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WO1998018914A1 - Recepteur tie2 soluble - Google Patents

Recepteur tie2 soluble Download PDF

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Publication number
WO1998018914A1
WO1998018914A1 PCT/US1997/019597 US9719597W WO9818914A1 WO 1998018914 A1 WO1998018914 A1 WO 1998018914A1 US 9719597 W US9719597 W US 9719597W WO 9818914 A1 WO9818914 A1 WO 9818914A1
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WIPO (PCT)
Prior art keywords
tumor
6his
protein
fusion protein
exflk
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PCT/US1997/019597
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English (en)
Inventor
Kevin G. Peters
Charles Lin
Prema S. Rao
Mark W. Dewhirst
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Duke University
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Priority to AU51540/98A priority Critical patent/AU5154098A/en
Publication of WO1998018914A1 publication Critical patent/WO1998018914A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/022—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from an adenovirus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • the present invention relates to a soluble receptor endothelium specific and to the use thereof as an antiangiogenic agent.
  • Angiogenesis the formation of new capillaries from pre-existing blood vessels, is a fundamental process required for both normal embryonic development and the development of pathologic conditions such as cancer (Folkman, J. Natl. Cancer Inst. 82:4-6 (1990), Folkman et al, J. Biol. Chem. 267:10931-10934 (1992)).
  • Tumor growth is an angiogenesis-dependent process that requires stimulation of new vessel growth (Viglietto et al, Oncogene 1 1 :1569- 1579 (1995), Kandel et al, Cell 66:1095-1104 (1991)).
  • Tumor angiogenesis is most likely mediated by growth factors produced by tumor cells and/or by tumor infiltrating inflammatory cells such as macrophages or mast cells (Folkman et al, J. Biol. Chem. 267:10931-10934 (1992), Sunderkotter et al, J. Leukoc. Biol. 55:410-422 (1994)). These factors stimulate the proliferation, migration and morphogenesis of endothelial cells as a result of interaction with specific cell surface receptors. Although many factors likely contribute to vascular growth in tumors, VEGF (vascular endothelial growth factor) is currently the best candidate for an endogenous mediator of vascular growth (Connolly, J. Cell Biochem.
  • VEGF and VEGF receptors A role for VEGF and VEGF receptors in tumor angiogenesis is supported by a growing number of reports demonstrating expression of VEGF and VEGF receptors in a number of different tumor types
  • Tie2 (a.k.a. Tek) was identified (Dumont et al, Oncogene 8:1293-1301 (1993), Iwama et al, Res. Comm. 195:301 (1993)). Tie2 was expressed predominantly in endothelial cell precursors (angioblasts) and in endothelial cells participating in angiogenesis (Schnurch et al, Development 119:957-968 (1993), Dumont et al, Dev. Dynamics 203:80-92 (1995)).
  • Tie2 function in transgenic mice resulted in embryonic lethality at day 8.5 due to defects in vascular development characterized by a reduction in endothelial cell number and a defect in the formation of microvessels (Sato et al, Nature 376:70-74 (1995), Dumont et al, Genes and Development 8:1897-1909 (1994)). Similar vascular defects occured following the disruption of a recently cloned Tie2 ligand (Suri et al, Cell 87:1171-1180 (1996), Davis et al, Cell 87:1161-1169 (1996)). These findings indicated that the Tie2 pathway was essential for the formation ofthe embryonic vasculature and suggested a role for Tie2 in pathologic angiogenesis including tumor neovascularization.
  • the present invention relates to a endothelium specific receptor inhibitor comprising an extracellular portion (domain) of an endothelium specific receptor, essentially free of transmembrane domain sequences, and a non-endothelium specific receptor sequence that is essentially non-immunogenic and/or that does not mediate the formation of dimers or oligomers.
  • a endothelium specific receptor inhibitor comprising an extracellular portion (domain) of an endothelium specific receptor, essentially free of transmembrane domain sequences, and a non-endothelium specific receptor sequence that is essentially non-immunogenic and/or that does not mediate the formation of dimers or oligomers.
  • FIG. 1 A and IB Design and production of ExTek.6His. Schematic diagrams ofthe full-length Tie2 receptor and CSF-1 receptor, c-fms, together with their respective soluble extracellular domains fused at the carboxy-terminus to a 6 histidine tag (ExTek.6His and ExFms.6His) (Fig. 1 A). Recombinant baculoviruses were constructed for the production of soluble, recombinant ExTek.6His and ExFms.6His by insect cells. ExTek.6His and ExFms.6His were purified from the conditioned media of baculovirus infected SF9 cells by one step
  • FIG. 3 Inhibition of tumor vascularization by ExTek.6His protein.
  • Tumor vessel length density was measured as an indicator of tumor vascularization from photomicrographs of window chambers bearing 10 day old ExTek.6His treated tumors or control treated tumors.
  • ExTek.6His protein did not adversely effect R3230AC tumor cell proliferation or viability.
  • Cells were either maintained in the presence of ExTek.6His protein at 3 ⁇ M, which was approximately equivalent to the protein concentration used in the window chamber, or control solution for three days. Live cells were counted after suspension in PBS with 0.02%) trypan blue on each following day. Each point represents the mean of 3 experiments.
  • the ExTek concentration was calibrated by using purified recombinant ExTek protein produced in Baculoviral expression vector. High level of ExTek (more than 1 mg/ml) in blood was achieved two days after viral infection. The expression was transient, which slowed diminished to baseline in about 10 days.
  • FIGS 6 A and 6B Inhibition of well established primary tumor growth by AdExTek.
  • the effect of AdExTek on primary tumor growth was tested in two different kind of well established murine tumors.
  • Murine mammary tumor 4T1 (5x10 ⁇ cells per mouse) and melanoma F10.9 (5xl0-> cells per mouse) were implanted into the right flank of Balb/c and C57/BL mouse, respectively. After a palpable tumor was achieved (day 0), the mice were divided into two groups in each different tumor implanted animals. One group of mice were injected with control virus Ad ⁇ -gal directing the expression of ⁇ -galactosidase and the other group mice were injected with AdExTek virus iv through retro-obital sinus.
  • the tumor size was measured afterward for 12 days.
  • the ExTek expression was tested 2 days after viral injection.
  • a significant inhibition on tumor growth was observed in AdExTek treated either 4T1 tumor group and F10.9 tumor group vs. control Ad ⁇ -gal treated animals.
  • the inhibition was stronger during the first 6 days which correlated with high level expression of ExTek in the blood.
  • the tumor growth inhibition decreased with the decrease of ExTek expression in blood 6 days after viral injection.
  • FIGS 7A-7D AdExTek suppresses the tumor lung metastases growth.
  • the inhibition of AdExTek on tumor lung metastasis was analyzed by measuring the lung weight and counting metastases on lung surface under a dissecting microscopy from both 4T1 tumor group (Figs. 7A and 7B) and F10.9 tumor group (Figs. 7C and 7D).
