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WO1997010841A1 - Proprietes anti-angiogeniques du polypeptide ii activant les monocytes endotheliaux - Google Patents

Proprietes anti-angiogeniques du polypeptide ii activant les monocytes endotheliaux Download PDF

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Publication number
WO1997010841A1
WO1997010841A1 PCT/US1996/015007 US9615007W WO9710841A1 WO 1997010841 A1 WO1997010841 A1 WO 1997010841A1 US 9615007 W US9615007 W US 9615007W WO 9710841 A1 WO9710841 A1 WO 9710841A1
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WIPO (PCT)
Prior art keywords
emap
endothelial
tumor
monocyte activating
activating polypeptide
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PCT/US1996/015007
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English (en)
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WO1997010841A9 (fr
Inventor
David Stern
Margaret Schwarz
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The Trustees Of Columbia University In The City Of New York
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Priority to AU71618/96A priority Critical patent/AU7161896A/en
Publication of WO1997010841A1 publication Critical patent/WO1997010841A1/fr
Publication of WO1997010841A9 publication Critical patent/WO1997010841A9/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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • 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
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5064—Endothelial cells
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • G01N33/5088—Supracellular entities, e.g. tissue, organisms of vertebrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2510/00—Detection of programmed cell death, i.e. apoptosis

Definitions

  • VEGF vascular endothelial growth factor
  • angiostatin angiostatin
  • EMAP II has been described in PCT International Publication No. WO 95/09180, published April 6, 1995, the contents of which are hereby incorporated by reference.
  • EMAP II has anti-angiogenic properties and results in suppression of tumor growth, likely due to perivascular apoptotic tissue injury and targeting of EMAP II to proliferating endothelial cells. These results demonstrate that endogenous or exogenously administered EMAP II controls blood vessel formation in a range of pathophysiologically relevant situations .
  • WO 95/09180 discloses that EMAP II administered in one intratumoral dose followed by one intravenous dose reduces the size of a tumor.
  • WO 95/09180 also discloses that EMAP II has inflammatory activity. On the basis of its inflammatory activity one would have expected that EMAP II would be toxic and therefore inappropriate for multiple administrations over a long period of time. Surprisingly, it has been found that multiple administrations of EMAP II decrease tumor size even without an intratumoral dose and without observed toxic effect. The ability to administer a therapeutically effective regimen of EMAP II without an intratumoral injection makes it possible to treat tumors whose small size makes it difficult or impossible to administer an intratumoral injection.
  • Retinal neovascularization is a major cause of blindness in the United States.
  • Pathologic retinal angiogenesis is a common pathway leading to vision loss in disease processes such as retinopathy of prematurity, diabetic retinopathy, sickle cell retinopathy, and age related macular degeneration.
  • Factors associated with retinopathy vascularization include hypoxia (cause of retinopathy of prematurity) , diabetes, and known angiogenic factors such as Vascular endothelial growth factor (VEGF) .
  • VEGF Vascular endothelial growth factor
  • the use of an established model of hypoxic induced retinopathy (Pierce, E. Jan. 1995; Smith, L., Jan. 1994) demonstrates that EMAP II, a protein associated with tumor antiangiogenesis, inhibits the neovascularization associated with retinopathy.
  • This invention provides a method of treating a tumor in a subject, comprising administering to the subject an amount of an agent, selected from endothelial monocyte activating polypeptide II and an endothelial monocyte activating polypeptide II-derived polypeptide, effective to treat the tumor, wherein the endothelial monocyte activating polypeptide II is administered subcutaneously, intraperitoneally, or intravenously.
  • an agent selected from endothelial monocyte activating polypeptide II and an endothelial monocyte activating polypeptide II-derived polypeptide
  • This invention provides a method of inhibiting the growth of endothelial cells, comprising contacting the endothelial cells with an amount of an agent, selected from endothelial monocyte activating polypeptide II and an endothelial monocyte activating polypeptide II-derived polypeptide, effective to inhibit growth of the endothelial cells.
  • FIG. 1 SDS-PAGE of recombinant EMAP II.
  • E. coli homogenate and pools of fractions containing EMAP II (See Fig. 2, below) were subjected to reduced SDS-PAGE (10-20% Tricine gels; 1-2 ⁇ g/lane) and protein visualized by silver staining.
  • Lane 1 E. coli cell homogenate after centrifugation (12, OOOxg) ;
  • lane 2 polyethylene imine supernatant; lane 3, Heparin Sepharose pool; lane 4, SP Sepharose pool; lane 5, Phenyltoyopearl pool; and lane 6, EMAP II formulated into PBS.
  • FIGS. 2A, 2B and 2C Chromatographic steps in the purification of recombinant EMAP II.
  • Fig. 2A Heparin Sepharose. The polyethylene imine supernatant was applied to Heparin Sepharose in Tris buffer, washed and eluted with an ascending salt gradient . Fractions were monitored for absorbance at 280 nm and analyzed on SDS-PAGE and/or immunoblotting to identify the EMAP II pool (designated by the arrow and labeled EMAP II) .
  • FIG. 2B SP Sepharose. The Heparin Sepharose pool from (Fig. 2A) was concentrated, desalted and applied to SP Sepharose High Performance in MOPS buffer.
  • EMAP II was eluted by an ascending salt gradient and pooled as above. Phenyl toyopearl.
  • the SP Sepharose pool was adjusted to 2 M (NH 4 ) 2 S0 4 and applied to Phenyl toyopearl in phosphate buffer with salt, washed, and EMAP II eluted with a descending salt gradient.
  • the salt gradients are shown as ( ) , 0-1 M in
  • FIGS. 3A, 3B, 3C , 3D and 3E Matrigel angiogenesis model: effect of EMAP II on bFGF-induced neovascularization. Mice received subcutaneous Matrigel implants and were sacrificed after 14 days to analyze new vessel formation by histologic examination and hemoglobin assay.
  • Fig. 3A and 3C implant containing bFGF (100 ⁇ g/ml) /heparin (40 U/ml) shown at low and high power, respectively;
  • Fig. 3B and 3D implant containing bFGF/heparin + EMAP II (100 ng/ml) shown at low and high power, respectively; Fig.
  • FIGS 4A, 4B and 4C Disappearance of 125 I-EMAP II from mouse plasma after IV or IP injection (Fig. 4A) , precipitability of the tracer in trichloroacetic acid (Fig. 4B) , and tissue accumulation (Fig. 4C) .
  • Fig. 4A Mice received 125 I-EMAP II (0.26 ⁇ g) by either IV or IP injection and plasma was sampled at the indicated time points. The methods for data fitting and parameters of clearance are described in the text.
  • Fig. 4B Trichloroacetic acid precipitability of 125 I-EMAP II in spleen and B16 tumor harvested 12 hrs after IP injection as above.
  • Tissue was homogenized, weighed, counted, and subjected to precipitation in trichloroacetic acid (20%) .
  • FIGS. 5A, 5B, 5C, 5D, 5E, 5F and 5G Effect of EMAP II on Lewis Lung Carcinoma (LLC) .
  • LLC Lewis Lung Carcinoma
  • Mice were injected subcutaneously on day 1 with LLC cells, and then on days 3- 15 received every 12 hrs IP either: vehicle alone (control) , EMAP II (100 or 1000 ng) or heat-inactivated EMAP II (1000 ng) .
  • Histology of LLC tumors harvested from the indicated above groups on day 15 Figure 5B and 5C, vehicle alone, high and low power, respectively; Figure 50C and 5E, EMAP II (100 ng) high and low power, respectively; Figs. 5F-G. DNA fragmentation by in situ nick translation: Fig. 5F, vehicle alone and Fig. 5G, EMAP II (1000 ng) .
  • Figures 6A, 6B, SC, 6D, 6E and 6F Effect of EMAP II on cultured endothelial cells (ECs) .
  • Figures 6A-6D Effect on EC monolayer wound repair in vitro. A postconfluent monolayer of ECs was wounded (wound margin at upper right) , and then either vehicle ( Figures 6A, 6C) or rEMAP II (10 ng/ml; Figures 16B, 16D) was added.
  • Figures 6A-6B DNA fragmentation by ELISA of subconfluent ECs in normoxia or hypoxia (p0 2 «14 torr) , exposed to rEMAP .II as indicated. Data shown represent mean and, in each case, S.E. was less than 10%. These experiments were repeated a minimum of three times.
  • FIG. 7 PCR analysis of EMAP II transcripts in normal murine tissue. RNA was harvested from normal murine tissues as indicated, and processed for PCR as described in the text. The bands corresponding to the amplicons for EMAP II (400 bp) and ⁇ -actin (560 bp) are shown by the arrows. A 100 bp ladder was used as the standard in the far left lane.
  • Figures 9A, 9B, 9C, 9D, 9E and 9F Effect of rEMAP II on C6 gliomas implanted intracranially into rats and subcutaneously into mice.
  • Figures 9A-9D Intracranial C6 gliomas in rats.
  • Figure 9A C6 glioma cells were implanted stereotactically as described, and rats were maintained for 10 days, at which time they were divided into eight treatment groups as indicated. Tumor volume was evaluated on day 26 (after 16 days of treatment) . ** and * indicate p ⁇ 0.0001 and p ⁇ 0.005, respectively, by Kruskal-Wallis .
  • Fig. 9A the mean ⁇ SE is shown.
