9 plaque forming units to about 10 10 plaque forming units.
  • Adenoviral vectors in the quantities set out above, are preferred.
  • infectious vector particles can be administered systemically, such as, for example, by intravenous administration (such as, for example, via peripheral vein injection) or administered via the portal vein, to the bile duct, intramuscularly, intraperitoneally, or intranasally.
  • infectious vector particles can be administered locally, by, e.g, intraocular injection or injection at the site of a tumour.
  • Intraocular injection can be either into the anterior or posterior chamber of the eye, e.g., into the aqueous humor or vitreous humor.
  • the injection can be subretinal, e.g., by injection of aliquots (e.g., 1 to 10 microliters per aliquot) of vector-containing solution behind the retina, after which the solution is absorbed and the infectious vector particles infect local cells of the ocular tissues and produce the active polypeptide.
  • aliquots e.g., 1 to 10 microliters per aliquot
  • Such administration can comprise either a single injection, multiple injections administered on the same day, single injections administered over a period of weeks or months, or multiple injections administered over a period of weeks or months.
  • the vector particles may be administered in combination with a pharmaceutically acceptable carrier suitable for administration to a patient.
  • the carrier may be a liquid carrier (for example, a saline solution), or a solid carrier, such as, for example, mirocarrier beads.
  • Adeno-associated virus has also been proposed for use in gene therapy, including e.g. endostatin gene therapy for tumours (see, e.g., Nguyen et al., Cancer Research, 58, 5673-5677 (1998)).
  • Methods for producing and utilizing AAV are described, e.g., in U.S. Patents 5,173,414; 5,252,479; 5,552,311; 5,658,785; 5,763,416; 5,773,289; 5,843,742; 5,869,040; 5,942,496; and 5,948,675.
  • Lentiviral vectors are also preferred, especially bovine immunodeficiency viral vectors, such as those disclosed in PCT publication WO 01/44458.
  • Another approach to gene therapy involves transferring a gene to cells in tissue culture by such methods as electroporation, lipofection, calcium phosphate mediated transfection, or viral infection.
  • the method of transfer includes the transfer of a selectable marker to the cells.
  • the cells are then placed under selection to isolate those cells that have taken up and are expressing the transferred gene. Those cells are then delivered to a patient. ln this embodiment, the nucleic acid is introduced into a cell prior to administration in vivo of the resulting recombinant cell.
  • Such introduction can be carried out by any method known in the art, including but not limited to transfection, electroporation, microinjection, infection with a viral or bacteriophage vector containing the nucleotide sequences, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, etc..
  • Numerous techniques are known in the art for the introduction of foreign genes into cells and may be used in accordance with the present invention, provided that the necessary developmental and physiological functions of the recipient cells are not disrupted.
  • the technique should provide for the stable transfer of the nucleic acid to the cell, so that the nucleic acid is expressible by the cell and preferably heritable and expressible by its cell progeny.
  • cells producing a polypeptide of the invention are delivered in microencapsulated form, e.g., in the form of cells microencapsulated in sodium alginate or calcium alginate poly L-lysine alginate (see, e.g., Read et al., Nature Biotechnology 19, 29-34 (Jan 2001) and Joki etal., Nature Biotechnology 19, 35-39 (Jan 2001)).
  • the microencapsulated cells can be implanted proximally to the eye or the tumour, or at a site where a polypeptide of the invention produced by the cells will most quickly enter the subject's bloodstream, e.g., in the liver.
  • the amount of cells envisioned for use depends on the desired effect, patient state, etc., and can be determined by one skilled in the art.
  • Cells into which a nucleic acid can be introduced for purposes of gene therapy encompass any desired, available cell type, and include but are not limited to epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or progenitor cells, in particular hematopoietic stem or progenitor cells, e.g., as obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver, etc.
  • the cell used for gene therapy is autologous to the patient.
  • a nucleic acid encoding a polypeptide of the invention is introduced into the cells such that it is expressible by the cells or their progeny, and the recombinant cells are then administered in vivo for therapeutic effect.
  • stem or progenitor cells are used. Any stem-and/or progenitor cells which can be isolated and maintained in vitro can potentially be used in accordance with this embodiment of the present invention.
  • Such stem cells include but are not limited to hematopoietic stem cells (HSCs), stem cells of epithelial tissues such as the skin and the lining of the gut, embryonic heart muscle cells, liver stem cells (see, e.g., WO 94/08598), and neural stem cells.
