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WO1997010841A9 - Proprietes anti-angiogeniques du polypeptide ii activant les monocytes endotheliaux - Google Patents

Proprietes anti-angiogeniques du polypeptide ii activant les monocytes endotheliaux

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Publication number
WO1997010841A9
WO1997010841A9 PCT/US1996/015007 US9615007W WO9710841A9 WO 1997010841 A9 WO1997010841 A9 WO 1997010841A9 US 9615007 W US9615007 W US 9615007W WO 9710841 A9 WO9710841 A9 WO 9710841A9
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WO
WIPO (PCT)
Prior art keywords
emap
endothelial
tumor
monocyte activating
activating polypeptide
Prior art date
Application number
PCT/US1996/015007
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English (en)
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WO1997010841A1 (fr
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Publication date
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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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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. These studies show that 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 antiangiogenesi ⁇ , 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 (12,000xg) ; 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 SO 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 25 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, 6C, 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.
  • FIG. 7 PCR analysis of EMAP II transcripts in normal murine tissue. RNA was harvested from normal murine tissues a ⁇ indicated, and processed for PCR as described in the text. The bands corresponding to the amplicons for EMAP II
  • 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 C ⁇ 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 was initiated for the next 24 days a ⁇ 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. 10A, 10B, IOC, 10D and 10E Effect of rEMAP II on vascular ingrowth into Matrigel implants impregnated with VEGF.
  • Figures 11A, 11B, 11C, 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 m 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 11C Quantitation of apoptotic endothelial nuclei as a ratio of labelled nuclei/cells counted m each of ten high power fields in the presence of the indicated concentration of rEMAPII. * denotes P ⁇ x.
  • 11D Quantitation of labelled nuclei as m
  • This invention provides a method of treating a tumor m a sub ect, comprising administering to the subject an amount of an agent, selected from endothelial monocyte activating polypeptide II and an endothelial monocyte activating polypeptide II-der ⁇ ved polypeptide, effective to treat the tumor, wherein the endothelial monocyte activating polypeptide II is administered subcutaneously, mtraperitoneally, or intravenously.
  • an agent selected from endothelial monocyte activating polypeptide II and an endothelial monocyte activating polypeptide II-der ⁇ ved polypeptide, effective to treat the tumor, wherein the endothelial monocyte activating polypeptide II is administered subcutaneously, mtraperitoneally, 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 m ammo acid sequence or in ways that do not involve sequence, or both. Variants m ammo acid sequence are produced when one or more amino acids in naturally occurring EMAP II is substituted with a different natural ammo acid, an ammo acid derivative or non-native ammo 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 ammo acid substitutions, deletions or insertions which do not abolish the EMAP II biological activity.
  • Conservative substitutions typically include the substitution of one ammo acid for another with similar characteristics such as substitutions with the followmg groups: valine, glycine; glycine, alanine, valine, isoleucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanme, 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.
  • Arginine R D-Arg, Lys, homo-Arg, D-homo- Arg, Met,D-Met, He, D-Ile, Orn, D-Orn
  • Glutamic Acid E D-Glu,D-A ⁇ p,A ⁇ p, Asn, D-Asn, Gin, D-Gln
  • 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.
  • the peptides of this invention may also be modified by various changes such a,s insertions, deletions and substitutions, either conservative or nonconservative where such changes might provide for certain advantages their use .
  • variants with amino acid substitutions which are less conservative may also result m 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 cham for a residue havmg 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 withm the ⁇ cope of the invention include protems and peptides with ammo 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 m vitro chemical derivatization of portions of naturally occurring EMAP II, as well as changes m acetylation, methylation, phosphorylation, carboxylation or glycosylation.