  • Figure 9B-9C the mean ⁇ SE is shown.
  • Intracranial tumors derived from C6 glioma cells, were harvested from animals treated with vehicle (Fig. 9B and 9D; IT/IP) alone or EMAP II (Fig. 9C and 9E; IT/IP) . Sections were stained with hematoxylin and eosin (9B, 9C) ) or subjected to the TUNEL procedure (9D, 9E) .
  • Figure 9F Subcutaneous C6 gliomas in nude mice. Tumor cells were implanted, animals were maintained for 3 days, and treatment with EMAP II wa ⁇ initiated for the next 24 days as described. At the end of the experiment, tumor volume was measured and data shown represent the mean ⁇ SE. The intracranial tumor experiments were repeated three times and the subcutaneous tumor studies were repeated twice.
  • FIGS. IOA, IOB, IOC, 10D and 10E Effect of rEMAP II on vascular ingrowth into Matrigel implants impregnated with VEGF.
  • Implants were evaluated by hematoxylin and eosin staining ( Figures
  • Figures IIA, IIB, IIC, 11D and HE Interaction of rEMAP II with cultured endothelial and C6 glioma cells.
  • Figure 11A- 11B Human umbilical vein endothelial cells or C6 glioma cells in Medium 199 containing fetal calf serum (10%) were exposed to rEMAP II (10nM;A) or medium alone (B) for 24 hrs at 37 ' C, samples were harvested and subjected to TUNEL analysis as described.
  • Figure IIC Quantitation of apoptotic endothelial nuclei as a ratio of labelled nuclei/cells counted in each of ten high power fields in the presence of the indicated concentration of rEMAPII.
  • This invention provides a method of treating a tumor in a subject, comprising administering to the subject an amount of an agent, selected from endothelial monocyte activating polypeptide II and an endothelial monocyte activating polypeptide II-derived polypeptide, effective to treat the tumor, wherein the endothelial monocyte activating polypeptide II is administered subcutaneously, intraperitoneally, or intravenously.
  • an agent selected from endothelial monocyte activating polypeptide II and an endothelial monocyte activating polypeptide II-derived polypeptide, effective to treat the tumor, wherein the endothelial monocyte activating polypeptide II is administered subcutaneously, intraperitoneally, or intravenously.
  • the tumor is a carcinoma.
  • EMAP II refers to Endothelial Monocyte Activating Polypeptide II.
  • rEMAP II refers to recombinant Endothelial Monocyte Activating Polypeptide II.
  • EMAP II may also include variants of naturally occurring EMAP II. Such variants can differ from naturally occurring EMAP II in amino acid sequence or in ways that do not involve sequence, or both. Variants in amino acid sequence are produced when one or more amino acids in naturally occurring EMAP II is substituted with a different natural amino acid, an amino acid derivative or non-native amino acid.
  • Particularly preferred variants include naturally occurring EMAP II, or biologically active fragments of naturally occurring EMAP II, whose sequences differ from the wild type sequence by one or more conservative amino acid substitutions, which typically have minimal influence on the secondary structure and hydrophobic nature of the protein or peptide. Variants may also have sequences which differ by one or more non- conservative amino acid substitutions, deletions or insertions which do not abolish the EMAP II biological activity.
  • Conservative substitutions typically include the substitution of one amino acid for another with similar characteristics such as substitutions within the following groups: valine, glycine; glycine, alanine; valine, isoleucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
  • the non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine.
  • the polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine.
  • the positively charged (basic) amino acids include arginine, lysine and histidine.
  • the negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
  • Phenylalanine F D-Phe,Tyr, D-Thr,L-Dopa,His,D- His, Trp, D-Trp, Trans 3,4 or 5-phenylproline, cis 3,4 or 5 phenylproline
  • variants within the invention are those with modifications which increase peptide stability.
  • Such variants may contain, for example, one or more non-peptide bonds (which replace the peptide bonds) in the peptide sequence.
  • variants that include residues other than naturally occurring L-amino acids, such as D- amino acids or non-naturally occurring or synthetic amino acids such as beta or gamma amino acids and cyclic variants are also included. Incorporation of D- instead of L-amino acids into the polypeptide may increase its resistance to proteases. See, e.g., U.S. Patent 5,219,990.
  • the peptides of this invention may also be modified by various changes such as insertions, deletions and substitutions, either conservative or nonconservative where such changes might provide for certain advantages in their use.
  • variants with amino acid substitutions which are less conservative may also result in desired derivatives, e.g., by causing changes in charge, conformation and other biological properties.
  • substitutions would include for example, substitution of hydrophilic residue for a hydrophobic residue, substitution of a cysteine or proline for another residue, substitution of a residue having a small side chain for a residue having a bulky side chain or substitution of a residue having a net positive charge for a residue having a net negative charge.
  • the derivatives may be readily assayed according to the methods disclosed herein to determine the presence or absence of the desired characteristics.
  • Variants within the scope of the invention include proteins and peptides with amino acid sequences having at least eighty percent homology with EMAP II. More preferably the sequence homology is at least ninety percent, or at least ninety-five percent.
  • Non-sequence modifications may include, for example, in vivo or in vitrc chemical derivatization of portions of naturally occurring EMAP II, as well as changes in acetylation, methylation, phosphorylation, carboxylation or glycosylation.
  • the protein is modified by chemical modifications in which activity is preserved.
  • the proteins may be amidated, sulfated, singly or multiply halogenated, alkylated, carboxylated, or phosphorylated.
  • the protein may also be singly or multiply acylated, such as with an acetyl group, with a farnesyl moiety, or with a fatty acid, which may be saturated, monounsaturated or polyunsaturated.
  • the fatty acid may also be singly or multiply fluorinated.
  • the invention also includes methionine analogs of the protein, for example the methionine sulfone and methionine sulfoxide analogs.
  • the invention also includes salts of the proteins, such as ammonium salts, including alkyl or aryl ammonium salts, sulfate, hydrogen sulfate, phosphate, hydrogen phosphate, dihydrogen phosphate, thiosulfate, carbonate, bicarbonate, benzoate, sulfonate, thiosulfonate, mesylate, ethyl sulfonate and benzensulfonate salts.
  • ammonium salts including alkyl or aryl ammonium salts, sulfate, hydrogen sulfate, phosphate, hydrogen phosphate, dihydrogen phosphate, thiosulfate, carbonate, bicarbonate, benzoate, sulfonate, thiosulfonate, mesylate, ethyl sulfonate and benzensulfonate salts.
  • Variants of EMAP II may also include peptidomimetics of EMAP II .
  • Such compounds are well known to those of skill in the art and are produced through the substitution of certain R groups or amino acids in the protein with non-physiological, non-natural replacements. Such substitutions may increase the stability of such compound beyond that of the naturally occurring compound.
  • the subject is a mammal.
  • suitable mammalian subjects include, but are not limited to, murine animals such as mice and rats, hamsters, rabbits, goats, pigs, sheep, cats, dogs, cows, monkeys and humans.
  • the agent is administered intraperitoneally.
  • the agent is administered in at least twenty doses. In a specific embodiment the agent is administered in about twenty-four doses. In an embodiment the agent is administered over a period of at least ten days.
  • the agent is administered over a period of about twelve days. In an embodiment of this invention, the frequency of administration is at least about one dose every twelve hours. In an embodiment the effective amount is from about 2.4 micrograms to about 24 micrograms. In an embodiment the effective amount is from about 100 nanograms to 24 micrograms per dose. In a more specific embodiment the effective amount is from about 100 nanograms to about 1000 nanograms per dose.
  • the endothelial monocyte activating polypeptide II-derived polypeptide is at least about ninety percent homologous to the sequence (S/M/G) KPIDASRLDLRIG
  • QIQPDLHTNAECVATYKGAPFEVKGKGVCRAQTMANSGIK (SEQ I.D. No. ) , wherein the sequence is truncated by from zero to about three amino-terminal residues and from zero to about one hundred thirty-six carboxy-terminal residues. In a preferred embodiment the homology is at least about ninety- five percent.
  • the endothelial monocyte activating polypeptide I I - derived polypeptide is at least about ninety percent homologous to the sequence ( S/M/G) KPIDVSRLDLRIG ( C/R ) I ITARK ⁇ PDADSLYVEEVDVGEIAPRTVVSGLVNHVPLEQMQNRMVILLCNLK
  • QIQPDLHTNDECVATYKGVPFEVKGKGVCRAQTMSNSGIK SEQ I . D . No . ) , wherein the sequence is truncated by f rom zero to about three amino-terminal residues and from zero to about one hundred thirty-six carboxy-terminal residues. In a preferred embodiment the homology is at least about ninety- five percent.
  • the agent is endothelial monocyte activating polypeptide II.
  • the EMAP II is murine EMAP II or human EMAP II.
  • the endothelial monocyte activating polypeptide II is recombinant endothelial monocyte activating polypeptide II.
  • tumors that are too small for intratumoral injection can be treated before they grow to a larger size. Accordingly, in an embodiment of this invention the tumor is too small for intratumoral injection.
  • the diameter of the tumor is less than or equal to about two millimeters.
  • This invention provides a method of inhibiting the growth of endothelial cells, comprising contacting the endothelial cells with an amount of an agent, selected from endothelial monocyte activating polypeptide II and an endothelial monocyte activating polypeptide II-derived polypeptide, effective to inhibit growth of the endothelial cells.