  • HSCs hematopoietic stem cells
  • stem cells of epithelial tissues such as the skin and the lining of the gut
  • embryonic heart muscle cells embryonic heart muscle cells
  • liver stem cells see, e.g., WO 94/08598
  • neural stem cells neural stem cells.
  • Epithelial stem cells (ESCs) or keratinocytes can be obtained from tissues such as the skin and the lining of the gut by known procedures. In stratified epithelial tissue such as the skin, renewal occurs by mitosis of stem cells within the germinal layer, the layer closest to the basal lamina. Stem cells within the lining of the gut provide for a rapid renewal rate of this tissue.
  • ESCs or keratinocytes obtained from the skin or lining of the gut of a patient or donor can be grown in tissue culture. If the ESCs are provided by a donor, a method for suppression of host versus graft reactivity (e.g., irradiation, drug or antibody administration to promote moderate immunosuppression) can also be used.
  • HSCs any technique which provides for the isolation, propagation, and maintenance in vitro of HSCs can be used in this embodiment of the invention.
  • Techniques by which this may be accomplished include (a) the isolation and establishment of HSC cultures from bone marrow cells isolated from the future host, or a donor, or (b) the use of previously established long-term HSC cultures, which may be allogeneic or xenogeneic.
  • Non-autologous HSCs are used preferably in conjunction with a method of suppressing transplantation immune reactions of the future host/patient.
  • human bone marrow cells can be obtained from the posterior iliac crest by needle aspiration (see, e.g., Kodo et al., 1984, J.
  • HSCs can be made highly enriched or in substantially pure form. This enrichment can be accomplished before, during, or after long-term culturing, and can be done by any techniques known in the art.
  • Long-term cultures of bone marrow cells can be established and maintained by using, for example, modified Dexter cell culture techniques (Dexter et al., 1977, J. Cell Physiol. 91 :335) or Witlock-Witte culture techniques (Witlock and Witte, 1982, Proc. Natl. Acad. Sci. USA 79:3608-3612). It is understood, however, that the scope of the present invention is not to be limited to the specific embodiments described above. The invention may be practiced other than as particularly described and still be within the scope of the accompanying claims.
  • the pharmacological useful properties of the polypeptides of the present invention as therapeutics against tumour diseases and ocular neovascularization can be demonstrated for example by using known animal models for said diseases, such as those described e.g. in the Examples below, and applying the polypeptides, nucleic acid molecules or recombinant vectors of the present invention in these models.
  • Fig. 1 Growth of A375 tumours in the flanks of immunosuppressive mice, measured as tumour weight 14 days after application of tumour cells.
  • Control Tumours which develop within 14 days after application of 1x10 6 A375 tumour cells, that are stably transfected with the empty pCDNA3 vector.
  • s-EphB4 Tumours which develop within 14 days after application of 1x10 6 A375 tumour cells, which are stably transfected with the pCDNA3 vector containing the coding sequence for the extracellular domains of the Eph B4 receptor and which secrete the soluble s-EphB4 polypeptide.
  • s-ephrinB2 Tumours which develop within 14 days after application of 1x10 6 A375 tumour cells, which are stably transfected with the pCDNA3 vector containing the coding sequence for the extracellular domains of the ephrin B2 ligand and which secrete the soluble s-ephrinB2 polypeptide.
  • Fig. 2 Growth of A375 tumours in the flanks of immunosuppressive mice.
  • Immunosuppressive mice are injected either in both flanks with A375 control tumour cells (animal group 1), or are injected in both flanks with A375 tumour cells which secrete s-EphB4 (aminal group 2), or are injected in the left flank with A375 control tumour cells and in the right flank with A375 tumour cells which secrete s-EphB4 (aminal group 3).
  • the figure shows the tumour weight after 14 days (A) and the growth of the tumours (B).
  • the tumour volume of tumours from both flanks is determined. Examples:
  • Example 1 Inhibition of tumour angiogenesis and tumour growth by expression of the soluble extracellular domains of murine Eph B4 (murine s-EphB4) or ephrin B2 (murine s- ephrinB2) in tumour cells.
  • L38847 are amplified by means of sequence-specific oligonucleotide primers and Reverse Transcription Polymerase Chain Reaction (RT PCR) from isolated total RNA from mouse cerebral tissue or mouse kidney tissue, and modified with corresponding restriction cleavage sites as well as a stop codon.