  • the protein is modified by chemical modifications in which activity i ⁇ 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 II-derived polypeptide is at least about ninety percent homologous to the sequence (S/M/G) KPIDVSRLDLRIG ( C/R ) I ITARKHPDADSLYVEEVDVGEIAPRTWSGLVNHVPLEQMQNRMVILLCNLK
  • QIQPDLHTNDECVATYKGVPFEVKGKGVCRAQTMSNSGIK (SEQ I . D . No . ) , wherein the sequence is truncated by f rom zero to about three ammo-termmal 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 mvention 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-der ⁇ ved 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 m 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 H-derived polypeptide, thereby inhibiting the formation of blood vessels m 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, ⁇ heep, 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 Il-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 re ⁇ idue ⁇ . In a preferred embodiment the homology i ⁇ at least about ninety- five percent.
  • the endothelial monocyte activating polypeptide Il-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 H-derived polypeptide, thereby treating the condition involving the presence of exces ⁇ blood vessels.
  • an agent selected from endothelial monocyte activating polypeptide II and an endothelial monocyte activating polypeptide H-derived polypeptide
  • the condition involves the presence of excess blood vessel ⁇ 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, compri ⁇ ing administering to the subject an amount of an agent, selected from endothelial monocyte activating polypeptide II and an endothelial monocyte activating polypeptide Il-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 microinfusion.
  • 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.
  • an agent selected from endothelial monocyte activating polypeptide II and an endothelial monocyte activating polypeptide II-derived polypeptide
  • 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 H-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 ves ⁇ els 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 sy ⁇ tem, 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.
  • Example 1 Endothelial-Monocyte Activating Polypeptide II, A Novel Antiangiogenic Protein, Suppresses Tumor Growth and Induces Apoptosis of Growing Endothelial Cells.
  • Bovine aortic endothelial cells were isolated from calf aortae, grown in culture and characterized, based on the presence of von Willebrand factor and thrombomodulm, as described previously (Nawroth P. , 1988) .
  • Bovme vascular smooth muscle cells were prepared by additional scraping of the aortae followmg removal of the endothelium, and were characterized based on the pre ⁇ ence 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 (F10) cells were all maintained in high glucose defined minimal es ⁇ ential 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) .
  • ATCC American Type Culture Collection
  • NIH 3T3 cells ATCC
  • B16 (F10) cells were all maintained in high glucose defined minimal es ⁇ ential 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) .
  • DMEM minimal es ⁇ ential medium
  • Recombinant EMAP II was prepared from E. coli (host HMS174 [DE3] ) transformed with a plasmid containing the coding ⁇ equence for mature EMAP II, as described previously
  • 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 mto 3-
  • EMAP II-containing fraction ⁇ from SP Sepharose chromatography were adjusted to 2 M (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 sodium phosphate (20 mM; pH 7) containing 1 M (NH ) 2 S0 ⁇
  • a descending gradient of salt (2 to 0M) in sodium phosphate (20 mM) was applied EMAP H-contammg 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 mto 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 fraction ⁇ obtained during the purification procedure wa ⁇ ⁇ ub ected to N-termmal sequence analysis, mass ⁇ pectrometry and SDS-PAGE.
  • Immunoblottmg was performed followmg SDS-PAGE by transferring prote 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) . Prestamed, low molecular weight markers (Bio-Rad) were u ⁇ ed to follow the transfer. Immunoreactive protem was visualized using rabbit anti- mature EMAP II N-termmal 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 immunoblottmg of plasma and cell extracts. This antibody was used to develop an ELISA to detect EMAP II antigen; cells or tissues were homogenized m the presence of protese inhibitors (phenylmethylsulfonyl fluoride, 1 M; trasylol, 0.1%) , centrifuged to remove debris, and the supernatant wa ⁇ diluted in carbonate/bicarbonate buffer (pH 9.6) and incubated Maxisorb microtiter plates (Nunc) overnight at 4°C.