  • the endothelial cells are aortic endothelial cells, for example bovine aortic endothelial cells.
  • This invention provides a method of inhibiting the formation of blood vessels in a subject, comprising administering to the subject an effective amount of an agent, selected from endothelial monocyte activating polypeptide II and an endothelial monocyte activating polypeptide II-derived polypeptide, thereby inhibiting the formation of blood vessels in the subject.
  • an agent selected from endothelial monocyte activating polypeptide II and an endothelial monocyte activating polypeptide II-derived polypeptide, thereby inhibiting the formation of blood vessels in the subject.
  • the subject is a mammal.
  • suitable mammalian subjects include, but are not limited to, murine animals such as mice and rats, hamsters, rabbits, goats, pigs, sheep, cats, dogs, cows, monkeys and humans .
  • the agent may be administered according to techniques well known to those of skill in the art, including but not limited to subcutaneously, intravascularly, intraperitoneally, topically, or intramuscularly.
  • the effective amount is from about 10 nanograms to about 24 micrograms. In a specific embodiment the effective amount is from about 100 nanograms to about 1 microgram.
  • the endothelial monocyte activating polypeptide II-derived polypeptide is at least about ninety percent homologous to the sequence (S/M/G) KPIDASRLDLRIG
  • QIQPDLHTNAECVATYKGAPFEVKGKGVCRAQTMANSGIK (SEQ I.D. No. ) , wherein the sequence is truncated by from zero to about three amino-terminal residues and from zero to about one hundred thirty-six carboxy-terminal residues. In a preferred embodiment the homology is at least about ninety- five percent .
  • the endothelial monocyte activating polypeptide II-derived polypeptide is at least about ninety percent homologous to the sequence (S/M/G) KPIDVSRLDLRIG
  • QIQPDLHTNDECVATYKGVPFEVKGKGVCRAQTMSNSGIK (SEQ I.D. No. ) , wherein the sequence is truncated by from zero to about three amino-terminal residues and from zero to about one hundred thirty-six carboxy-terminal residues. In a preferred embodiment the homology is at least about ninety- five percent .
  • the agent is endothelial monocyte activating polypeptide II.
  • the EMAP II is murine EMAP II or human EMAP II.
  • the endothelial monocyte activating polypeptide II is recombinant endothelial monocyte activating polypeptide II.
  • This invention provides a method of treating a condition involving the presence of excess blood vessels in a subject, comprising administering to the subject an effective amount of an agent, selected from endothelial monocyte activating polypeptide II and an endothelial monocyte activating polypeptide II-derived polypeptide, thereby treating the condition involving the presence of excess blood vessels.
  • an agent selected from endothelial monocyte activating polypeptide II and an endothelial monocyte activating polypeptide II-derived polypeptide
  • the condition involves the presence of excess blood vessels in the eye.
  • One such condition is retinopathy.
  • the retinopathy is diabetic retinopathy, sickle cell retinopathy, retinopathy of prematurity, or age related macular degeneration.
  • the present invention provides for a method of treating a tumor in a subject, comprising administering to the subject an amount of an agent, selected from endothelial monocyte activating polypeptide II and an endothelial monocyte activating polypeptide II-derived polypeptide, effective to treat the tumor, wherein the endothelial monocyte activating polypeptide II is administered subcutaneously or intraperitoneally; and intravenously, intracranially, or intramorally.
  • the tumor may be a glioblastoma.
  • the agent may be administered intratumorally by positive pressure microinf sion.
  • the present invention further provides for a method for evaluating the ability of an agent to inhibit growth of endothelial cells, which includes: (a) contacting the endothelial cells with an amount of the agent, selected from endothelial monocyte activating polypeptide II and an endothelial monocyte activating polypeptide II-derived polypeptide; (b) determining the growth of the endothelial cells, and (c) comparing the amount of growth of the endothelial cells determined in step (b) with the amount determined in the absence of the agent, thus evaluating the ability of the agent to inhibit growth of endothelial cells.
  • the present invention provides for a method for evaluating the ability of an agent to inhibit the formation of blood vessels in a cellular environment, which comprises: (a) contacting the cellular environment with an amount of the agent, selected from endothelial monocyte activating polypeptide II and an endothelial monocyte activating polypeptide II-derived polypeptide; (b) determining whether or not blood vessels form in the cellular environment, and (c) comparing the amount of growth of blood vessels determined in step (b) with the amount determined in the absence of the agent, thus evaluating the ability of the agent to inhibit formation of blood vessels is the cellular environment.
  • a cellular environment includes but is not limited to a cell culture system, cells in vivo, cells in vitro, an organ culture, an animal model system.
  • a cellular environment may include a cells growing in a subject, a tumor cell culture system, an endothelial cell culture system, an embryonic cell culture system, an angiogenic cell culture system.
  • a cellular environment may be either in vitro or in vivo.
  • a cellular environment may include a hybridoma cell culture system.
  • the present invention provides for a pharmaceutical composition which comprises an agent capable of inhibiting blood vessel formation and a pharmaceutically acceptable carrier.
  • the carrier may include but is not limited to a diluent, an aerosol, a topical carrier, an aquous solution, a nonaqueous solution or a solid carrier.
  • Bovine aortic endothelial cells were isolated from calf aortae, grown in culture and characterized, based on the presence of von Willebrand factor and thrombomodulin, as described previously (Nawroth P. , 1988) .
  • Bovine vascular smooth muscle cells were prepared by additional scraping of the aortae following removal of the endothelium, and were characterized based on the presence of smooth muscle cell actin (Gown A., 1985) .
  • Lewis Lung carcinoma cells obtained from American Type Culture Collection (ATCC) , NIH 3T3 cells (ATCC) , and B16 (FlO) cells were all maintained in high glucose defined minimal essential medium (DMEM; Gibco) containing fetal bovine serum. Meth A tumor cells were provided by Dr. Lloyd Old (Center for Cancer Research, NY) , and grown as described (Old L., 1987; Old L., 1961) .
  • Recombinant EMAP II was prepared from E. coli (host HMS174 [DE3] ) transformed with a plasmid containing the coding sequence for mature EMAP II, as described previously (Kao J., 1994) .
  • a procedure was developed for purification of recombinant EMAP II on a preparative scale. Frozen (- 80°C) E. coli cell paste was mixed 1:10 (w/v) with Tris-HCl (20mM; pH 7.4) containing octyl- ⁇ -glucoside (0.1%) and an homogeneous suspension formed by agitation with a TURRAX ® for 20 min (speed 60) at 4°C.
  • the suspension was then disrupted by three passes through a TURRAX ® Microfluidizer (Mode 110F) at 4°C.
  • Polyethylene imine at pH 7 was then added to the homogenate to a concentration of 0.25%.
  • the homogenate was left for 30 min on ice to precipitate cell debris and DNA. Solids were removed from the homogenate by centrifugation (5000xg; 30 min) , the polyethylene imine supernatant was retained and filtered (0.2 ⁇ m) , and applied
  • the Heparin Sepharose pool was concentrated using an Amicon Stirred Cell (Amicon) with an Amicon YM10 Diaflo Ultrafilter to less than 100 ml.
  • the retentate was desalted into 3- (Morpholino) -propane-sulfonic acid (MOPS; 25 mM; pH 6.9) on a Sephadex G25 (Medium Grade, Pharmacia) size exclusion column (480 ml bed volume) .
  • the pool was then applied to an SP Sepharose High Performance (Pharmacia) cation exchange column (55ml bed volume) run at a flow rate of 10 ml/min. After washing the column with MOPS buffer, EMAP II- containing fractions were eluted by application of a 0 to 0.5 M ascending linear salt gradient in MOPS.
  • the pool was identified, assayed for total protein an biological activity as above .
  • EMAP II-containing fractions from SP Sepharose chromatography were adjusted to 2 M in (NH 4 ) 2 S0 4 with solid (NH 4 ) 2 S0 4 and applied to a Phenyl Toyopearl 650 M (Tosohaas) column (90 ml bed volume) equilibrated in sodium phosphate (20 mM; pH 7) containing 1 M (NH 4 ) 2 SO . After washing with the above buffer, a descending gradient of salt (2 to 0M) in sodium phosphate (20 mM) was applied. EMAP II-containing fractions were pool and characterized as above.
  • EMAP II from in the Phenyl Toyopearl column eluate was concentrated to 3-5 mg/ml, and formulated into phosphate- buffered saline (PBS; pH 7.4) by buffer exchange on a Sephadex G25 column (as above) .
  • Lipopolysaccharide (LPS) was removed using filtration through a Posidyne filter (Pall Corp.) , and LPS levels were estimated using the Endospecy chromogenic assay (limit of detection ⁇ 10pg/ml) .
  • Purified EMAP II, as well as EMAP II in fractions obtained during the purification procedure was subjected to N-terminal sequence analysis, mass spectrometry and SDS-PAGE.
  • Immunoblotting was performed following SDS-PAGE by transferring protein to nitrocellulose in Tris-HCl (12 mM) , glycine (96 mM; final pH 8.3) containing methanol (20%) using the Novex Western Transfer Apparatus at constant voltage (30 V) for 2-4 hr (4°C) . Prestained, low molecular weight markers (Bio-Rad) were used to follow the transfer. Immunoreactive protein was visualized using rabbit anti- mature EMAP II N-terminal peptide IgG (0.1 ⁇ g/ml) followed by the Amplified Alkaline Phosphatase Goat Anti-Rabbit Immuno-Blot Assay Kit (Bio-Rad) .