  • the amplified DNA is cloned by means of Hindlll and BamHI into the vector pCDNA3 (Invitrogen) and the accuracy of the sequence is checked.
  • the pCDNA3 vector contains the cytomegalovirus (CMV) promoter region and the G418 resistance gene. With this vector, the corresponding polypeptides s-EphB4 and s-ephrinB2 can be constitutively expressed in mammal cells under the control of the CMV promoter.
  • CMV cytomegalovirus
  • the recombinant vectors with the specific murine s-EphB4 and s-ephrinB2 nucleotide sequences, as well as the empty vector, are transfected with lipofectin (Gibco, LifeScience) into A375 human melanoma cells (ATCC No.: CRL-1619). Stable cell clones are generated by selection with 0.5 mg/ml G418 (Gibco, LifeScience). The expression of s-EphB4 and s- ephrinB2 is verified by Northern analysis and by a ligand receptor binding test [to this end, each 2 ml of conditioned tumour cell medium is concentrated by about five times (Amicon, Zentriprap).
  • 0.1 ml of concentrate is applied to a nitrocellulose membrane, and the membrane is subsequently incubated for one hour at room temperature in Tris-buffered saline (TBS) with 1% BSA.
  • TBS Tris-buffered saline
  • s-ephrinB2 is detected by incubation with biotinilated s-EphB4- Fc fusion protein (1 ⁇ g/ml; R&D Systems, Minneapolis, MN) and streptavidin coupled alkaline phosphatase.
  • s-EphB4 is detected by incubation with biotinilated s-ephrinB2-Fc fusion protein (1 ⁇ g/ml; R&D Systems, Minneapolis, MN) and streptavidin coupled alkaline phosphatase.
  • the transfected cell clones did not show differing growth kinetics (for the proliferation test, 8000 cells were seeded in a 24-well cell culture plate and the cell count was determined after 24, 48, 72 and 96 hours).
  • tumour growth and tumour angiogenesis 1x10 6 transfected A375 tumour cells are each injected intradermally into one or both flanks of immunosuppressive mice. Tumour growth is monitored over 14 days. After 14 days, the test animals are killed, the tumours prepared, weighed and examined histologically and by means of immunohistochemistry.
  • tumour cells Animals which are injected with tumour cells (A375), which constitutively secrete either s-
  • EphB4 or s-ephrinB2 develop considerably smaller tumours than animals which are injected with control cells, which are transfected with the empty vector (Fig. 1).
  • the tumours which are formed by A375 tumour cells and constitutively secrete either s-EphB4 or s- ephrinB2, have a disordered vessel structure compared to control tumours.
  • tumours Animals that are injected with control tumour cells in both flanks develop tumours of equal size on both flanks. The kinetics of tumour growth is unchanged on both flanks ⁇ for the kinetics of tumour growth, the tumour volume (greatest diameter and perpendicular on this diameter) is measured by a slide gauge. The volume is determined by the factor
  • tumour diameter [mm] x perpendicular 2 [mm 2 ] ⁇ Animals which are injected in both flanks with s-EphB4 secreting tumour cells, develop tumours of equal size on both sides, but the tumour size is significantly smaller than that of the corresponding control tumours.
  • tumour growth is unchanged on both flanks.
  • Example 2 Therapeutical application of recombinant s-EphB4 (SEQ ID NO:2) and s- ephrinB2 (SEQ ID NO:6..
  • the cDNA sequences coding for the soluble extracellular domains of the murine Eph B4 receptor (SEQ ID NO:1) or for those of the murine ephrin B2 ligand (SEQ ID NO:5), are cloned into a eukaryotic expression vector (for example pCDNA3, see also Example 1 ).
  • a eukaryotic expression vector for example pCDNA3, see also Example 1 .
  • "Chinese Hamster Ovarian" (CHO) cells are transfected with these recombinant vectors and stable cell clones are isolated by selection markers.
  • the expression levels of secreted soluble s-EphB4 protein and s-ephrinB2 protein from stably transfected CHO cell clones are determined by Western Blot Analysis (anti-EphB4 antibodies, Santa Cruz Ltd; anti-EphrinB2 antibodies, R&D Systems, Minneapolis, MN). Functionality is tested in a binding test, in which cell supernatants of stably transfected CHO cells are applied to a nitrocellulose membrane, the membrane is subsequently incubated for one hour at room temperature in TBS with 1% BSA.