  • lung weight in control Ad ⁇ -gal treated 4T1 tumor group (Fig. 7A) or F10.9 tumor group (Fig. C) was more than twice ofthe AdExTek treated tumor groups or normal, uninjected mouse lung. There was no significant difference in lung weight between AdExTek treated group vs. normal mouse lung.
  • FIG. 8A Schematic diagrams ofthe full-length VEGF receptor, flk-1, and the full length CSF-1 receptor, c-fms, compared to the recombinant soluble extracellular domains of flk-1 (ExFlk.6His) and c-fms (ExFms. ⁇ His) fused to a 6 histidine tag at the carboxy-terminus.
  • Fig. 8A Schematic diagrams ofthe full-length VEGF receptor, flk-1, and the full length CSF-1 receptor, c-fms, compared to the recombinant soluble extracellular domains of flk-1 (ExFlk.6His) and c-fms (ExFms. ⁇ His) fused to a 6 histidine tag at the carboxy-terminus.
  • FIGS 9A-9C ExFlk.6His binds VEGF and inhibits VEGF mediated endothelial cell mitogenesis and migration in vitro.
  • Fig. 9A Saturation binding and Scatchard analysis of ExFlk.6His binding to VEGF. Purified ExFlk.6His was radiolabelled with Na 125 I. Increasing amounts of I25 I labeled ExFlk.6His were then added to micro- wells pre-coated with VEGF (lOng/well) for 1 hour at room temperature. The wells were washed and radioactivity was detected by a r- counter. Nonspecific binding was determined by incubation of 125 I-labeled
  • Fig. 9B ExFlk.6His blocked VEGF -induced thymidine incorporation in endothelial cells. Human umbilical vein endothelial cells(HUVECs) starved for 24 hours in quiescence medium were stimulated with VEGF (lOng/ml), VEGF plus ExFlk.6His (2.5 ⁇ g/ml) or VEGF plus ExFms.6His (2.5 ⁇ g/ml).
  • the filter inserts with HUVECs were then placed in each well of a 24 well culture plate(lower chamber) containing 600 ⁇ l of DMEM/BSA alone as control, DMEM/BSA plus human recombinant VEGF(10ng/ml), DMEM/BSA/VEGF plus either ExFlk.6His or ExFms.6His and incubated at room temperature for 30 min.
  • the migrated cells on the lower surface ofthe filter insert were counted after 4 hours incubation at 37°C.
  • Single asterisk indicates significant difference from control (p ⁇ 0.0005) and double asterisk indicates significant difference from VEGF-stimulated (p ⁇ 0.0005).
  • the experiment was performed in triplicate and repeated once with similar results. Error bars indicate standard error of all samples.
  • FIGS 10A and 10B ExFlk.6His formed heterodimers with endogenous, cell surface Flk-1 in the presence of VEGF.
  • ExFlk.6His could function as a "dominant negative" inhibitor, 125j_rf x F V ]
  • ⁇ 6His was pre-incubated with either buffer, or an equimolar ratio of VEGF, VEGF plus excess ExFlk.6His, or FGF, and then allowed to bind to the cell surface of cultured endothelial cells(ECRF24).
  • Bound ⁇ - ⁇ 1-E Flk .6His was measured by counting lysed cells using a g-counter (Fig. 10A).
  • a chemical cross-linker BS3 was added prior to cell lysis. After cross-linking, cell lysates were analyzed on 5%> SDS-PAGE followed by autoradiography (Fig. 10B).
  • ExFlk.6His Mechanisms by which ExFlk.6His might inhibit VEGF receptor activation. Endogenous VEGF receptors are activated by ligand- mediated dimerization. Excess ExFlk.6His could function as competitive inhibitor, preventing receptor activation by simply competing for VEGF binding. Alternatively, ExFlk. ⁇ His could also undergo VEGF-mediated heterodimerization with the endogenous receptors on the cell surface and function as a "dominant- negative" inhibitor. It is likely that both mechanisms contribute to the inhibitory action of ExFlk.6His on VEGF stimulated angiogenesis.
  • the present invention relates to a soluble endothelium specific receptor inhibitor.
  • the inhibitor is a fusion protein comprising an extracellular domain of an endothelium specific receptor fused to a non endothelium specific receptor sequence.
  • the present invention further relates to a method of inhibiting angiogenesis using such a fusion protein.
  • Endothelium specific receptors of particular relevance to the present invention include those that play a role in endothelial cell proliferation, migration and that are otherwise involved in vascular growth. Examples include Tie-1 (Partanen et al, Mol. Cell. Biol.
  • the fusion protein ofthe invention generally comprises an extracellular domain of an endothelium specific receptor, or ligand-binding portion thereof (see, for example, Davis-Smyth, EMBO J. 15:4919 (1996)), advantageously, Tie-2, and a non-endothelium specific receptor sequence.
  • the extracellular domain is, preferably, essentially free of transmembrane domain sequences ofthe endothelium specific receptor.
  • the non-endothelium specific receptor sequence ofthe fusion protein does not mediate formation of dimers or oligomers and/or is of low immunogenicity (eg is biologically inert, eg compared to Fc).
  • the non endothelium specific receptor sequence is, preferably, not Fc.
  • Preferred non endothelium specific receptor sequences include polyhistidine (eg a 6-histidine tag) and a strep tag.
  • the non receptor sequence is, advantageously, C-terminal to the receptor sequence.
  • the present invention also relates to a nucleic acid (DNA or RNA) that encodes the above-described fusion protein.
  • the present invention relates to a nucleic acid that encodes a fusion protein comprising the extracellular domain of Tie-2 or KDR (advantageously, human Tie-2 or KDR) fused N-terminal to a 6-His tag.
  • the present invention also relates to a recombinant molecule comprising the nucleic acid described above and to a host cell transformed therewith.
  • a recombinant molecule comprising a vector and a nucleic acid encoding the fusion protein of the invention can be constructed.
  • Vectors suitable for use in the present invention include plasmid and viral vectors.
  • Vectors into which a nucleic acid encoding the fusion protein can be cloned include any vector compatible with introduction into a selected host cell.
  • Such vectors include any of a wide variety of commercially available plasmids, as well as baculoviruses, retroviruses, and adenoviruses.
  • the nucleotide sequence ofthe invention can be present in the vector operably linked to regulatory elements, for example, a promoter. Suitable promoters include strong promoters, including inducible promoters. Specific examples include CMV, PGK and tumor cell specific and/or endothelial cell specific promoters such as Tie-1 or Tie-2 promoters.
  • the recombinant molecule ofthe invention can be constructed so as to be suitable for introduction a host cell.
  • Suitable host cells include prokaryotic cells, such as bacteria, lower eukaryotic cells, such as yeast, and higher eukaryotic cells, such as mammalian cells, and insect cells.
  • the recombinant molecule ofthe invention can be introduced into appropriate host cells by one skilled in the art using a variety of known methods.
  • the present invention further relates to a method of producing the fusion protein as defined above.
  • the method comprises culturing the above-described host cells under conditions such that the fusion protein encoding sequence is expressed and the fusion protein thereby produced.