  • Antibody to EMAP II was prepared by standard methods (30) , and was found to be monospecfic based on immunoblotting of plasma and cell extracts. This antibody was used to develop an ELISA to detect EMAP II antigen; cells or tissues were homogenized in the presence of protese inhibitors (phenylmethylsulfonyl fluoride, 1 mM; trasylol, 0.1%) , centrifuged to remove debris, and the supernatant was diluted in carbonate/bicarbonate buffer (pH 9.6) and incubated in Maxisorb microtiter plates (Nunc) overnight at 4°C.
  • protese inhibitors phenylmethylsulfonyl fluoride, 1 mM; trasylol, 0.1%)
  • Matrigel model Matrigel (Kleinman H., 1986; Passaniti A.,
  • Balb/c mice received 125 I-EMAP II (0.26 ⁇ g) either intravenously (IV) via the tail vein or intraperitoneally (IP) . Plasma samples were taken at the indicated times, and animals were sacrificed at 24 hours.
  • Plasma 125 I-EMAP II in the tissue was corrected for residual blood based on the presence of 51 Cr-labelled microsperes .
  • Plasma 125 I-EMAP II concentration data were fit to a two-compartment open model using nonlinear regression by extended leat squares analysis (Siphar, SIMED, Creteil, France) .
  • residual analysis an examination of the standard deviation
  • Akaike, Leonard and Schwarz criteria were tested to select the most appropriate model (Yamoaka K., 1978) .
  • t 1/2 C-, t 1/2 ⁇ , t 1/2 r denote half-lives for distribution, elimination and resorption half-lives, respectively.
  • Tumor models LLC and B16 (FlO) cells were rinsed with Hanks buffered saline solution, trypsinized, counted, resuspended in phosphate-buffered saline, and injected subcutaneously into backs of C57BL6/J mice (2 x 10° cells/animal) .
  • animals Under the third day following administration of tumor cells, animals underwent IP injection of EMAP II every 12 hrs for 12 days of either vehicle alone (serum albumin, 1%) , vehicle + EMAP II (at 100 or 1000 ng) , or vehicle + hea -inactivated EMAP II (1000 ng) .
  • Tumor volume data was analyzed using the Kruskal-Wallis one way ANOVA and a Mann-Whitney mean rank test. Animals were sacrificed and tumors analyzed histologically at day 15.
  • LLC an Meth A
  • Paraffin embedded tumor slices were deparaffinized and digoxigenin-11-UTP was used to label fragmented DNA according to the Genius 1 kit (Amersham, location) .
  • tissue was treated with proteinase K (1 ⁇ g/ml) , and incubated with digoxigenin-11- UTP, klenow, and DNTP's overnight. Nitroblue tetrazolium and alkaline phosphatase were used to reveal the digoxigenin labelled DNA fragments.
  • sequential sections of Meth A tumors underwent analysis for DNA fragmentation and EMAP II. Apoptosis was analyzed in cultured cells exposed to EMAP II (10 and 100 ng) .
  • lysis buffer Tris-borate buffer, 45 mM; EDTA, ImM; pH 8.0; NP-40, 0.25%
  • proteinase K 1 mg/ml
  • RNAase A 0.1 mg/ml
  • DNA was purified by phenol-chloroform extraction.
  • DNA (10 ⁇ g/lane) was subjected to agarose gel (1.8%) electrophoresis at 40 volts using an 100 bp ladder as standard (Boehringer Mannheim) . Gels were stained with ethidium bromide.
  • EMAP II Preparative scale purification of recombinant EMAP II. Previous studies have employed FPLC Mono Q followed by preparative SDS-PAGE to prepare homogenous EMAP II (Kao J., 1992; Kao J. , 1994) , resulting in microgram quantities of purified EMAP II. In order to obtain the larger amounts of polypeptide necessary for a range of studies to characterize properties of EMAP II, using in vitro and in vivo systems, a larger scale approach was employed. E. coli paste, from bacteria transformed with the a plasmid expressing mature EMAP II, was disrupted using a microfluidizer, the latter method found to be 100% effective based on light microscopy.
  • Protein yields of over 98% were obtained by the concentration and formulation of EMAP II into phosphate- buffered saline. Posidyne filtration caused no loss of protein and reduced endoxoin levels to ⁇ 10 pg/3-5 mg purified EMAP II protein. The final formulated pool was seen as an apparently diffuse band at 21 kDa by gel electrophoresis (Fig. 1, lane 6) . The faint band at Mr -40 kDa was probably due to aggregation of EMAP II, as indicated by the characterization of the purified material below. Mass spectrometry gave measured mass of 18,006 which is close to the expected mass of 17,970.
  • N-terminal sequence analysis showed a single sequence with an 100% match between purified murine EMAP II and the published sequence (Kao J. , 1992; Kao J. , 1994) .
  • the purified material was also recognized by anti-mature EMAP II amino terminal peptide IgG by immunoblotting, and in the endothelial cell tissue factor induction assay gave activities of 0.3-0.4 units/ng of protein. The latter is what has been observed with nonrecombinant EMAP II prepared from meth A-induced murine fibrosarcomas (Kao J., 1992) or recombinant EMAP II prepared by a non-preparative scale method (Kao J., 1994) .
  • FIG. 3A which closely parallelled the appearance of implants from animals whose gel content either bFGF/herapin + vehicle (albumin) or bFGF/herapin + heat-treated EMAP II (Fig. 3B) .
  • This induction of blood vessel formation is similar to that reported previously with bFGF in this model (Passaniti A. , 1992) .
  • Plasma clearance and tissue deposition of infused EMAP II In order to perform in vivo studies with EMAP II, its plasma clearance and tissue deposition was evaluated (Fig. 4A) . Clearance studies were performed using 125 I-EMAP II administered either IV or IP. The fall in plasma concentration of)I-EMAP II after IV injection fit best to a bi-exponential function; the distribution and elimination half-lives were 0.47+0.17 and 103 ⁇ 5 min, respectively. Following IP injection, 125 I-EMAP II was detected in plasma after 1 min, and the maximum concentration was reached by 35 ⁇ 10min. The resorption phase of EMAP II handling in vivo was best described as a first-order process. The elimination phase following IP administration fit to a onoexponential decline, and the resorption and elimination half-lives were 50.1 ⁇ 0.10 and 102 ⁇ 6 min, respectively.
  • EMAP II Effect of EMAP II on endothelium.
  • EMAP II was initially isolated from Meth A tumors, due to their known thrombohemorrhage, resulting in spontaneously occurring areas of apparent necrosis/apoptosis (Old L. , 1986) .
  • These data along with examination of multiple LLC and B16 melanomas following treatment with EMAP II (in which apoptosis followed a perivascular pattern) , suggested that EMAP II might modulate endothelial cell growth.
  • Vasculature in tumors is known for its prothrombotic diathesis, increased permeability, exaggerated response to cytokines, and increased number of growing/migrating endothelial cells (Folkman, J., 1995; Old L., 1986; Asher A., 1987; Constantinidis I., 1989; Watanable N., 1988; Senger D., 1983) .
  • These properties which distinguish vessels in the tumor bed from these in normal tissues, suggest parameters to be exploited in defining agents to selectively target tumor neovasculature.
  • EMAP II was first studied based on its modulation of endothelial properties, such as induction of leukocyte adherence molecules and the procoagulant cofactor tissue factor. Further studies on mononuclear phagocytes and polymorphonuclear leukocytes confirmed its ability to induce cell migration and activation. These data suggested that EMAP II had properties of an inflammatory cytokine, at least based on in vitro findings.
  • EMAP II The effect of EMAP II is less likely to be mediated by direct action on the tumor cells, as EMAP II does not impact adversely on tumor cell growth and viability in vitro.
  • experiments with cultured endothelium demonstrated induction of apoptosis of rapidly growing cultures, whereas there was a less pronounced effect on cultures approaching confluence.
  • mitoses in just-confluent endothelium were markedly diminished, induction of programmed cell death was minimal, possibly to a cell cycle-dependence of EMAP II-induced cellular effects.
  • hypoxia is an important stimulus for angiogenesis, it was of interest to note that EMAP II had an exaggerated apoptotic effect in endothelial cultures subjected to oxygen deprivation.
  • a hypoxia induced retinal neovascularization model has been well established by the "Association for Research in Vision and Ophthalmology Statement for the use of Animals in Ophthalmic and Vision Research," is followed.
  • To produce retinal neovascularization litters of 7 day old (postnatal day seven -P7) C57BL/6J mice with nursing mothers are exposed to 75% oxygen for 5 days and returned to room air at age P12 (room air will mimic hypoxia in the mouse) . Animals receive IP vehicle (mouse serum albumin) - control, EMAP II 100-lOOOng or heat inactivated EMAP II (lOOOng) every twelve hours beginning on P7 and continuing until evaluation of retina. Mice of the same age kept in room air are used as controls.
  • mice are evaluated on days P13-18 in room air for the development of retinopathy. This is accomplished by humane euthanasia of the mice, the infusion of a fluorescein-dextran solution and the use of fluorescence microscopy for the viewing of the eye vasculature. By assessing the amount of new vascularization, inhibition of retinal angiogenesis is demonstrated.