  • s-ephrinB2 is detected by incubation with biotinilated s-EphB4-Fc fusion protein (1 ⁇ g/ml, R&D Systems, Minneapolis, MN) and streptavidin coupled alkaline phosphatase.
  • s-EphB4 is detected by incubation with biotinilated s-ephrinB2-Fc fusion protein (1 ⁇ g/ml, R&D Systems, Minneapolis, MN) and strepdavidin coupled alkaline phosphatase.
  • Evidence of binding is found by enzymatic detection of the alkaline phosphatase.
  • CHO cell clones which noticeably secrete functionally active s-EphB4 protein or s-ephrinB2 protein are fermented and the recombinant proteins from the cell supernatant are purified. Purification is carried out by column chromatography with ConcavalinA affinity chromatography (Amersham/Pharmacia) followed by DEAE sepharose (Amersham/Pharmacia). Separation according to size is then carried out by gel filtration. The purified protein is dissolved in "Hanks buffered salt solution" (HBSS), sterile-filtered and stored in lyophilised state. Directly before therapeutical usage, the protein is dissolved in e.g. sterile water in a concentration of 2 mg/ml.
  • HBSS "Hanks buffered salt solution”
  • A375 tumours are induced by intradermal injection of 1x10 6 A375 melanoma cells into the flanks of immunosuppressive mice. Animals which develop a readily tangible tumour are treated by daily intraperitoneal injection of 0.1 mg/animal of recombinant protein and compared with tumour-bearing control animals which are given the same intraperitoneal volume of HBSS. The success of therapy is determined by the tumour growth and histochemical and immunohistochemical characterization.
  • tumour-bearing, immunosuppressive mice which receive injections of recombinant, purified s-EphB4 polypeptid or s-ephrinB2 polypeptid, develop considerably smaller tumours with disorderd vessel structure compared to control animals which are given corresponding volumes of HBSS.
  • Example 3 Generation and characterization of an adenoviral vector containing the DNA encoding for the soluble extracellular fragment of the human Eph B4 receptor.
  • adenoviral vector expressing the extracellular domain of human Eph B4 receptor (human s-EphB4):
  • the cDNA encoding the extracellular domain of human Eph B4 is PCR-amplified from human fetus whole Marathon-Ready cDNA (Clonetech Laboratories, Inc. Palo Alto, CA) with the primers; sense (5'- ATC ACT GCT AGC AAC TCA GTT CGG ATC CTA CC-3') and antisense (5'- ATC ATA GCT AGC TCA CTG CTC CCG CCA GCC CTC GC -3').
  • the PCR amplifies the region encoding the first 537 amino acids coding region of human Eph B4 (aa 1-537 of SEQ ID NO:4), including the signal peptide (the first 15 amino acids) and extracellular region of the mature Eph B4 receptor protein (16 to 537 amino acids).
  • PCR is carried out with Pfu DNA polymerase (Stratagene, La Jolla, CA) in the presence of 1 ⁇ M primers as described, 0.4 mM dNTP, 1.5 mM MgCI 2 , and 1 ng cDNA.
  • the reaction is run for 35 cycles with the following conditions: 94 °C hot start for 1 min, 95 °C denaturation for 1 min, 62.5 to 67.5 °C with 0.5 °C thermal gradient annealing for 1 min, 72 °C extension for 3 min, and ended at 72 °C extension for 10 min.
  • the 1674 base pairs (bp) s-EphB4 containing DNA fragments are pooled together, gel-purified and subcloned into pPCR-script plasmid according to the manufacturer's procedure (Stratagene, La Jolla, CA).
  • the Nhe1 digested s-EphB4 DNA fragment is gel purified and cloned into the Spe1 site of the adenoviral shuttle plasmid, pAvsCvXlx [this vector contains the left end of the adenoviral vector including the left inverted terminal repeat (ITR), a CMV promoter, an adenovirus major late promoter tripartite leader sequence, a multiple cloning site for inserting the transgene of interest, a SV40 early polyadenylation signal, and a loxP site], downstream of the simian CMV promoter and upstream of the SV40 polyadenylation signal to generate pAvCshs- EphB4-C16.
  • the DNA sequence of the entire PCR-amplified region of the pAvCshs-EphB4- C16 is confirmed by sequence analysis by the Gene Therapy Core Technologies Molecular Core Laboratory.