  • the fusion proteins ofthe invention can be used as antigens to generate fusion protein specific antibodies, which are also within the scope ofthe invention. Methods of antibody generation are well known in the art. Both monoclonal and polyclonal antibodies are included, as are binding fragments thereof.
  • the present invention also relates to methods of using the fusion proteins ofthe invention to screen compounds for their ability to bind to the extracellular domain of an endothelium specific receptor and thus to identify compounds that can serve, for example, as agonists or antagonists of angiogenesis.
  • a fusion protein ofthe invention comprising a 6-His tag and bound via the tag to a solid support (eg to an affinity column) is contacted with a test compound and the ability ofthe compound to bind the fusion protein determined.
  • Test compounds that bind are potential agonists or antagonists of angiogenesis.
  • the contacting can be effected in the presence or absence of a natural ligand for the receptor the extracellular domain of which is present in the fusion protein.
  • competitive binding assay conditions can be used to determine whether the test compound enhances or inhibits binding ofthe ligand to the fusion protein.
  • Compounds that inhibit binding ofthe natural ligand to the fusion protein can be expected to be antagonists of angiogenesis and the reverse for compounds that enhance binding.
  • Screening procedures such as those described above are useful for identifying agents for their potential use in the treatment of various forms of pathologic angiogenesis, such as cancer, retinal neovascularization, arthritis and atherosclerosis as well as conditions characterized by reduced vascular density such as diabetes and hypertension.
  • pathologic angiogenesis such as cancer, retinal neovascularization, arthritis and atherosclerosis
  • Agonists identified in accordance with the above screen can also be used to enhance wound healing.
  • the present invention also relates to pharmaceutical compositions comprising, as active agent, the fusion proteins (and nucleic acids) ofthe invention.
  • compositions comprising, as active agent, compounds selected using the above-described screening protocols.
  • Such compositions include the active agent in combination with a pharmaceutically acceptable carrier.
  • the amount of active agent in the composition can vary with the agent, the patient and the effect sought. Likewise, the dosing regimen will vary depending on the composition and the disease/disorder to be treated.
  • the present invention also relates to methods of treating pathologic angiogenesis by modulating (e.g., inhibiting) binding of endothelial growth factors to endothelium specific receptors so as to preclude receptor activation (eg dimerization).
  • This embodiment ofthe invention includes the use in gene therapy regimens of DNA sequences encoding the above-described fusion proteins.
  • the encoding sequences can be present in a construct which, when introduced into target cells, results in expression ofthe DNA sequence and production ofthe fusion protein.
  • Target cells include endothelial cells, particularly endothelial cells present at the site of pathologic angiogenesis (eg, at a tumor site, retina, synovium, atherosclerotic plaque.)
  • target cells can be distant from the site of pathologic angiogenesis so long as expression ofthe DNA results in circulating levels ofthe fusion protein sufficiently high to exert the effect sought (that is, enhancement or inhibition of angiogenesis).
  • a DNA transfer method that: (1) directs the therapeutic sequence into specific target cell types, (2) is highly efficient in mediating uptake ofthe therapeutic polynucleotide into the target cell population, and (3) is suited for use in vivo for therapeutic application.
  • Delivery ofthe fusion protein encoding sequence can be effected using any of a variety of methodologies including transfection with a viral vector, fusion with a lipid, and cationic supported DNA introduction (see generally Verma et al, Nature 389:239 (1997)).
  • transfection with a viral vector fusion with a lipid
  • cationic supported DNA introduction see generally Verma et al, Nature 389:239 (1997).
  • Adenoviral vectors have been described for use in human gene therapy. Advantages of adenovirus vectors, particularly replication defective adenoviruses, include safety, the potential to carry large insert polynucleotide sequences, very high viral titres, ability to infect non-replicating cells, and suitability for infecting tissues in situ.
  • adenoassociated viruses which integrate, can be used, as can other viral systems depending on the target site, or natural/engineered tissue tropism.
  • Gene transfer can be effected using replication-defective retroviral vectors harboring the therapeutic polynucleotide sequence as part ofthe retroviral genome (Miller et al, Mol. Cel. Biol. 10:4239 (1990); Kolberg, J. NIH Res. 4:43 (1992); Cornetta et al, Hum. Gene. Ther. 2:215 (1991)).
  • Advantages of retroviral vectors for gene therapy include the high efficiency of gene transfer into replicating cells, the precise integration ofthe transferred genes into cellular DNA, and the lack of further spread ofthe sequences after gene transduction.
  • plasmid DNA can be purified to homogeneity thereby reducing the potential for pathogenic contamination.
  • Liposome-mediated DNA transfer has been described by various investigators (Wang and Huang, Biochem. Biphys. Res. Commun. 147:980 (1987); Wang and Huang, Biochemistry 28:9508 (1989); Litzinger and Huang, Biochem. Biophys. Acta 1113:201 (1992); Gao and Huang, Biochem. Biophys. Res. Commun. 179:280 (1991); Feigner, WO 91/17424; WO 91/16024).
  • Liposomal compositions may not possess the specificity necessary to deliver the exogenous DNA to all target cell types and non-physiological pH conditions may be necessary to effect fusion.
  • Immunoliposomes have also been described as carriers of exogenous polynucleotides (Wang and Huang, Proc. Natl. Acad. Sci. USA 84:7851 (1987); Trubetskoy et al, Biochem. Biophys. Acta 1131 :311 (1992)). Immunoliposomes can be expected to have improved cell type specificity as compared to liposomes due to the inclusion of specific antibodies that bind to surface antigens on target cell types.
  • Low molecular weight polylysine and other polycations are carriers that can be used to effect DNA-mediated transfection into cells.
  • Zhou et al (Biochem. Biophys. Acta 1065:8 (1991)) have reported synthesis of a polylysine - phospholipid conjugate, a lipopolylysine comprising PL linked to N-glutarylphosphatidylethanolamine, which reportedly increases the transfection efficiency of DNA as compared to lipofectin, a commercially used transfection reagent.
  • any suitable DNA delivery method can be used in the context ofthe present invention, including direct physical application of naked DNA comprising the expression construct/transgene to the target cell population.
  • the nucleic acid-containing compositions ofthe invention can be stored and administered in a sterile physiologically acceptable carrier, where the nucleic acid is dispersed in conjunction with any agents which aid in the introduction of the DNA into cells.
  • a sterile physiologically acceptable carrier including water, PBS, ethanol, lipids, etc.
  • concentration ofthe DNA will be sufficient to provide a therapeutic dose, which will depend on the efficiency of transport into the cells.
  • compositions containing the present fusion protein encoding sequences can be administered for prophylactic and/or therapeutic treatments.
  • compositions are administered to a patient already affected by the particular disease/disorder, in an amount sufficient to cure or at least partially arrest the condition and its complications.