  • protese inhibitors phenylmethylsulfonyl fluoride, 1 M
  • trasylol trasylol
  • Matrigel model Matrigel (Klem an H , 1986; Passaniti A.,
  • EMAP II 100 ng
  • bFGF 100 ng/ml
  • heparin 40 U/ml; Sigma
  • EMAP II 100 ng
  • vehicle alone 1% BSA
  • heat- activated EMAP II alone or with bFGF/heparm
  • the angiogemc response was analyzed at 7 and 14 days post-moculation by routme histology and hemoglobin assay (Sigma) .
  • tissue associated radioactivity was determined on weighed samples either after drying (for total radioactivity) , or followmg homogenization of tissue and trichloroacetic acid precipitation (20%) 125 I-EMAP II 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) In order to asse ⁇ s the "goodness of fit,” residual analysi ⁇ (an examination of the ⁇ tandard deviation) was performed.
  • t 1/2 Qt, t 1/2 ⁇ , t 1/2 r denote half-lives for distribution, elimination and re ⁇ orption half-lives, respectively.
  • Tumor models LLC and B16 (F10) cells were rinsed with Hank ⁇ buffered saline solution, trypsinized, counted, resuspended m phosphate-buffered salme, and injected subcutaneously mto backs of C57BL6/J mice (2 x IO 6 cells/animal)
  • 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 + heat-inactivated EMAP II (1000 ng)
  • Tumor volume data was calculated according to the formula
  • HistoloQic analysis was performed on formalin fixed, paraffin embedded ti ⁇ sue, using hematoxylm and eos staining. DNA nick translation was used in tumor tissue
  • LLC an Meth A
  • Paraffin embedded tumor slices were deparaffmized and digoxigenin-11-UTP was used to label fragmented DNA accordmg 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' ⁇ overnight. Nitroblue tetrazolium and alkaline pho ⁇ phata ⁇ e were used to reveal the digoxigenin labelled DNA fragments.
  • sequential sections of Meth A tumors underwent analysis for DNA fragmentation and EMAP II.
  • DAP-1 6- diamidmo-2 -phenylmdoledilactate
  • DNA laddering for apoptosis in cultured cells exposed to EMAP II was performed as de ⁇ cribed (Gorczyca W., 1993) Briefly, after 12 hour ⁇ of exposure to EMAP II, cultures were treated with lysi ⁇ buffer (Tris-borate buffer, 45 mM; EDTA, ImM; pH 8.0; NP-40, 0.25%) , digested with proteinase K (1 mg/ml) and RNAase A (0.1 mg/ml) , and then 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 (Boehrmger Mannheim) . Gels were stained with ethidium bromide.
  • N-termmal 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 al ⁇ o recognized by anti-mature EMAP II ammo terminal peptide IgG by immunoblottmg, and in the endothelial cell tissue factor induction assay gave activities of 0.3-0.4 units/ng of protem.
  • EMAP II Effect of EMAP II on bFGF-induced angiogenesis.
  • bFGF and herapin were mixed with a gel of basement membrane protems produced by Engelbreth-Holm-Swarm tumor cells (Matrigel) to serve as a model angiogemc stimulus (Kleinman H , 1986, Pas ⁇ amti A., 1992)
  • Metrigel basement membrane protems produced by Engelbreth-Holm-Swarm tumor cells
  • Subcutaneous Matrigel implants m C57BL6/J mice were evaluated 14 days after inoculation for ves ⁇ el formation, cellular infiltration and hemoglobin content.
  • FIG. 3A Histologic analysis of the gel showed formation of vessel and white cell infiltration to be most pronounced m implants from animals treated w th bFGF and heparin
  • FIG. 3B This induction of blood vessel formation is similar to that reported previously with bFGF in this model (Passaniti A. , 1992) .
  • implants from animals treated with bFGF/herapin + EMAP II displayed marked reduction in vessel ingrowth (Fig.
  • Plasma clearance and tissue deposition of infused EMAP II In order to perform in vivo studie ⁇ with EMAP II, its plasma clearance and tissue deposition was evaluated (Fig. 4A) . Clearance studie ⁇ were performed u ⁇ ing 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 monoexponential 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) .