  • Endothelial-monocyte Activating Polypeptide II a Novel Anti-tumor Cytokine That Suppresses Primary and Metastatic Tumor Growth, and Induces Apoptosis in Growing Endothelial Cells
  • Neovascularization is essential for growth and spread of primary and metastatic tumors.
  • murine methylcholanthrene A-induced fibrosarcomas well-known for their spontaneous vascular insufficiency
  • a novel cytokine has been identified and purified, Endothelial-Monocyte Activating Polypeptide (EMAP) II, that potently inhibits tumor growth in vivo, and appears to have anti-angiogenic activity in vivo and in vitro.
  • EMAP II Endothelial-Monocyte Activating Polypeptide
  • EMAP II suppressed the growth of primary Lewis Lung Carcinomas, with a reduction in tumor volume of 65% compared with controls (p ⁇ 0.003 by Mann-Whitney) .
  • EMAP II blocked outgrowth of Lewis lung carcinoma macrometastases; total surface metastases were suppressed by 65%, and of the 35% metastases present, about 80% of these were inhibited with maximum diameter ⁇ 2 mm (p ⁇ 0.002 compared with controls) .
  • EMAP II In growing capillary endothelial cultures, EMAP II induced apoptosis in a time- and dose-dependent manner; an effect enhanced by concomitant hypoxia, whereas other cell types, such as Lewis Lung carcinoma cells, were unaffected.
  • meth A methylcholanthrene A-induced fibrosarcoma
  • EMAP Endothelial-Monocyte Activating Polypeptide
  • TNF Tumor Necrosis Factor
  • EC endothelial cell
  • SMC smooth muscle cell
  • LPS lipopolysaccharide
  • r recombinant
  • BFGF basis fibroblast growth factor
  • LLC Lewis Lung Carcinoma
  • IV intraperitoneal
  • IP intraperitoneal
  • DAP-1 6-diamidino- 2phenylindoledilactate
  • TUNEL terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling
  • VEGF Vascular Endothelial Growth Factor.
  • Murine methylcholanthrene A-induced (meth A) fibrosarcomas which exhibit spontaneous vascular insufficiency manifested by a heterogeneous pattern of thrombohemorrhage and central necrosis, as well as their failure to form metastatic lesions (Old, L., 1986; Old, L., 1961) , provide an ideal starting point for isolation of tumor-derived mediators which perturb the vasculature (Clauss, M. , 1990; Clauss, M., 1990; Kao, J., 1992; Kao, J., 1994) .
  • EMAP II Endothelial-Monocyte Activating Polypeptide II
  • meth A-conditioned medium meth A-conditioned medium based on its capacity to induce activation of endothelial cells and mononuclear phagocytes.
  • EMAP II showed no significant homology to other known protein ⁇ , such as cytokines or growth factor ⁇ .
  • EMAP II induced endothelial release of von Willebrand factor translocation of P-selectin to the cell surface, synthesis and expression of E-selectin and procoagulant tissue factor (Kao, J., 1992; Kao, J., 1994) ; these, and EMAP II-mediated activation of cultured monocytes, resulting in production of cytokines and stimulation of cell migration, suggested phlogogenic properties.
  • TNF tumor necrosis factor
  • Interleukin 1 Interleukin 1
  • EMAP II has anti-angiogenic properties preventing blood vessel ingrowth in an experimental angiogenesis model, and suppressing the growth of primary and metastatic tumors without toxicity in normal organs. Consistent with this hypothesis, EMAP II appears to target growing endothelial cells; exposure of growing cultured capillary endothelium to EMAP II induces apoptosis, which is magnified by concomitant hypoxia.
  • EMAP II is a polypeptide with anti-angiogenic properties which targets rapidly growing vascular beds, and ⁇ ugge ⁇ ts that, in addition to its effects on tumor neovessels, it may contribute to phases of normal development and wound repair in which cessation of blood vessel growth and tissue resorption are critical.
  • Bovine aortic and capillary endothelial cells were isolated from calf aortae and adrenal, respectively, grown in culture and characterized, based on the presence of vonWillebrand factor and thrombomodulin, as described previously (Gerlach, H., 1989) .
  • Bovine vascular smooth muscle cells were prepared by additional scraping of the aortae following removal of the endothelium, and were characterized based on the presence of smooth muscle cell actin (Gown, A., 1985) .
  • Lewis Lung Carcinoma (LLC) and B16(F10) melanoma cells obtained from American Type Culture Collection (ATCC) , were maintained in high glucose-defined Minimal Essential Medium (DMEM; Gibco) containing fetal bovine serum (10%) . Meth A tumor cells (Center for Cancer Research, NY) were grown as described (Old, L., 1986) . EMAP II-induced apoptosis was studied in subconfluent endothelial culture ⁇ (Gerlach, H., 1989) . DNA fragmentation was quantified using a 5-bromodeoxyuridine (Brdu) incorporation kit from Boehringer-Mannheim according to the manufacturer's instructions.
  • Prdu 5-bromodeoxyuridine
  • cells were incubated for 12 hrs with BrdU, were plated for 24 hrs on 96-well plates, and were then treated with either vehicle (fetal bovine serum, 10%) alone or vehicle + rEMAP II, as indicated. After 12 or 24 hrs at 37°C, cells were lysed, centrifuged (250xg) for 10 min, and then the top 0.1 ml was aspirated and applied to an ELISA plate with pre-adsorbed anti-DNA antibody. Site of primary antibody binding were identified using peroxidase-conjugated anti-BrdU antibody. Where indicated, EC ⁇ were incubated with rEMAP II and/or exposed to hypoxia
  • Recombinant EMAP II was prepared from E. coli (host HMS174 [DE3] ) transformed with a plasmid containing the coding sequence for mature EMAP II, as described previously (Kao, J., 1994) .
  • Frozen (-80 * C) E. coli cell paste was mixed 1:10 (w/v) with Tris-HCl (20 mM; pH 7.4) containing octyl- ⁇ -glucoside (0.1%) and an homogeneous suspension was formed by agitation using a microfluidizer for 20 min (speed 60) at 4°C.
  • the Heparin Sepharose pool was concentrated using an Amicon Stirred Cell (Amicon) , the retentate was desalted into 3- (Morpholino) -propane-sulfonic acid (MOPS, 25 mM; pH 6.9) , and was then applied to an SP Sepharose High Performance (Pharmacia) cation exchange column (55 ml bed volume) .
  • MOPS 3- (Morpholino) -propane-sulfonic acid
  • SP Sepharose High Performance (Pharmacia) cation exchange column 55 ml bed volume
  • the column was eluted by application of a 0 to 0.5 M ascending linear salt gradient in MOPS, and EMAP II-containing fractions were adjusted to 2 M in (NH ) 2 S0 4 , applied to a Phenyl Toyopearl 650 M (Tosohaas) column (90 ml bed volume) , equilibrated in sodium phosphate (20 mM; pH 7) containing 1 M (NH 4 ) 2 S0 4 .
  • the column was eluted with a descending salt gradient (2 to 0 M) in sodium phosphate (20 mM) , and EMAP II in the Phenyl Toyopearl column eluate was concentrated to 3-5 mg/ml, and formulated into phosphate-buffered saline (PBS; pH 7.4) by buffer exchange on a Sephadex G25 column (as above) .
  • Lipopolysaccharide (LPS) was removed using filtration through a Posidyne filter (Pall Corp.) , and LPS levels were estimated using the Endospecy chromogenic assay (limit of detection ⁇ 10 pg/ml) .
  • EMAP II was subjected to N-terminal sequence analysis, mass spectrometry and SDS-PAGE; the current material was found to be homogeneous according to these criteria.
  • Antibody to rEMAP II was prepared by standard methods in rabbits (Vaitukatis, J., 1981) and was found to be monospecific, based on immunoblotting of plasma and cell extracts, and anti-EMAP II IgG blocked the activity of rEMAP II in cell culture assays (Kao, J., 1994) .
  • This antibody wa ⁇ used to develop an ELISA to detect EMAP II antigen by the general protocol described previously (Kao, J. , 1994) .
  • RNA Stat-60 kit Teltest
  • Taq polymerase Perkin-Elmer-Cetus
  • Matrigel model Matrigel (Kleinman, H., 1986; Passaniti, A., 1992) (Collaborative Research) containing either vehicle (1% BSA) , rEMAP II (100 ng/ml) + vehicle; basic Fibroblast Growth Factor (bFGF; 100 ng/ml; Collaborative Research) + heparin (40 U/ml; Sigma) + vehicle; rEMAP II (100 ng/ml) + bFGF/heparin + vehicle, or heat-inactivated rEMAP II (100 ng/ml; alone or with bFGF/heparin) + vehicle was mixed at 4° C. Matrigel mixtures were injected subcutaneously into
  • Balb/c mice received 125 I-rEMAP II (0.26 ⁇ g) either intravenously (IV) via the tail vein or intraperitoneally (IP) . Plasma samples were taken, and animals were sacrificed at 24 hours. Organs were then dried, weighed and radioactivity assessed.
  • mice bearing 14 day old subcutaneous B16 tumors received 125 I-rEMAP II (0.26 ⁇ g/animal; IP) , and, 1 hour before sacrifice, were infused with 51 Cr-labelled microspheres (10 ⁇ ) in normal saline (the latter to monitor residual blood in the tis ⁇ ue) .