  • An amount adequate to accomplish this is defined as a "therapeutically effective dose” or "efficacious 98/18914
  • Amounts effective for this use will depend upon the severity ofthe condition, the general state ofthe patient, and the route of administration. It will be appreciated that combinations of fusion protein encoding sequences ofthe invention can be used, for example, sequences encoding the ExTek.6His and ExFlk.6His fusion proteins described in the Examples that follow.
  • the fusion protein ofthe invention can be used directly to inhibit angiogenesis.
  • the fusion protein can be administered, for example, systemically or locally, by injection, infusion, via a slow release device, or via a pump. While the amount administered and the dosing regimen will vary with the protein, the mode of administration, the patient and the effect sought, cancer treatment may be effected using for example, 50 ⁇ g-2mg doses, administered by IV infusion over 30 min to 1 hr, twice weekly, similarly arthritis treatment. Arthritis treatment can also be effected via local administration to the involved synovium.
  • the fusion protein can be administered intraocularly via injection (eg at doses of 100 ng to 100 ⁇ g), scleral pumps can also be used.
  • Atherosclerosis can be treated with systemic administration ofthe fusion protein or administration via a stent or balloon angioplasty.
  • the invention includes the administration of more than one fusion protein ofthe invention.
  • the rat endothelial marker MRC-OX43 and biotinylated anti-mouse immunoglobulin was purchased from Harlan Bioproducts for Science and Dako Corporation, respectively.
  • HRP conjugated Streptavidin and liquid DAB substrate kits were from Biogenex Corporation.
  • Avidin Biotin blocking and DAB enhancing solution were from Vector.
  • mice Tek-specific cDNA primers one : 5' GGATCCATGGACCTGATC 3', an Bam H I site was introduced in front ofthe start codon; the other: 3* CGTCTGGAGCCTAGCTA 5', a Cla I site was introduced at 5' end
  • the cDNA from nucleotide 124 to 2346 or amino acids 1- 741 of mouse Tie2 was amplified by RT-PCR from 9-12 day mouse embryo as previously described (Dumont et al, Oncogene 8:1293-1301 (1993)).
  • BSK Cla-/6His is a modified BSK vector (Stratagene). The original Cla I site was deleted and a new Cla I site with an in frame 6-histidine tag followed by a stop codon was inserted into the BamH I and Xba I site. A 2.1 kb EcoR I/Not I fragment from
  • BSK ExTek.6His containing the mouse extracellular domain coding region followed by a six histidine tag was subcloned into the same sites of pVL 1393, a baculoviral expression vector (Pharmingen).
  • the ExTek.6His transfer plasmid and Baculogold baculoviral DNA (Pharmingen) were co-transfected into SF9 cells for production ofthe recombinant baculovirus BvExTek. ⁇ His according to the manufacturer's instructions.
  • Second passage virus was used to infect serum- free SF9 cells for ExTek.6His protein expression.
  • the same approach was used to generate a recombinant baculovirus (BvExFms. ⁇ His) expressing the entire extracellular domain of human c-fms receptor fused to a 6-histidine tag at C terminus.
  • Mock control solution was generated from the supernatant of uninfected SF9 cells following the same purification procedure as for ExTek.6His protein. Aliquots of purified ExTek.6His and ExFms.6His protein and mock control solution were analyzed by SDS-PAGE on a 7.5% gel.
  • Tumor window chamber model Tumors were grown in cutaneous window chambers in Fischer 344 rats
  • a 0.1 mm3 piece of R3230AC tumor from a donor rat was then placed onto the fascial plane and an additional 100 ⁇ l of ExTek.6His protein or control solution was added and the chambers were sealed with glass cover slips.
  • the tissue within the chamber is approximately 200 microns thick and is semi-transparent.
  • a pair of tumor window chambers were done at each time, one treated with ExTek.6His and the other with mock control solution.
  • the tumor implants in each pair were tailored in similar size from the same larger piece of grossly viable tumor tissue.
  • the areas of tumor implants were measured by using an image analysis software (JAVA, Jandel Co.). The baseline host vasculature, the blood flow and the proximity of tumor tissue to vessels were scored.
  • Tumor growth and neovascularization was photographed using a dissecting microscope (Zeiss, Stemi SV6) on days 5, 7 and 10 and window chambers were harvested for H & E staining on day 10.
  • a window chamber bearing a 5 day old untreated R3230AC tumor was freshly frozen in OCT.
  • Tumor volumes which were assumed to approximate a flat cylinder in shape, were calculated using the formula:
  • Tumor vascular length density as an indicator of tumor vasculature was measured from photographs of 10 day old tumors within the window chamber using a previously described method (Dewhirst et al, Radiat. Res. 130:345-354 (1992)). 3-5 areas inside the tumor were randomly selected for measurement. The vascular length density in mm/rrnrP was calculated using the formula:
  • pellets were rehydrated with a drop of PBS buffer and then placed in a surgically created pocket within the cornea stroma, 1.5 mm from the limbus. Corneas were observed every other day until day 5 or 7 when the animals were anesthetized and perfused with lactated ringers solution followed by colloid carbon solution to enumerate the vessels. Responses were scored as positive when vigorous and sustained directional ingrowth of capillary sprouts and hairpin loops toward the implant were detected. Negative responses were recorded when no growth was detected or when there was only an occasional sprout or hairpin loop with no evidence of sustained growth. Positive controls consisted of Hydron pellets containing 25 ng/5 ⁇ l pellet.
  • Negative controls consisted of sham implants and Hydron pellets containing media alone. Media was incorporated into pellets at a concentration of 1 ⁇ g of total protein per cornea. Representative corneas were examined histologically and except for occasional neutrophils found in the limbus of both control and test corneas, nonspecific inflammation was not a contributing factor in any ofthe corneal responses.
  • Tumor windows to be processed for H & E staining were fixed in 4%> paraformaldehyde overnight at 4°C and embedded in paraffin.
  • Samples for immunohistochemistry were freshly frozen in liquid nitrogen and embedded in OCT.
  • serial sections of 10 microns were cut, fixed in ice cold acetone for 10 minutes, and blocked with 0.03 %> H2O2, 1% horse serum and avidin/biotin blocking reagents (Vector). After blocking, sections were incubated with a monoclonal anti-Tie2 antibody or an antibody specific for rat endothelium (MRC-OX43) in a humidified chamber for 50 minutes at room temperature.
  • MRC-OX43 monoclonal anti-Tie2 antibody or an antibody specific for rat endothelium
  • ExTek.6His and ExFms.6His were purified from the supernatant of baculovirus infected SF9 cells by one-step
  • ExTek.6His blocks angiogenesis stimulated by tumor cell conditioned media
  • n m (x):m is the number of rats used in each experiment; n is the number of rats with the indicated response; x is the percentage of rats with the indicated response.
  • Tie2 is expressed in tumor vessels at the onset of tumor angiogenesis
  • ExTek. ⁇ His protein was used as an inhibitor in a rat cutaneous window chamber bearing a R3230AC mammary tumor.
  • small fragments of tumor O.lmm ⁇
  • vascularization of tumors in the window chamber is first detected at about 5 days after implantation and is followed by a rapid growth phase ofthe tumor.