  • EMAP II-induced apoptosis of rapidly growing ECs was further analyzed by electrophoresi ⁇ for DNA fragmentation, characte ⁇ tic ladder formation wa ⁇ observed in growing ECs exposed to EMAP II, whereas vascular smooth muscle cell DNA was unaffected.
  • 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; Con ⁇ tantinidi ⁇ I., 1989; Watanable N. , 1988; Senger D., 1983) .
  • the ⁇ e propertie ⁇ which di ⁇ tinguish 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 fir ⁇ t studied based on its modulation of endothelial properties, such as induction of leukocyte adherence molecules and the procoagulant cofactor tissue factor. Further studie ⁇ on mononuclear phagocyte ⁇ and polymorphonuclear leukocyte ⁇ confirmed it ⁇ ability to induce cell migration and activation. The ⁇ e data suggested that EMAP II had properties of an inflammatory cytokine, at least based on in vitro finding ⁇ .
  • EMAP II endogenously, suggests that vasculature was a target of the cytokine.
  • 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.
  • EMAP II had an exaggerated apoptotic effect in endothelial cultures subjected to oxygen deprivation. This was not observed in either smooth muscle cells or fibroblast ⁇ under ⁇ imilar hypoxic conditions. Data, ⁇ howing high affinity endothelial binding sites for EMAP II, contrasted to the absence of such sites on tumor cells, would be consi ⁇ tent with differential expression of EMAP II receptors on these cell types. Although the basis for this apparent specificity of EMAP II at the cellular level is at present unclear, this might reflect difference ⁇ receptor expression or post-receptor signalling. Such specificity is clearly critical for guiding future work directed at mechanisms underlying actions of EMAP II on the cellular and molecular levels.
  • a hypoxia induced retinal neovascularization model has been well established by the "Association for Research m 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 m 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 u ⁇ ed a ⁇ 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.
  • Endothelia -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 Intraperitoneally administered recombinant 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 apopto ⁇ i ⁇ in a time- and do ⁇ e-dependent manner; an effect enhanced by concomitant hypoxia, whereas other cell types, such as Lewis Lung carcinoma cells, were unaffected.
  • EMAP II is a tumor suppressive mediator with anti-angiogenic properties allowing it to target growing endothelium and limit establishment of neovasculature .
  • 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 spontaneou ⁇ va ⁇ cular in ⁇ ufficiency manife ⁇ ted by a heterogeneous pattern of thrombohemorrhage and central necrosi ⁇ , 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 showed no significant homology to other known proteins, such a ⁇ cytokines or growth factors.
  • 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.
  • EMAP II administered in vivo, locally or sy ⁇ temically, gave rise to, at most, mild and transient inflammation (Kao, J., 1994) , suggesting that its effects were quite different from those of tumor necrosis factor (TNF) or Interleukin 1 (Old, L., 1961; Sherry, B. , 1988; Dinarello, C. , 1993) .
  • 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 target ⁇ rapidly growing vascular beds, and ⁇ ugge ⁇ t ⁇ 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 ⁇ craping of the aortae following removal of the endothelium, and were characterized based on the presence of smooth muscle cell actin (Gown, A., 1985) .
  • LLC Lewis Lung Carcinoma
  • B16(F10) melanoma cells obtained from American Type Culture Collection (ATCC) , were maintained in high glucose-defined Minimal Essential Medium
  • DMEM fetal bovine serum
  • Meth A tumor cells Center for Cancer Research, NY
  • Meth A tumor cells were grown as described (Old, L., 1986) .
  • EMAP H-induced apoptosis was studied in subconfluent endothelial cultures (Gerlach, H., 1989) .
  • BrdU 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.