  • I-rEMAP II I-rEMAP II
  • I-rEMAP II plasma clearance, volume of distribution, and accumulation in tumor tissue.
  • tissue as ⁇ ociated radioactivity was determined on weighed samples either after drying (for total radioactivity) , or following homogenization of tissue and trichloroacetic acid precipitation (20%) .
  • Plasma 125 I-rEMAP II in the tissue was corrected for residual blood based on the presence of 51 Cr-labelled microspheres.
  • Plasma 125 I-rEMAP II concentration data were fit to a two-compartment open model using nonlinear regression by extended least squares analysis (Siphar, SIMED, Creteil, France) .
  • Siphar, SIMED, Creteil, France extended least squares analysis
  • mice were subcutaneously injected with meth A cells and on day 9 ⁇ tarted on a course of IP injections every third day of either nonimmune rabbit IgG (400 g/dose) or rabbit anti-murine EMAP II IgG (200 or 400 ⁇ g/dose) .
  • This regimen of IgG administration was based on pilot studie ⁇ in which 125 I-rabbit anti-EMAP II IgG infu ⁇ ed into mice demonstrated a half-life of elimination of 29.4 ⁇ 2.67 hrs. Animals were sacrificed at day 14 and tissue was analyzed for evidence of apoptosi ⁇ a ⁇ de ⁇ cribed below.
  • Tumor volume data were analyzed using the Kruskal-Wallis one way ANOVA and a Mann-Whitney mean rank test. Data is expressed as a dimensionless ratio of observed tumor volume divided by initial (day 3) tumor volume. Animals were sacrificed and tumors analyzed histologically at day 15.
  • mice received LLC cells subcutaneou ⁇ ly and were observed until tumor volume reached ⁇ l .5 cm 3 .
  • Animals then received rEMAP II (1000 ng/dose) in vehicle or vehicle alone IP every 12 hrs for 72 hrs prior to resection of the primary tumor.
  • mice were observed for an additional 15 days, during which time they received rEMAP II (1000 ng IP every 12 hrs) in vehicle or vehicle alone (same schedule) .
  • lungs were injected intratracheally with India ink (15%) to visualize lung surface nodules, and tissue was fixed in Fekete's solution (70% alcohol; 5% glacial acetic acid; 3.7% formaldehyde) .
  • Fekete's solution 70% alcohol; 5% glacial acetic acid; 3.7% formaldehyde
  • Surface meta ⁇ tatic le ⁇ ions were counted by gross inspection of the tissue under 4X-magnification, and macrometastases were defined based on a smallest surface nodule diameter >2 mm.
  • Tissue analysis histology, apoptosis, immunohistology.
  • TUNEL terminal deoxynu leotidyl transferase-mediated dUTP-biotin nick end labeling
  • EMAP II antigen showed virtually undetectable levels in the above normal tissues (limit of detection ⁇ 250 pg/ml) and no peak of EMAP II in the plasma after LPS administration or hind limb ischemia.
  • EMAP II Effect of EMAP II on bFGF-induced angiogenesis.
  • bFGF and hepeirin were mixed with a gel of ba ⁇ ement membrane proteins produced by Engelbreth-Holm-Swarm tumor cells (Matrigel) to serve as a model angiogenic stimulus (Klein an, H., 1986; Pas ⁇ aniti, A. , 1992) .
  • Subcutaneou ⁇ Matrigel implants in C57BL6/J mice were evaluated 14 days after inoculation for vessel formation, cellular infiltration and hemoglobin content.
  • Histologic analysi ⁇ of the gel showed formation of vessels to be most pronounced and comparable in implants from animals treated with either bFGF/heparin + vehicle (albumin) or bFGF/heparin + heat-inactivated rEMAP II + vehicle; higher magnification confirmed the presence of neovessels in these implants.
  • This induction of blood ves ⁇ el formation is similar to that reported previously with bFGF in this model (Passaniti, A., 1992) .
  • Plasma clearance and tissue deposition of infused rEMAP II Plasma clearance and tissue deposition of infused rEMAP II.
  • the resorption phase of rEMAP II handling in vivo was be ⁇ t described as a first-order proces ⁇ .
  • the elimination phase following IP administration fit to a monoexponential decline, and the resorption and elimination half-lives were 50.1 ⁇ 0.1 and 102+6 min, respectively.
  • the precipitability of the tracer in trichloroacetic acid (20%) was greater in the tumor compared with other tissues, consi ⁇ tent with a relative accumulation of apparently intact rEMAP II in tumor ti ⁇ ue.
  • mice receiving active rEMAP II showed a striking reduction in tumor volume (Fig. 5A-G) Differences between tumor volume in control and EMAP Il-treated animals were statistically significant using either the Kruskal-Walli ⁇ one way ANOVA analy ⁇ is (p ⁇ 0.034) or comparing control versus high dose rEMAP II by Mann-Whitney analysis (p ⁇ 0.003) .
  • rEMAP II might suppress growth of metastatic lesions.
  • the LLC model was employed by allowing primary tumors to grow to a volume of ⁇ 1.5 cm 3 , at which time metastases are present (but suppressed by the primary the primary tumor; O'Reilly, M., 1994; Holmgren, L., 1995) . Then the primary lesion was resected (with no recurrence at the site of resection) , and analysis of surface lung nodules was undertaken 15 days later.
  • rEMAP II treatment was begun 72 hr prior to resection of the primary tumor and was continued through the end of the experiment (See Figs. 8A-E) .
  • Animal ⁇ receiving rEMAP II 1000 ng IP every 12 hrs
  • rEMAP Il-treated animals demon ⁇ trated 65% suppression (p ⁇ 0.009 by Mann Whitney) in outgrowth of the total number of surface metastases, compared with mice receiving vehicle alone (Fig. 8E) .
  • ELISA for DNA fragmentation was performed to more precisely delineate apoptotic effects of rEMAP II on endothelium: there was a dose-dependent increase in DNA fragmentation in cultured capillary endothelium, reaching 250% over that observed in controls within 24 hrs (Fig. 6E) .
  • tumor tissue is also known for the presence of areas of local tissue hypoxia/hypoxemia (Olive, P., 1992; Kalra, R. , 1994) , it wa ⁇ a ⁇ essed whether rEMAP II might display enhanced activity under oxygen deprivation.
  • Neovascularization is a critical regulator of the growth of both primary and metastatic neoplasms (Fidler, I., 1994; Folkman, J., 1989; Folkman, J. , 1995; Murray, C, 1995) .
  • vascular endothelial growth factor VEGF; Plate, K., 1992; Warren, R. , 1995; Kim, J., 1993
  • acidic fibroblast growth factor Maciag, T., 1984
  • bFGF basic fibroblast growth factor
  • angiogenin Fett, J. , 1985; King, T., 1991, Olson, K.
  • angiostatin O'Reilly, M. , 1994
  • thrombospondin TRIron, K., 1994
  • glioma-derived angiogenesis inhibitory factor Van Meir, E., 1994
  • they can inhibit tumor growth either at the primary tumor site (thrombospondin; Dameron, K., 1994) or at a site of di ⁇ tant metastases (angiostatin; O'Reilly, M., 1994; O'Reilly, M. , 1996) .
  • Carcinogen-induced murine meth A and similar tumors are ideally suited to the analysis of host-tumor interactions because short-term vascular insufficiency (exaggerated by concomitant administration of an agent such as TNF) , and longer-term immunologic mechanisms limit local tumor growth (Old, L., 1986; Old, L., 1961; Nawroth, P., 1988; Watanabe, N. , 1988; Freudenberg,
  • A-derived EMAP II with apoptosis in the tumor bed (the latter suppre ⁇ ed by anti-EMAP II IgG) and immunolocalization of the polypeptide to vascular and perivascular areas of the tumor, suggested a role for this cytokine in vascular dysfunction associated with meth A tumors. Consistent with the ability of EMAP II to modulate vessel growth and/or integrity was the observation that neovessel formation into bFGF-containing implants wa ⁇ blocked by rEMAP II.
  • cytokines such as tran ⁇ forming growth factor- ⁇ or TNF-a have been found to induce vascular ingrowth in angiogenesis models (Leibovich, S., 1987; Fraker-Schroder, M., 1987; Madri, J. , 1992) .
  • ⁇ tudie ⁇ have ⁇ hown rEMAP II to markedly attenuate growth of a human brea ⁇ t carcinoma line (MDA-MB 468) grown in nude mice and al ⁇ o to ⁇ uppress C6 gliomas in rats.
  • MDA-MB 4608 human brea ⁇ t carcinoma line
  • EMAP II action ⁇ of EMAP II were localized, under the ⁇ e condition ⁇ , to the tumor.
  • the data do not rule out the possbility that EMAP II may have other effects on the tumor beyond that on the vasculature.
  • the action of EMAP II on endothelium or other elements in the tumor microenviromment might release diffusible mediators toxic for tumor cells, thus causing tumor injury initially close to the vasculature, but then extending deeper into the tumor.
  • a salient feature of tumor vasculature which distinguishes vessels in the tumor stroma from those in normal tis ⁇ ue, is the increased fraction of growing/migrating endothelial cells (Fidler, I., 1994; Folkman, J., 1989; Folkman, J., 1995) .
  • Studies in cell culture suggested a selective effect of rEMAP II on growing/migratory endothelium; cells at the leading edge of a wound in the monolayer failed to effectively fill the gap and cell proliferation was suppressed.