  • immunohistochemical studies demonstrated expression of Tie2 in vessels surrounding and penetrating the tumor implant at 5 days after implantation.
  • ExTek inhibits tumor growth in cutaneous "window chambers"
  • Recombinant adenovirus was generated and propagated in monolayer cultured 293 cells maintained in DMEM supplemented with 10%> fetal bovine serum and 1% penicillin/streptomycin (Gibco/BRL) at 37°C with 5% C ⁇ 2-
  • the murine mammary carcinoma cell line 4T1 and murine melanoma cell line F10.9 were maintained in DMEM plus 10%> fetal bovine serum at 37°C with 5% CO2.
  • a novel adeno viral vector system in which partial El region and all the E3 region were deleted, derived from the in340 strain of adenovirus type 5 (ad5) was used in construction of AdPac ⁇ -gal (Channon et al, Cardiovascular Res. 32:962-972 (1996)). The ⁇ -gal gene was inserted in El region. The resulting viral vector, AdPac ⁇ -gal, served as a control virus and was used to generate AdExTek by replacing the ⁇ -galactosidase gene with one containing ExTek cDNA.
  • the ExTek cDNA containing the mouse extracellular domain coding region fused to a strep-tag at C terminal was subcloned into BamH I and Nhe I site of the transfer plasmid pGEM CMV/BGH poly(A).
  • the plasmid pGEM CMV/BGH poly(A).
  • CMV ExTek/BGH poly (A) was then digested with Xba I and Bst B 1 , and the 3.6 kb plasmid fragment consistent with the early CMV promotor and enhancer, ExTek and BGH was directly ligated to the Xba I site of parent virus Ad.Pac ⁇ -gal DNA.
  • the ligation mixture was purified by phenol-chloroform extraction and ethanol precipitation, then transfected into 293 cells using the calcium phosphate method. After observation of a cytopathathic effect (7-10 days), the cells were lysed by multiple freeze/thaw cycles, and the recombinant virus was isolated by plaque assay on 293 cells.
  • Plaque purified AdExTek or Ad.Pac ⁇ -gal virus was used to generate high titer virus stock by infecting forty 150 mm plates of confluent 293 cells at a multiplicity of infection of 2 in DMEM plus 2%o FBS.
  • the viruses were purified from the infected cell lysates as described previously (Channon et al, Cardiovascular Res. 32:962-972 (1996)).
  • the virus was stored in virus storage buffer (VSB; 20 mM Tris pH 7.4, 150 mM NaCl, 5 mM KCL, 1 mM MgCl 2 ) plus
  • ExTek concentration in blood was determined by a simple ELISA assay.
  • AdExTek adenovirus (5x10° pfu) was injected into Balb/c mouse circulation through retro-obital sinus. Two days later, small amount of blood was collected in a heparinized microcapiUary tube from tail vein. Mouse plasma was recovered after brief centrifugation to remove cells. Serial dilutions (in PBS) of the plasma were incubated in micro wells overnight at 40°C. The coated wells were blocked with 5 % milk in TBST (lOmM of Tris Cl, pH 8.0, 150mM of NaCl, 0.05% of Tween 20) for 30 minute.
  • a biotinylated mouse anti Tie2 monoclonal antibody (Ab33) diluted in TBST (0.5 ⁇ g/ml) was incubated for 1 hour followed by incubation with 1 :2000 diluted strep-avidin alkaline phosphatase conjugate (Gibco/BRL) in TBST for 30 minutes.
  • the phosphatase activity was determined by addition of Sigma @104 phosphatase substrate dissolved in diethanolamine and 0.5 mM MgCl2, pH 9.8, and absorbency at 405nm was measured in V max Kinetic Microplate Reader
  • a murine mammary tumor cell line 4T1 or a murine melanoma cell line F10.9 was implanted into the flank of female Balb/c mouse or
  • mice lungs to be processed for H & E staining were fixed in Bouin's solution for a few days, decolorized in 70%) ethanol several times before being embedded in paraffin. Serial sections of 8 microns were cut. The sections were stained with H&E for histoligic examination.
  • AdExTek a recombinant adenovirus for gene transfer of ExTek
  • AdExTek a recombinant adenovirus
  • ExTek cDNA was inserted in El region driven by the CMV promotor.
  • AdExTek was administered to Balb/c mice in intravenous injection into the retro- orbital sinus. The plasma was collected every other day after viral injection. Serial dilutions ofthe plasma were then incubated in ELISA plates. The ExTek content was determined using biotinylated anti-Tie2 antibody (Ab33). The concentration of ExTek was calibrated using purified recombinant ExTek (Lin et al, J. Clin. Invest. 100:2072-2078 (1997)). High level expression could be detected in the plasma (1-2 mg/ml) two days after viral injection. The high level expression was transient with levels falling to baseline within 12 days (Fig 5B). The mice with high levels of circulating ExTek showed no apparent ill effects, no weight loss and no behavioral abnormalities. AdExTek inhibits the growth rate of two well established primary murine tumors
  • Tumor cells 5x10 ⁇ in 50 ⁇ l of PBS were implanted into the flank of female Balb/c mice (for 4T1) or female C57/BL (for F10.9). Following the development of an easily palpable tumor (7-
  • AdExTek suppress tumor metastasis
  • two highly metastatic cell lines 4T1 and F10.9 (5x10 ⁇ ) were mixed with 5x10 ⁇ pfu of either the AdExTek virus or the control AdPac ⁇ -gal virus and then co-injected into the circulation through retro-obital sinus.
  • 5x10 ⁇ 5x10 ⁇
  • 3 mice injected with control virus died from massive lung metastases but all ofthe mice that received AdExTek were alive.
  • the remaining animals were sacrificed and the lungs were removed, weighed and fixed in Bouin's solution. Lungs were grossly examined under dissecting microscopy.
  • Recombinant baculovirus was generated and propagated in monolayer cultured SF9 cells maintained in Grace's Insect Medium Supplemented (Gibco/BRL) at 28°C. Protein expression was carried out in suspension cultured SF9 cells in Protein-Free Insect Medium (Gibco/BRL).
  • the R3230AC rat adenocarcinoma cell line was maintained in DMEM plus 10%> fetal bovine serum (FBS, Gibco/BRL) at 37°C with 5% CO2- Human umbilical vein endothelial cells (HUVEC) were purchased from Clonetics Inc.
  • ECRF24 an immortalized HUVEC, was provided by Dr. Hans Pannekoek (Fontijn et al, DNA Exp. Cell.