  • the recombinant adenoviral vector Av3Cshs-EphB4 (with E1 , E2a, and E3-deletions) is generated in the following manner.
  • the pAvCshs-EphB4-C16 and pSQ3 (a 31 '574 bp plasmid vector containing the right hand portion of the adenoviral vector genome and a loxP site that allows Cre-mediated recombination with pAvCshs-EphB4-C16) are first linearized with Notl and Clal restriction enzymes, respectively.
  • S8 cells are derived from A549 cells (ATCC No. CCL-185) that have been transfected with plasmids containing the E1 and E2a regions, respectively, of the adenoviral vector (see also: Gorziglia et al., J. Virology 70, 4173-4178, 1996] per well using the LipofectAMINE PLUS reagent (Life Technologies, Rockville, MD) according to the manufacturer's procedure.
  • the cells are cultured with media in the presence of 0.3 ⁇ M of dexamethasone the day before transfection.
  • the DNA-PLUS is prepared with 0.5 ⁇ g of linearized pAvCshs-EphB4-C16, 1 ⁇ g of linearized pSQ3 and 0.5 ⁇ g of the expression plasmid pC-Cre3.1 encoding the Cre recombinase, in a 100- ⁇ l Optimen (Life Technologies) with 6- ⁇ l of PLUS reagent. After 15 min of incubation, the DNA-PLUS is mixed with 0.8-ml of 1 to 200-fold diluted lipofectAMINE Reagent. The DNA-PLUS- LipofectAMINE complex is added to the cells and incubated for 3 hours. The medium is then replenished with fresh medium.
  • CPE cytopathic effect
  • the cells and the medium are harvested by scraping.
  • a crude viral lysate (CVL) is prepared from 1 ml resuspended cell pellet by five cycles of freezing and thawing.
  • the Av3Cshs-EphB4 vector is re-amplified in S8 cells with 0.3 ⁇ M dexamethasone in Richter's CM medium containing 5% FBS until CPE was observed.
  • the CVL is plaque- purified.
  • the harvested single plaque free of replication competent adenovirus (RCA) is selected to generate purified vector.
  • the research seedlot purified Av3Cshs-EphB4 vector is scaled up by the GTI Gene Therapy Core Technologies.
  • the adenoviral vector titer (particles/ml) and biological titer (pfu/ml) are determined as described (Mittereder, N., March, K.L., and Trapnell, B.C. (1996) J. Viro. 70, 7498-7509) by GTI Gene Therapy Core Technologies.
  • the ratio of total particles to infectious particles is calculated.
  • Genomic DNA is prepared from the purified Av3Cshs-EphB4 vector using DNA isolation kit (Gentra Systems, Minneapolis, MN) according to the manufacturer's procedure.
  • DNA isolation kit Genetra Systems, Minneapolis, MN
  • Various restriction digests by BamHI, Xba1 , and Xhol of the purified viral DNA of Av3Cshs-EphB4 are compared on 1.0% agarose-TAE gel and stained with ethidium bromide to visulize the various digests for the verification of the correct genome structure and purity.
  • Hep3B (ATCC No. HB-8064) and A549 cells are transduced with Av3Cshs-EphB4, or the control Av3CsNull (same genome structure with no transgene in the expression cassette) vectors at the particles to cell ratio of 750. 72 hours after transduction, the cell pellets are harvested. Total RNA is isolated using the RNA-DNA STAT 60 (Tel-Test) extraction method. Northern blot analysis is carried out according the standard procedure. A 20 ⁇ g aliquot of total RNA is analyzed on 1 % agarose formaldehyde/MOPS gel.
  • RNA is transferred to a nylon membrane and prehydridized in the solution of 6.75% SDS, 0.5M NaPO 4 , pH 7.0, 1.0 mM EDTA, pH 8.0, 5 ⁇ g/ml BSA, and 100 ⁇ g/ml sheared, denatured salmon sperm DNA.
  • the probe of 1368 bp s-EphB4 internal fragment is generated by EcoN1 digestion, gel purified and 3 P-labeled.
  • the membrane is hybridized with a [ 32 P]-labeled s-EphB4 internal probe at 68 °C overnight and washed in SSC/SDS containing buffers at 68 °C following the standard protocol.
  • the membrane is exposed to an x-ray film at -70 °C overnight.
  • the membrane is reprobed with [ 32 P]-labeled Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) internal probe to verify equal amount of RNA loaded on each lane.