  • the predominate affect appeared to be induction of apoptosis, especially in the actively dividing cell population.
  • postconfluent endothelium at a distance from the wound was not affected by rEMAP II.
  • cytokine to cultures of growing tumor cells (LLC, B16 melanoma or meth A) showed no change in cell proliferation or induction of apoptosi ⁇ , though rEMAP II suppres ⁇ ed these tumors in vivo.
  • Enhanced EMAP II-induced apoptosis in hypoxic endothelial cultures provided further support for the relevance of our finding to tumor biology, as the presence of hypoxic areas in tumors is well-established (Olive, P., 1992; Kalra, R., 1994) .
  • hypoxia could potentially sensitize endothelium to EMAP II by several mechanisms, including arrest of cells at the Gl/S interface (Shreeniwa ⁇ , R., 1991) or increa ⁇ ed ⁇ ensitivity to subsequent encounters with oxidizing stimuli.
  • hypothesi ⁇ pilot ⁇ tudie ⁇ suggest that EMAP II has an important effect on cellular redox status as addition of N-acetylcysteine blocks EMAP II-mediated endothelial apoptosi ⁇ .
  • Mechanism ⁇ through which EMAP II induces possible cellular oxidant stress, as well as elucidation of the cell surface receptor for EMAP II will provide more definitive answers to questions concerning the specificity and selectivity of its cellular effects.
  • EMAP II the suppressive effect of rEMAP II on tumors without, apparently, an adverse affect on the function of normal organs. This may be due to EMAP II's effect on the endothelium; EMAP II could perturb endothelium in vivo not only by direct effects on endothelial apoptosis, but also by other means.
  • EMAP II-mediated induction of endothelial tissue factor could trigger local activation of clotting in the tumor bed, thereby diminishing blood flow and enlarging the volume of tumor at risk for ischemia.
  • EMAP II might also modulate the expre ⁇ sion of other mediators which control the local angiogenic balance, including enhanced activity of pathways regulating production of angiostatic peptides, such as angiostatin or thrombospondin, and/or might suppress expression of pro-angiogenic factors in the tumor bed. Furthermore, EMAP II might elicit endothelial production of mediators which directly impair tumor cell viability (as mentioned above) . Though there are many mechanistic, physiologic and practical questions to be explored in future studies (Will EMAP II affect well-established vessels in human tumors which grow over much longer times than the accelerated murine. model ⁇ ? Will an optimal anti-tumor regimen of EMAP II induce tumor regression or will it just be static? etc.) , the data support the potential of EMAP II, a cytokine with apparent anti-angiogenic properties, to suppress primary and metastatic tumor growth, and to induce apoptosis in the tumor without apparent adverse affects on normal organs.
  • Example 3 ENDOTHELIAL-MONOCYTE ACTIVATING POLYPEPTIDE II SUPPRESSES GROWTH OF C6 GLIOMAS BY TARGETING THE VASCULATURE
  • Endothelial-Monocyte Activating Polypeptide (EMAP) II is a novel mediator initially purified from methylcholanthrene A-induced fibrosarcomas, well-known for spontaneous vascular insufficiency and thrombohemorrhage. Testing the effect of EMAP II on C6 gliomas which elicit a characteristic angiogenic response, largely due to expres ⁇ ion of Vascular Endothelial Growth Factor (VEGF) was therefore carried out.
  • EMAP II Endothelial-Monocyte Activating Polypeptide
  • rEMAP II had a striking effect on C6 gliomas grown subcutaneously in nude mice, causing a six-fold decrease in tumor volume, without evidence of systemic toxicity.
  • rEMAP II blocked the angiogenic response to locally administered VEGF, demonstrating a direct effect of EMAP II on VEGF-driven vascular ingrowth.
  • VEGF Vascular Endothelial Growth Factor
  • EMAP Endothelial Monocyte Activating Polypeptide II
  • r recombinant
  • IP Intraperitoneal
  • IT Intratumoral
  • TUNEL Deoxynucleotidyl Transferase-mediated DUTP-biotin nick end labelling.
  • Vascularization of solid tumors is critical for their growth beyond a small collection of neoplastic cells (Fidler, I., 1994; Folkman, J. , 1989; Folkman, J. , 1995) .
  • vasculature i ⁇ insulated from the neuronal compartment by the blood-brain barrier
  • effective mechanisms for induction of neovasculature have evolved to support tumor growth.
  • Glioblastoma the most frequently occurring intracranial neoplasm, displays characteristic vascularization with evidence of endothelial proliferation and a complex vascular network, thereby providing an especially relevant example of ongoing angiogenesis (San Galli, F., 1989; Plate, K.
  • VEGF Vascular Endothelial Growth Factor
  • the secreted isoform of VEGF (residues 1-165) is produced by glioblastoma/glioma at the tumor margin, especially at sites of local necrosis (and presumably, hypoxia) , enhancing neovessel formation by attracting endothelium which has been shown to selectively express the VEGF receptor Flk-1 (Plate, K. , 1992; Shweiki, D. 1992; Weindel, K., 1994; Plate, K. , 1994; Samoto, K. , 1995) .
  • Direct evidence of a role for VEGF in glioma growth derives from experiments demonstrating that antibodies to VEGF (Kim, K. , 1993) , a dominant negative mutant of Flk-1/VEGF (Millauer, B., 1994), and VEGF antisense introduced into gliomas suppresses the tumors (Saleh, M., 1996) .
  • VEGF vascular endothelial growth factor
  • VEGF has been show to have other properties associated with inflammatory mediators in vitro, including induction of the procoagulant tissue factor on endothelial cells and mononuclear phagocyte ⁇ (Clauss, M. , 1990) . Although the relevance of these findings to the biology of VEGF in vivo has not been clarified, it has been speculated that this could account for pathologic findings in the vasculature of gliomas, including evidence of vascular leakage and local thrombi. The role of VEGF as a central angiogenic mediator has been demonstrated more directly. Deletion of the VEGF gene results in an embryonic lethal, with failure of vasculogenesis (Harpal, K.
  • VEGF ha ⁇ been implicated in neovascularization associated with diabetic retinopathy, ischemic events, and tumor growth (Shweiki, D. 1992; Weindel, K. , 1994; Plate, K., 1994; Samoto, K. , 1994; Miller, J., 1994; Aiello, L., 1994) .
  • Endothelial-Monocyte Activating Polypeptide (EMAP) II is a novel mediator initially identified in meth A tumors, well-known for their spontaneous vascular insufficiency
  • EMAP II directly into tumors elicited thrombohemorrhage and sensitized tumor vasculature to subsequent sy ⁇ temic infu ⁇ ion of tumor necrosis factor.
  • EMAP II might target tumor vasculature.
  • the goal of the studies was to determine if EMAP II could antagonize the angiogenic effects of glioma-derived angiogenic factors, especially VEGF, thereby implying its potential to block pathologic vascular ingrowth.
  • the result ⁇ herein indicate that systemically administered EMAP II blocks neovessel formation in response to VEGF in a Matrigel model, and that it potently suppresses growth of C6 gliomas.
  • C6 glioma cell ⁇ (Benda, P., 1971) were obtained from ATCC and were grown in Dulbecco's Modified Eagle Medium containing fetal bovine serum (10%; Gemini, Gibco, Grand Island NY) .
  • Mouse brain endothelial cells were characterized and grown as described (Gumkowski, F., 1987) .
  • Human umbilical vein endothelial cells were grown and characterized as described (Kao, J., 1992) . DNA fragmentation was evaluated by agarose gel electrophoresi ⁇ (xBorczyca et al., 1993- a ⁇ k dave p.) .
  • Radioligand binding ⁇ tudies employed 12r> I-rEMAP II and cultured C6 glioma or endothelial cells.
  • rEMAP II was prepared from E.
  • the protocol for binding included washing cultured endothelium (2xl0 4 cells/well) in Hanks' balanced salt solution, and then adding Minimal Essential Medium containing fetal bovine serum (10%) at 4°C containing 125 I-rEMAP II alone or in the presence of an 100-fold molar excess of unlabelled rEMAP II. Wells were incubated for 2 hrs at 4 ° C, unbound material was removed by six rapid washe ⁇ (for a total of 6 ⁇ ec/well) with pho ⁇ phate-buffered saline, and cell-associated radioactivity was eluted with phosphate-buffered saline containing Nonidet P-40 (1%) .
  • Matrigel model Matrigel (Collaborative Research) (Kleinman, H., 1986; Passaniti, A., 1992) containing either vehicle (1% bovine serum albumin) , VEGF (100 ng/ml; Collaborative Re ⁇ earch) + vehicle, or heat-inactivated VEGF (15 min at 100 'C) + vehicle (mouse serum albumin, 1 mg/ml) was mixed at 4°C. Matrigel mixtures (0.25 ml/site; two sites per animal) were injected subcutaneously into C57BL6/J mice (0.25 ml/site) at two sites per animal.
  • C6 glioma cells were implanted into the frontal lobe of Wistar rats (250-300 grams; Charles River) by a modification of methods described in the literature (San- Galli, F. , 1989; Bernstein, J., 1990) .