  • Endothelial cells were maintained at 37°C, 5% CO 2 in complete endothelial cell growth medium (EGM, Clonetics, Inc) and grown on 2% gelatin (Sigma) coated plates. Endothelial cells were serum starved in endothelial cell basal medium (EBM, Clonetics, Inc). Anti-Flk antibodies (C-20 and 1158) and protein-A agarose were purchased from Santa Cruz Biotechnology. Anti phosphotyrosine antibody (4G10) was purchased from Upstate Biotechnology. Human recombinant VEGF 165 was purchased from R & D Systems. Construction ofthe ExFlk.6His and ExFms.6His baculovirus vectors and production of recombinant viruses
  • VEGF vascular endothelial growth factor
  • flk-1 and flt-1 Two closely related endothelium-specific receptor tyrosine kinases, flk-1 and flt-1 (De Vries et al, Science 255:989-991 (1992), Terman et al, Biochem. Biophys. Res. Comm. 187:1579-1586 (1992), Shibuya, Adv. Cancer Res. 67:281-316 (1995)).
  • the entire extracellular domain of murine flk-1 was generated by PCR from a plasmid containing a cDNA encoding the extracellular and transmembrane domains of flk-1.
  • the forward primer consisted of nucleotides 208 to 223 with a BamHI site introduced upstream ofthe start codon; the reverse primer consisted of nucleotides 2493-2478 in the reverse orientation with a Cla I site introduced at the 5' end.
  • the resulting PCR product was digested with BamHI/Clal and ligated to the same sites of an intermediate vector (BSK Cla-/6His) to generate a cDNA encoding a fusion protein consisting ofthe entire extracellular domain of flk-1 with a 6 histidine tag at the C terminus
  • BSK/ExFlk.6His A 2.3 kb EcoR I/Not I fragment from BSK/ExFlk.6His was subcloned into the same sites of pVL 1393, a baculoviral expression transfer vector (Pharmingen).
  • pVL 1393/ExFlk.6His and Baculogold baculoviral DNA were co-transfected into SF9 cells for production ofthe recombinant baculovirus (BvExFlk.6His) according to the manufacturer's instructions. Second passage virus was used to infect serum-free SF9 cells for ExFlk.6His protein production.
  • Suspension cultured SF9 serum free insect cells (1 liter) were infected with approximately lpfu cell of second passage BvExFlk.6His or BvExFms.6His for 54 hours at 28°C. Cells were removed by centrifugation at 3000 rpm(Sorvall) for 20 minutes at 4°C. The supernatant was dialyzed against 8 liters of PBS pH 8.0 for 48 hours with one change of buffer. The dialyzed supernatant was then incubated with 4ml of Ni ++ NTA resin (Qiagen). After 1 hour at room temperature, the resin-bound ExFlk.6His protein or ExFms.6His protein was loaded onto a 10ml column.
  • wash buffer 50mM NaH2PO. ⁇ , 300mM NaCl and 20mM imidazole pH 8.0
  • the protein was eluted with elution buffer (wash buffer plus 250mM imidazole) followed by a buffer change to PBS pH 7.2 by ultra filtration (Centricon 10 from Amicon Co.).
  • Mock control material used in the tumor study was generated from the supernatant of uninfected SF9 cells following the same purification procedure. Aliquots of purified ExFlk.6His and ExFms.6His proteins were analyzed by SDS- PAGE on a 7.5% gel.
  • the wells were washed once with blocking buffer (25mM Hepes pH 7.4, lOOmM NaCl, 0.5% gelatin, 20 ⁇ g/ml BSA) and then blocked with the same buffer for 2 hours at room temperature.
  • Increasing amounts of 125j labeled ExFlk. ⁇ His (0.03-300nM) in binding buffer (25mM Hepes pH7,4, lOOmM NaCl, 20 ⁇ g/ml BSA, 0.5 ⁇ g/ml heparin) were added to each well and incubated for 1 hour at room temperature.
  • Nonspecific binding was determined by incubation of 125 I-labeled ExFlk. ⁇ His in the presence of 60-fold excess of unlabeled ExFlk. ⁇ His.
  • the wells were washed 3 times with binding buffer and counted in a gamma counter. Scatchard analysis of binding was performed with the aid ofthe Scatchard Fit program.
  • ECRF cells a transformed HUVEC line expressing Flk-1, were grown in EGM in a 60 mm dish (Fontijn et al, DNA Exp. Cell. Res. 216:199-207 (1995)). Upon reaching confluence, the cells were serum starved in EBM overnight followed by stimulation with human recombinant VEGF at 20 ng/ml plus different amounts of ExFlk. ⁇ His at 37°C for 5 minutes.
  • the cells were then washed with ice cold PBS 3 times on ice and then lysed with lysis buffer (20 mM Tris, pH 8.0, 137 mM NaCl, 10% glycerol, 1 % triton X- 100, 2 mM EDTA) supplemented with 1 mM PMSF (a protease inhibitor), 10 ng/ml leupeptin, 1 mM sodium vanadate.
  • Flk was immunopreciptated with anti-Flk antibody (antibody 1158, Santa Cruz) for 4 hours at 4°C, and immuncomplexes were collected by addition of protein A agarose beads.
  • HUVECs were plated in 24 well plates at a density of 25000 cells per well. After 24 hours, the media was replaced by endothelial cell basal media (EBM as quiescence media, Clonetics, Inc) and incubated for an additional 24 hours. The old media was then replaced with either fresh quiescence media alone as a control, or quiescence media plus human recombinant VEGF at 1 Ong/ml, or quiescence media plus 1 Ong/ml VEGF and purified ExFlk. ⁇ His at 2.5 ⁇ g/ml, or quiescence medium plus lOng/ml VEGF and purified control ExFms. ⁇ His at 2.5 ⁇ g/ml.
  • the cells were incubated for 24 hours followed by a 3 hour pulse-labeling with 2 ⁇ Ci/ml 3 H-thymidine (Amersham). The reaction was stopped by aspirating the media and washing the cells with Hanks Balanced Salt Solution (Gibco/BRL). The DNA was precipitated by treating the cells with cold 10% TCA at 4°C for 30 min followed by an absolute ethanol wash. The precipitated material was resuspended in 0.5ml of 0.5M NaOH, and ⁇ H-thymidine incorporation was determined from a 400 ⁇ l aliquot using Beckman LS6000SC scintillation counter.
  • the rate of migration of HUVECs was determined by using a modified Boyden chamber assay as described by Clyman et al (Cell Adhesion and Commun. 1 :333 (1994)). Briefly, Polycarbonate filter wells (Costar Transwell with an 8 ⁇ m pore size) were coated with 2% gelatin in PBS for 30 min at room temperature and subsequently incubated at 37°C for 1 hour with DMEM containing 0.1% BSA (DMEM/BSA). Confluent HUVECs were trypsinized, pelleted by centrifugation, washed with DMEM/BSA to remove residual serum and resuspended in fresh DMEM BSA to a final concentration of 2 x 10 6 cells/ml.
  • the lysate was recovered after centrifugation to remove cell nuclei and either directly analyzed by 5%> SDS- PAGE followed by auto radiography or immunopreciptated with an anti-Flk antibody raised against a c-tail peptide of Flk (antibody c-20, Santa Cruz) before the SDS-PAGE analysis.