  • GPDH Glyceraldehyde-3-phosphate dehydrogenase
  • S8 and Hep3B cells are transduced with Av3Cshs-EphB4 or the control, Av3CsNull vector at the particles to cells ratio of 100 and 500, respectively.
  • 72 hours post transduction the conditioned media are collected and subjected to SDS-PAGE analyses.
  • the resolved protein bands are then electrotransferred to PVDF membrane using the Mini Tans-blot Electrophoretic Transfer Cell (Cat# 165-3165, Bio-Rad).
  • Western blot analyses are carried out according to the following procedure.
  • the membrane blot is blocked with blocking buffer (5% powdered milk, 0.1% Tween-20 and PBS buffer) for 30 min, probed with 1 :1000 diluted rabbit anti-EphB4 antiserum (generated at GTI through outside service by Affinity Bioreagents Inc., Golden, CO) in blocking buffer at 4 °C overnight, probed with 1 :5000 diluted Horse Radish Peroxidase (HRP)-conjugated anti-rabbit IgG antibody in TPBS (0.1% Tween-20 and PBS buffer) (Amersham pharmacia Biotech) at 4°C for 1 hour and developed in chemiluminescence detection mixture (ECL kit, Ammersham Pharmacia Biotech) for 1 min followed by x-ray film exposure.
  • blocking buffer 5% powdered milk, 0.1% Tween-20 and PBS buffer
  • 1 diluted rabbit anti-EphB4 antiserum generated at GTI through outside service by Affinity Bioreagents Inc., Golden, CO
  • HRP Horse Rad
  • Polyclonal anti-EphB4 antiserum is generated according to the following procedure.
  • the rabbits are immunized with KLH conjugated synthetic peptide of (C)DGQWEELSGLDEEQH (amino acids 39-43 of internal region of human Eph B4) followed by two boost injections every other month.
  • Sera are collected and confirmed with reactivity directed against peptide antigen by ELISA and against Eph B4 protein by Western blot using purified recombinant Mouse EphB4/Fc chimera protein (Cat#446-B4, R&D Systems, Minneapolis, MN).
  • mouse EphB4/Fc chimera (Cat#446-B4-200, R&D Systems) is immobilized on a 96-well Nunc- ImmunoTM plate with polySorpTM surface (Fisher) at 4°C overnight. After 4 washes with TPBS (0.5% Tween-20 in DPBS), the plate is blocked with 10% FBS in DPBS for 30 min followed by 6 washes with TPBS.
  • the colored product is determined by absorbance at 415 nm by an ELISA reader.
  • 100 ng/ml (2.1 nM) of biotinylated mouse ephrinB2/Fc chimera is incubated for 30 min to 1 hour with increasing amounts of conditioned medium from untreated control S8 cells or S8 cells transduced with either null vector or s-EphB4 expressing vector prior to incubation of the mixture with immobilized EphB4/Fc.
  • Data is analyzed using Prism nonlinear regression curve fit to determine the best-fit curve and Kd, concentration of ligand required to reach half-maximal binding.
  • Liquid Matrigel (a sterile extract of basement membrane proteins) is obtained from Fisher Scientific, Part number CB40234C, mixed with test substances and preloaded into a 1.0 ml tuberulin syringes with G27 needles.
  • Male nude mice (Harlan) weighing about 20 g are anesthesized and 0.45 ml of the undiluted Matrigel alone or the undiluted Matrigel containing 1 ⁇ g/ml of FGF-2 (Fibroblast Growth Factor 2) or 3 ⁇ g/ml of VEGF (Vascular Endothelial Growth Factor; R&D Systems, Minneapolis, Minnesota) and 5 x 10 5 S8 cells transduced at 100 MOI (multiplicity of infection) with either null vector or s-EphB4 expressing vector are injected subcutaneously into the dorsal caudal portion of the midline (1 injection/animal) using a G27 needle.
  • FGF-2 Fibroblast Growth Factor 2
  • VEGF Vascular End
  • VEGF-induced angiogenesis 20 U/ml heparin is also included.
  • the Matrigel will rapidly form a solid gel that persists for over 7 days. On day 7, all animals are euthanized by CO 2 and Matrigel plugs are harvested for histological analysis and hemoglobin measurement.
  • the gene therapy vector, Av3Cshs-EphB4 mediates s-EphB4 expression and secretion:
  • Northern blotting analysis indicates that Av3Cshs-EphB4 transduced Hep3B and A549 cells express a 1.8 Kb mRNA transcript that specifically hybridizes to the Eph B4 cDNA probe.