  • Intratumoral administration involved positive pressure microinfusion through the implanted rod at a volume of 40 ⁇ l infused over 133 min. Once the treatment regimen including rEMAP II was begun, it was continued for a total of either 7 or 14 days. There were 8 eight animal ⁇ in each treatment group. At the end of the experiment, animal ⁇ were ⁇ acrificed by humane euthanasia, the cranium wa ⁇ opened, the brain removed, incubated in formalin (4%) at 4°C for 72 hr ⁇ , and placed in a brain matrix to make serial 1 mm coronal slices.
  • Tumor volume was calculated according to the formula for a spherical segment (see below; Weast R., 1966) based on the largest cross-sectional tumor diameter, and serial images were evaluated by NIH image.
  • Initial tumor size (i.e., prior to treatment on day 3) in each of the groups was 12-14 mm 3 .
  • Tumor volume data were analyzed using the Kruskal-Walli ⁇ one way ANOVA and a Mann-Whitney mean rank te ⁇ t. Data i ⁇ expressed as a dimensionless ratio of observed tumor volume divided by initial (day 3) tumor volume. Animals were sacrificed and tumors analyzed histologically at the indicated times.
  • TUNEL terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling
  • tumor specimens were cut into small pieces and immediately fixed by immersion in glutaraldehyde (1.5%) in sodium cacodylate-HCl (0.1 M; pH
  • C6 gliomaB Effect of rEMAP II on growth of C6 gliomaB in vivo.
  • C6 glioma cells were implanted stereotactically in the right frontal lobe of Wistar rats. This model was selected based on previous studies demonstrating that histologic features of these tumors closely resemble findings in tumors of patients (San-Galli, F., 1989; Bernstein, J. , 1990) . Tumor growth occurred steadily up to about 28 days, when death resulted from increased intracranial pressure. For this reason, experiments were terminated at day 24; there were no fatalities at this time.
  • IT treatment via indwelling cannula according to our protocol, ha ⁇ been shown to effectively deliver therapeutic agents within the central nervous sy ⁇ tem without elevating intracranial pressure.
  • Animals receiving rEMAP II by the IT/IP routes showed the greatest suppression of tumor growth; at a dose of 100 ng
  • IP IP/10 ⁇ g (IT) to lower concentrations, either 10 ng (IT)/1 ⁇ g (IP) , or 1 ⁇ g (IP) or 100 ng (IT) alone, resulting in less effective suppression of tumor growth, histologic evidence of pyknosis and apoptosis decreased.
  • IP/IT rEMAP II
  • experiment ⁇ were performed with tumor cell ⁇ inoculated subcutaneously in nude mice. Tumor cells were implanted into immunocompromised mice, and growth wa ⁇ allowed to occur for three days, at which time palpable tumors were reproducibly evident (approximate volume prior to treatment was 12-14 mm 3 , in each group) . rEMAP II was then administered starting on day 3, and tumor volume was measured every four days thereafter; data are reported at each time point as fold-change in tumor volume (a dimensionless ratio comparing tumor volume on the indicated day with that on day 3) ; this method allowed a comparison of each animal with itself.
  • rEMAP Il-treated animals di ⁇ played 6-fold reduction in volume compared with tumors in mice receiving vehicle alone by day 31 (Fig. 9E) .
  • Hi ⁇ tologic appearance of rEMAP Il-treated C6 glioma ⁇ showed small tumors with evidence of pyknotic change ⁇ and apoptosis throughout the lesions, compared with larger tumors in vehicle-treated controls which displayed homogeneous central areas and apoptotic/necrotic change ⁇ limited to the periphery.
  • Matrigel i ⁇ a complex mixture of basement membrane proteins, a ⁇ well a ⁇ other cell product ⁇ , from Engelbreth-Holm-Swarm (EHS) tumor cell ⁇ (Kle man, H., 1986; Passaniti, A., 1992) .
  • EHS Engelbreth-Holm-Swarm
  • an exogenous growth factor such as basic fibroblast growth factor
  • This model was employed by mixing Matrigel with recombinant human VEGF and subcutaneously implanting the mixture into mice.
  • Il-treated tumors displayed attentuated endothelium, often with fenestration ⁇ and open interendothelial junctions
  • EMAP II has several properties which are con ⁇ i ⁇ tent with the hypothe ⁇ i ⁇ that it specifically affects tumor vasculature.
  • EMAP II may provide a broader spectrum of activities which impact negatively on tumor survival in the host, including inhibition of other angiogenic activities in the tumor, ⁇ uch as basic fibrobla ⁇ t growth factor (Stan, A., 1995) .
  • EMAP II exerts its affects on tumors from the pathologic picture in treated tumors bed of tumor ⁇ treated with EMAP II
  • a mechanism other than direct tumor cell cytotoxicity seems likely. If this proves to be true, the most effective therapy might be to combine EMAP II with agents directly targetting neoplastic cell ⁇ , such as cytotoxic agents or anti-sense to Insulin-like Growth Factor, the latter having been shown to ⁇ uppre ⁇ s glioma growth (Resnicoff, M., 1994) .
  • agents directly targetting neoplastic cell ⁇ such as cytotoxic agents or anti-sense to Insulin-like Growth Factor

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Abstract

L'invention concerne le traitement de tumeurs par injection sous-cutanée, intrapéritonéale ou intraveineuse de polypeptide II activant les monocytes endothéliaux, ou d'un polypeptide dérivé du polypeptide II. Le polypeptide II ou ses dérivés peuvent également servir à traiter des troubles se manifestant par une présence excessive de vaisseaux sanguins, par exemple les rétinopathies.
PCT/US1996/015007 1995-09-18 1996-09-18 Proprietes anti-angiogeniques du polypeptide ii activant les monocytes endotheliaux WO1997010841A1 (fr)

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Cited By (8)

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WO1998008950A1 (fr) * 1996-08-28 1998-03-05 Incyte Pharmaceuticals, Inc. Nouvelle cytokine activant les monocytes
EP1137805A1 (fr) * 1998-11-13 2001-10-04 Children's Hospital Of Los Angeles Procedes destines a faciliter la croissance vasculaire
WO2002005852A1 (fr) * 2000-07-14 2002-01-24 Meditech Research Limited Hyaluronan utilise comme agent cytotoxique, pre-sensibilisateur a des medicaments et chimio-sensibilisateur dans le traitement de maladies
USD719240S1 (en) 2013-08-23 2014-12-09 Kohler Co. Shower device
USD740917S1 (en) 2013-03-16 2015-10-13 Kohler Co. Shower faceplate for shower device
US20150377908A1 (en) * 2013-02-13 2015-12-31 Rajashekhar Gangaraju Methods of diagnosing, treating and monitoring diabetic retinopathy
USD759197S1 (en) 2012-03-12 2016-06-14 Kohler Co. Shower faceplate
US9468939B2 (en) 2012-03-12 2016-10-18 Kohler Co. Faceplate for shower device

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US5198423A (en) * 1989-05-26 1993-03-30 Takara Shuzo Co., Ltd. Functional polypeptide containing a cell binding domain and a heparin binding domain of fibronectin
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US5202116A (en) * 1989-04-10 1993-04-13 Oncogen Methods for controlling human endothelial cell proliferation and effector functions using oncostatin m
US5198423A (en) * 1989-05-26 1993-03-30 Takara Shuzo Co., Ltd. Functional polypeptide containing a cell binding domain and a heparin binding domain of fibronectin
US5382514A (en) * 1992-03-31 1995-01-17 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services In vivo angiogenesis assay
WO1995009180A1 (fr) * 1993-09-29 1995-04-06 The Trustees Of Columbia University In The City Of New York Polypeptide ii d'activation des monocytes endotheliaux, constituant un mediateur d'activation de la reponse d'hotes

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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998008950A1 (fr) * 1996-08-28 1998-03-05 Incyte Pharmaceuticals, Inc. Nouvelle cytokine activant les monocytes
US5885798A (en) * 1996-08-28 1999-03-23 Incyte Pharmaceuticals, Inc. DNA encoding a monocyte activating cytokine
US6090377A (en) * 1996-08-28 2000-07-18 Incyte Pharmaceuticals, Inc. Monocyte activating cytokine
US6875749B2 (en) 1998-11-13 2005-04-05 Childrens Hospital Los Angeles Methods of facilitating vascular growth
EP1137805A4 (fr) * 1998-11-13 2003-03-26 Los Angeles Childrens Hospital Procedes destines a faciliter la croissance vasculaire
EP1137805A1 (fr) * 1998-11-13 2001-10-04 Children's Hospital Of Los Angeles Procedes destines a faciliter la croissance vasculaire
WO2002005852A1 (fr) * 2000-07-14 2002-01-24 Meditech Research Limited Hyaluronan utilise comme agent cytotoxique, pre-sensibilisateur a des medicaments et chimio-sensibilisateur dans le traitement de maladies
USD759197S1 (en) 2012-03-12 2016-06-14 Kohler Co. Shower faceplate
US9468939B2 (en) 2012-03-12 2016-10-18 Kohler Co. Faceplate for shower device
US20150377908A1 (en) * 2013-02-13 2015-12-31 Rajashekhar Gangaraju Methods of diagnosing, treating and monitoring diabetic retinopathy
US10132815B2 (en) * 2013-02-13 2018-11-20 Indiana University Research & Technology Corporation Methods of diagnosing, treating and monitoring diabetic retinopathy
USD740917S1 (en) 2013-03-16 2015-10-13 Kohler Co. Shower faceplate for shower device
USD719240S1 (en) 2013-08-23 2014-12-09 Kohler Co. Shower device

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