  • pellets were rehydrated with a drop of PBS and then placed in a surgically created pocket within the corneal stroma, 1.5mm from the limbus. Corneas were observed every other day until day 5 or 7 when the animals were anesthetized and perfused with lactated ringers solution followed by colloidal carbon solution to enumerate the vessels. Responses were scored as positive when vigorous and sustained directional ingrowth of capillary sprouts and hairpin loops toward the implant were detected. Negative responses were recorded when no growth was detected or when there was only an occasional sprout or hairpin loop with no evidence of sustained growth. Negative controls consisted of Hydron pellets containing media alone. Media was incorporated into pellets at a concentration of 1 ⁇ g of total protein per pellet.
  • Soluble receptor proteins were incorporated into pellets at lOOng per pellet. Histological examination of representative corneas revealed that nonspecific inflammation was not a contributing factor in any ofthe corneal responses except for occasional neutrophils found in the limbus of both control and test corneas.
  • Tumor window chamber model R3230AC tumors were grown in cutaneous window chambers in Fischer
  • a 0.1mm 3 piece of tumor from a donor rat was then placed onto the fascial plane and an additional lOO ⁇ l of protein or control solution was added and the chambers were sealed with glass cover slips.
  • a pair of tumor window chambers were done at each time, one treated with ExFlk. ⁇ His and the other with control solution.
  • the tumor implants in each pair were taken from the same region of grossly viable donor tumor tissue.
  • Ten days after implantation, tumors in window chambers (200 ⁇ m thick) were photographed using transillumination and a dissecting microscope (Zeiss, Stemi SV6) for vascular length density measurement and were subsequently harvested for H & E staining.
  • Tumor volumes which were assumed to approximate a flat cylinder in shape, were calculated using the formula:
  • Tumor volume (mm3) 3.14t(d/2)2
  • Tumor vascular length density as an indicator of tumor vascularity was measured from photographs of 10 day old tumors within the window chamber using a previously described method (Dewhirst et al, Radiat. Res. 130:345-354 (1992)). 3-5 areas inside the tumor were randomly selected for measurement. The vascular length density in mm/mm 3 was calculated using the formula:
  • R3230AC tumor cells were seeded at 2X10 4 /well into 24-well plates and maintained in the presence of purified ExFlk. ⁇ His protein (3 ⁇ M) or control solution. Cell morphology was monitored each day by light microscopy. Cells were trypsinized, suspended in PBS containing 0.02% trypan blue (Gibco/BRL) and live cells were counted with a hemacytometer on each following day for three days.
  • Results are reported as mean ⁇ SE for tumor volume and tumor vascular length density for each group.
  • a two-tailed Student's t test was used to analyze statistical differences between control and ExFlk. ⁇ His treated groups. Differences were considered statistically significant at p ⁇ 0.05.
  • ExFms. ⁇ His was purified from the supernatant of SF9 cells infected with a baculovirus vector directing the expression of a fusion between the extracellular domain ofthe CSF-1 receptor, c-fms, fused to a 6 histidine-tag.
  • a mock control was purified from uninfected SF9 cells using the same protocol.
  • ExFlk. ⁇ His and ExFms. ⁇ His proteins were purified to near homogeneity, yielding a single major band with the expected molecular masses of approximately 105kD and 70kD, respectively (Figure 8B).
  • ExFlk.6His protein binds VEGF, blocks the activation of endothelial VEGF receptor and neutralizes VEGF stimulated endothelial proliferation and migration in vitro
  • binding assays were done by adding 125j labeled ExFlk. ⁇ His to microtiter wells pre- coated with human recombinant VEGF. Bound 125j ExFlk. ⁇ His was then detected by counting the washed wells in a Gamma counter. Under the conditions 8/18914
  • ECRF endothelial cells
  • ExFlk. ⁇ His protein (2.5 ⁇ g/ml) but was not blocked by ExFms. ⁇ His at the same concentration (p ⁇ 0.0005).
  • HUVEC migration rate in a modified Boyden chamber assay increased approximately 3 fold by the addition of VEGF at 1 Ong/ml (p ⁇ 0.0005), and this increase in migration was reduced to background levels after pre-incubation of VEGF with ExFlk. ⁇ His (2.5 ⁇ g/ml) but not with ExFms. ⁇ His at the same concentration (p ⁇ 0.0005, Fig. 9C).
  • ExFlk.6His forms a VEGF-dependent heterodimer with endogenous VEGF receptors on the surface of cultured endothelial cells
  • ' ⁇ ⁇ l-ExFlk. ⁇ iis was mixed with VEGF to form a 125j_r ⁇ Fik .6His/VEGF complex. This pre-formed complex was then tested for its ability to bind to VEGF receptors on the surface of cultured endothelial cells (Figure 10A).
  • the cross- linked complex was immunopreciptated with an antibody against the c-tail of Flk- 1 (antibody 1158, Santa Cruz) and analyzed by SDS-PAGE and autoradiography. Again, consistent with the formation of VEGF mediated receptor heterodimer, a single high molecular complex containing both the endogenous cell surface receptor and the radiolabeled soluble receptor was immunopreciptated only in the presence of VEGF.
  • ExFlk. ⁇ His could function as a "dominant negative" inhibitor of VEGF receptor activation and should be a potent inhibitor of angiogenesis in vivo.
  • ExFlk.6His inhibits corneal angiogenesis in vivo
  • ExFlk. ⁇ His (lOOng)
  • the angiogenic response induced by tumor conditioned media was totally blocked in 5 corneas (72%>) and a weak growth was seen in 2 corneas (28%).
  • Addition of a control protein, ExFms. ⁇ His, to pellets containing tumor cell conditioned media did not block vessel formation.
  • vascularization of tumors in the window chamber is first detected at about 5 days after implantation and is followed by a rapid growth phase ofthe tumor.
  • ExFlk.6His inhibits tumor vascularization
  • ExFlk. ⁇ His protein This finding is in accordance with the ability of ExFlk. ⁇ His protein to neutralize VEGF mediated endothelial responses in vitro, and to block tumor cell conditioned media stimulated angiogenesis in the rabbit cornea. It is also consistent with the notion that the primary action ofthe ExFlk. ⁇ His protein is to inhibit tumor neovascularization. ExFlk.6His does not directly affect tumor cell proliferation or viability

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Abstract

L'invention concerne un récepteur Tie2 soluble et son utilisation en tant qu'agent antiangiogénique.