  • Western blot analysis also reveals a band with an apparent molecular mass of 85 kDa. This band is not observed in cells transduced with Av3CsNull or in untreated cells.
  • the predicted molecular mass of the expressed s-EphB4 protein is considerably smaller (57 kDa), suggesting that at least one of the three potential glycosylation sites is glycosylated.
  • conditioned media prepared from Av3Cshs-EphB4 transduced S8 substantially inhibit the binding in a dose-dependent manner.
  • Adenoviral vector expressing the extracellular domain of Eph B4 can inhibit both VEGF and FGF-2 directed angiogenesis:
  • VEGF and FGF-2 are both able to substantially induce angiogenesis as measured by the increase in hemoglobin content in the Matrigel plug.
  • FGF-2 is a more potent inducer of angiogenesis in this model (Table 2). Histological analysis reveals that the increase in hemoglobin content correlates with an increase in the number of blood vessels.
  • the addition of Av3Cshs-EphB4 transduced cells substantially inhibits both VEGF- and FGF-2-induced angiogenesis.
  • Example 4 Application of Av3Csms-EphB4 or Av3Csms-ephrinB2 vector in a mouse tumour model.
  • the recombinant adenoviral vectors Av3Csms-EphB4 and Av3CshsephrinB2 which contain the DNA encoding the soluble extracellular fragment of murine EphB4 and ephrinB2, respectively, are prepared analogously to the preparation of Av3Cshs-EphB4 as described in Example 3 above using the murine s-EphB4 and s-ephrinB4 cDNAs, respectively, instead of the human s-EphB4 cDNA.
  • A375 tumours are induced by intradermal injection of 1x10 6 A375 melanoma cells into the flanks of immunosuppressive mice. Animals which develop a readily tangible tumour are treated by intravenous injection of Av3Csms-EphB4 or Av3CshsephrinB2 (1x10 8 "plaque forming units" (pfu)/animal - 1x10 9 pfu/animal) and compared with tumour-bearing control animals which are treated with recombinant control adenoviruses (same genome structure with no transgene in the expression cassette) vectors. The success of therapy is determined by the tumour growth. The resulting tumours are further examined by histochemistry and immunohistochemistry.
  • A375 tumour-bearing, immunosuppressive mice which receive recombinant adenoviruses, which contain either the coding sequence for the extracellular domains of the murine Eph

Abstract

L'invention concerne des polypeptides extracellulaires isolés de récepteurs Eph B et de ligands éphrine B, ainsi que les molécules d'acide nucléique isolées correspondantes comprenant les séquences codant pour de tels polypeptides. L'invention concerne également des vecteurs de recombinaison, tels que des vecteurs viraux, qui comprennent ces molécules d'acide nucléique. L'invention concerne aussi des cellules hôtes comprenant de tels vecteurs de recombinaison, ainsi que des compositions pharmaceutiques contenant ces polypeptides, molécules d'acide nucléique ou vecteurs de recombinaison. L'invention concerne enfin l'utilisation de ces vecteurs dans la production de compositions pharmaceutiques destinées au traitement d'une maladie mammalienne, notamment une maladie humaine, plus particulièrement des maladies tumorales et la néovascularisation oculaire, au moyen de la thérapie génique.
PCT/EP2001/011252 2000-09-29 2001-09-28 Polypeptides extracellulaires de recpteurs eph b et ligands ephrine b, et molecules d'acide nucleiques correspondantes WO2002026827A1 (fr)

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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

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US7741272B2 (en) 1998-04-13 2010-06-22 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
US7595044B2 (en) 1998-04-13 2009-09-29 California Institute Of Technology Artery-and vein-specific proteins and uses therefor
WO2003004057A1 (fr) * 2001-07-03 2003-01-16 The Hospital For Sick Children Immunomodulation induite par les recepteurs ephrin et eph
WO2004024773A1 (fr) * 2002-09-16 2004-03-25 The Queen Elizabeth Hospital Research Foundation Inc. Procede destine a reguler le cancer
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WO2004069264A1 (fr) * 2003-02-07 2004-08-19 Ludwig Institute For Cancer Research Modulation a mediation eph/ephrine de l'adherence cellulaire et des metastases cellulaires tumorales
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EP1605961A4 (fr) * 2003-03-12 2009-11-11 Vasgene Therapeutics Inc Composes polypeptidiques permettant d'inhiber l'angiogenese et la croissance tumorale
US7381410B2 (en) 2003-03-12 2008-06-03 Vasgene Therapeutics, Inc. Polypeptide compounds for inhibiting angiogenesis and tumor growth
JP2007509611A (ja) * 2003-10-17 2007-04-19 アクティス バイオロジクス， インコーポレイテッド 癌を処置するためのｍｓｐ３６のレンチウイルスベクター送達
EP1526380A1 (fr) * 2003-10-21 2005-04-27 Progenika Biopharma, S.A. Méthode de diagnostic in vitro et de pronostic in vitro du cancer du pancréas et méthode pour le développement de médicaments contre le cancer du pancréas
JP2007516727A (ja) * 2003-12-31 2007-06-28 アクティス バイオロジクス， インコーポレイテッド 癌を処置するためのネイティブｉｇｆｂｐ−３および変異型ｉｇｆｂｐ−３のレンチウイルスベクター送達
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
WO2006006079A2 (fr) * 2004-04-05 2006-01-19 Aqumen Biopharmaceuticals K.K. Procedes permettant d'eliminer la neovascularisation au moyen de l'ephrine b2
WO2006006079A3 (fr) * 2004-04-05 2006-05-26 Aqumen Biopharmaceuticals K K Procedes permettant d'eliminer la neovascularisation au moyen de l'ephrine b2
WO2005113596A2 (fr) * 2004-05-14 2005-12-01 Receptor Biologix, Inc. Isoformes de recepteurs de la surface cellulaire et methodes d'identification et d'utilisation de ces derniers
WO2005113596A3 (fr) * 2004-05-14 2007-05-03 Receptor Biologix Inc Isoformes de recepteurs de la surface cellulaire et methodes d'identification et d'utilisation de ces derniers
US9533026B2 (en) 2004-09-23 2017-01-03 Vasgene Therapeutics, Inc Polypeptide compounds for inhibiting angiogenesis and tumor growth
AU2005286662B2 (en) * 2004-09-23 2011-10-06 Vasgene Therapeutics, Inc. Polypeptide compounds for inhibiting angiogenesis and tumor growth
WO2006034455A3 (fr) * 2004-09-23 2006-07-20 Vasgene Therapeutics Inc Composes de polypeptides pour l'inhibition de l'angiogenese et de croissance tumorale
EP2301963A1 (fr) * 2004-09-23 2011-03-30 Vasgene Therapeutics, Inc. Composés de polypeptides pour l'inhibition de l'angiogénèse et de croissance tumorale
JP2008514618A (ja) * 2004-09-23 2008-05-08 バスジーン セラピューティクス， インコーポレイテッド 血管形成及び腫瘍増殖阻害用ポリペプチド化合物
WO2006081418A2 (fr) * 2005-01-27 2006-08-03 The Burnham Institute Peptides de liaison aux recepteurs ephb
US7999069B2 (en) 2005-01-27 2011-08-16 Sanford-Burnham Medical Research Institute EphB receptor-binding peptides
AU2006208068B2 (en) * 2005-01-27 2011-08-04 The Burnham Institute EphB receptor-binding peptides
US7582438B2 (en) 2005-01-27 2009-09-01 Burnham Institute For Medical Research EphB receptor-binding peptides
JP2008528614A (ja) * 2005-01-27 2008-07-31 ザ・バーンハム・インスティテュート ＥｐｈＢ受容体結合ペプチド
WO2006081418A3 (fr) * 2005-01-27 2006-12-07 Burnham Inst Peptides de liaison aux recepteurs ephb
WO2007005244A1 (fr) * 2005-07-05 2007-01-11 The Regents Of The University Of California Recepteur de l'henipavirus et ses utilisations
WO2007043629A1 (fr) * 2005-10-05 2007-04-19 Aqumen Biopharmaceuticals K.K. Procédé d’inhibition de l’angiogenèse à l’aide d’éphrine b2
WO2007135781A1 (fr) * 2006-05-24 2007-11-29 Aqumen Biopharmaceuticals K.K. Peptide capable d'accroître l'activité de l'éphrine-b2, sel de celui-ci, composition à des fins médicales et kit de thérapie
US8975377B2 (en) 2007-08-13 2015-03-10 Vasgene Therapeutics, Inc Cancer treatment using humanized antibodies that bind to EphB4

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