PCT/US1997/019597 1996-10-31 1997-10-31 Recepteur tie2 soluble WO1998018914A1 (fr)

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US6413932B1 (en) 1999-06-07 2002-07-02 Immunex Corporation Tek antagonists comprising soluble tek extracellular binding domain
US6521424B2 (en) 1999-06-07 2003-02-18 Immunex Corporation Recombinant expression of Tek antagonists
WO2006002854A2 (fr) * 2004-06-25 2006-01-12 Licentia, Ltd. Recepteurs tie et ligands pour le tie, et procedes de modulation de la fertilite feminine
US7521053B2 (en) 2001-10-11 2009-04-21 Amgen Inc. Angiopoietin-2 specific binding agents
US7658924B2 (en) 2001-10-11 2010-02-09 Amgen Inc. Angiopoietin-2 specific binding agents
US8071650B2 (en) 2000-08-21 2011-12-06 Pacific Corporation Thiourea derivatives and the pharmaceutical compositions containing the same
WO2012162561A2 (fr) 2011-05-24 2012-11-29 Zyngenia, Inc. Complexes plurispécifiques multivalents et monovalents, et leurs utilisations
EP2671891A2 (fr) 2008-06-27 2013-12-11 Amgen Inc. Inhibition d'ang-2 pour traiter la sclérose en plaques
US9795594B2 (en) 2006-06-27 2017-10-24 Aerpio Therapeutics, Inc. Human protein tyrosine phosphatase inhibitors and methods of use
US9926367B2 (en) 2006-04-07 2018-03-27 Aerpio Therapeutics, Inc. Antibodies that bind human protein tyrosine phosphatase beta (HPTPbeta) and uses thereof
US9949956B2 (en) 2009-07-06 2018-04-24 Aerpio Therapeutics, Inc. Compounds, compositions, and methods for preventing metastasis of cancer cells
US9994560B2 (en) 2014-03-14 2018-06-12 Aerpio Therapeutics, Inc. HPTP-β inhibitors
US10150811B2 (en) 2011-10-13 2018-12-11 Aerpio Therapeutics, Inc. Methods for treating vascular leak syndrome and cancer
EP3424530A1 (fr) 2013-03-15 2019-01-09 Zyngenia, Inc. Complexes multispécifiques monovalents et multivalents et leurs utilisations
US10220048B2 (en) 2013-03-15 2019-03-05 Aerpio Therapeutics, Inc. Compositions and methods for treating ocular diseases
US10336820B2 (en) 2008-02-20 2019-07-02 Amgen Inc. Antibodies directed to angiopoietin-1 and angiopoietin-2 and uses thereof
US10952992B2 (en) 2015-09-23 2021-03-23 Aerpio Pharmaceuticals, Inc. Methods of treating intraocular pressure with activators of Tie-2
US11253502B2 (en) 2019-04-29 2022-02-22 EyePoint Pharmaceuticals, Inc. Tie-2 activators targeting the Schlemm's canal

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US5447860A (en) * 1992-06-26 1995-09-05 Immunex Corporation Tyrosine kinase
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JP2003501090A (ja) * 1999-06-07 2003-01-14 イミュネックス・コーポレーション Ｔｅｋアンタゴニスト
US6521424B2 (en) 1999-06-07 2003-02-18 Immunex Corporation Recombinant expression of Tek antagonists
US6413932B1 (en) 1999-06-07 2002-07-02 Immunex Corporation Tek antagonists comprising soluble tek extracellular binding domain
US7067475B2 (en) 1999-06-07 2006-06-27 Immunex Corporation Tek antagonists
US8071650B2 (en) 2000-08-21 2011-12-06 Pacific Corporation Thiourea derivatives and the pharmaceutical compositions containing the same
US7658924B2 (en) 2001-10-11 2010-02-09 Amgen Inc. Angiopoietin-2 specific binding agents
US7521053B2 (en) 2001-10-11 2009-04-21 Amgen Inc. Angiopoietin-2 specific binding agents
WO2006002854A3 (fr) * 2004-06-25 2006-03-16 Licentia Ltd Recepteurs tie et ligands pour le tie, et procedes de modulation de la fertilite feminine
WO2006002854A2 (fr) * 2004-06-25 2006-01-12 Licentia, Ltd. Recepteurs tie et ligands pour le tie, et procedes de modulation de la fertilite feminine
US11814425B2 (en) 2006-04-07 2023-11-14 Eye Point Pharmaceuticals, Inc. Antibodies that bind human protein tyrosine phosphatase beta (HPTPbeta) and uses thereof
US9926367B2 (en) 2006-04-07 2018-03-27 Aerpio Therapeutics, Inc. Antibodies that bind human protein tyrosine phosphatase beta (HPTPbeta) and uses thereof
US9795594B2 (en) 2006-06-27 2017-10-24 Aerpio Therapeutics, Inc. Human protein tyrosine phosphatase inhibitors and methods of use
USRE46592E1 (en) 2006-06-27 2017-10-31 Aerpio Therapeutics, Inc. Human protein tyrosine phosphatase inhibitors and methods of use
US10463650B2 (en) 2006-06-27 2019-11-05 Aerpio Pharmaceuticals, Inc. Human protein tyrosine phosphatase inhibitors and methods of use
US10336820B2 (en) 2008-02-20 2019-07-02 Amgen Inc. Antibodies directed to angiopoietin-1 and angiopoietin-2 and uses thereof
EP2671891A2 (fr) 2008-06-27 2013-12-11 Amgen Inc. Inhibition d'ang-2 pour traiter la sclérose en plaques
US9949956B2 (en) 2009-07-06 2018-04-24 Aerpio Therapeutics, Inc. Compounds, compositions, and methods for preventing metastasis of cancer cells
WO2012162561A2 (fr) 2011-05-24 2012-11-29 Zyngenia, Inc. Complexes plurispécifiques multivalents et monovalents, et leurs utilisations
US10150811B2 (en) 2011-10-13 2018-12-11 Aerpio Therapeutics, Inc. Methods for treating vascular leak syndrome and cancer
US10815300B2 (en) 2011-10-13 2020-10-27 Aerpio Pharmaceuticals, Inc. Methods for treating vascular leak syndrome and cancer
US12043664B2 (en) 2011-10-13 2024-07-23 EyePoint Pharmaceuticals, Inc. Methods for treating vascular leak syndrome and cancer
US10220048B2 (en) 2013-03-15 2019-03-05 Aerpio Therapeutics, Inc. Compositions and methods for treating ocular diseases
EP3424530A1 (fr) 2013-03-15 2019-01-09 Zyngenia, Inc. Complexes multispécifiques monovalents et multivalents et leurs utilisations
US9994560B2 (en) 2014-03-14 2018-06-12 Aerpio Therapeutics, Inc. HPTP-β inhibitors
US10858354B2 (en) 2014-03-14 2020-12-08 Aerpio Pharmaceuticals, Inc. HPTP-Beta inhibitors
US11666558B2 (en) 2015-09-23 2023-06-06 EyePoint Pharmaceuticals, Inc. Methods of treating intraocular pressure with activators of Tie-2
US10952992B2 (en) 2015-09-23 2021-03-23 Aerpio Pharmaceuticals, Inc. Methods of treating intraocular pressure with activators of Tie-2
US12171751B2 (en) 2015-09-23 2024-12-24 EyePoint Pharmaceuticals, Inc. Methods of treating intraocular pressure with activators of Tie-2
US11253502B2 (en) 2019-04-29 2022-02-22 EyePoint Pharmaceuticals, Inc. Tie-2 activators targeting the Schlemm's canal
US12064420B2 (en) 2019-04-29 2024-08-20 EyePoint Pharmaceuticals, Inc. Tie-2 activators targeting the Schlemm's canal

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