  • cell ⁇ were fixed in paraformaldahyde (2.5%) , rinsed with phosphate-buffered saline, incubated with 6-diamidino-2-phenylindoledilactate (DAP-1; final concentration, 1 ng/ml) and mounted with glycerol (10%) .
  • DAP-1 6-diamidino-2-phenylindoledilactate
  • glycerol 10% .
  • F-actin was visualized in cultured cells by incubation with rhodamine-conjugated phalloidin (Molecular Probe ⁇ ) . Wounding of endothelial monolayer ⁇ was performed using a 2-mm cork borer (Selden, S., 1981) .
  • 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 pa ⁇ te was mixed 1:10 (w/v) with Tris-HCl (20 mM; pH 7.4) containing octyl- ⁇ -glucoside (0.1%) and an homogeneou ⁇ suspension wa ⁇ formed by agitation u ⁇ ing a microfluidizer for 20 min (speed 60) at 4°C.
  • the Heparin Sepharose pool was concentrated using an Amicon Stirred Cell (Amicon) , the retentate wa ⁇ desalted mto 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) .
  • Amicon Stirred Cell Amicon Stirred Cell
  • MOPS mto 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 m MOPS, and EMAP Il-contammg fractions were adjusted to 2 M in (NH 4 ) 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) m sodium phosphate (20 mM) , and EMAP II in the Phenyl Toyopearl column eluate was concentrated to 3-5 mg/ml, and formulated mto phosphate-buffered salme (PBS; pH 7.4) by buffer exchange on a Sephadex G25 column (as above) Lipopolysaccharide
  • EMAP II was subjected to N-termmal 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 m rabbits (Vaitukatis, J., 1981) and was found to be monospecific, based on immunoblottmg 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 was 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 Perkm-Elmer-Cetus
  • Thermocycl g parameter ⁇ for the experiment shown in Fig. 7 were: 94 " C for 30 sec; 55 ' C for 30 sec; and, 72 " C for 30 sec for a total of 35 cycles. Samples were subjected to agarose gel (1%) electrophoresis and bands were visualized by ethidium bromide staining. Identity of amplicons wa ⁇ confirmed by Southern blotting with the appropriate cDNA probes .
  • Matrigel model Matrigel (Klemman, H., 1986, Pas ⁇ aniti, 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/heparm + vehicle, or heat-mactivated rEMAP II (100 ng/ml; alone or with bFGF/heparm) + vehicle was mixed at 4° C.
  • vehicle 1% BSA
  • rEMAP II 100 ng/ml
  • heparin 40 U/ml; Sigma
  • Matrigel mixtures were injected subcutaneously mto C57BL6/J mice (0.25 ml/site) at two sites per animal.
  • the angiogemc response was analyzed at 7 and 14 days post-inoculation by routine histology and hemoglobin assay (Sigma) .
  • 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 ⁇ acrificed at 24 hours.
  • 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 started on a course of IP injections every third day of either nonimmune rabbit IgG (400 ⁇ g/dose) or rabbit anti-murme EMAP II IgG (200 or 400 ⁇ g/dose) .
  • This regimen of IgG administration was based on pilot ⁇ tudies in which 125 I-rabbit anti-EMAP II IgG infused mto 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 apoptosis as described 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 metastatic le ⁇ ion ⁇ 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.
  • Histologic analysis was performed on formalin fixed, paraffin-embedded tissue, using hematoxylin and eosin staining.
  • the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNED assay was used to evaluate apoptosis; paraffin embedded tumor slice ⁇ were deparaffinized and digoxigenin-11-dUTP was used to label fragmented DNA according to the Genius 1 kit (Amersham) .
  • tissue was treated with proteinase K (1 ⁇ g/ml) , and incubated with digoxigenin-lld-UTP, klenow, and dNTP's overnight.
  • EMAP II could impact on tumor viability, which led to an examination of its sequestration in normal tis ⁇ ues.
  • 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 heparin were mixed with a gel of basement membrane proteins produced by Engelbreth-Holm-Swarm tumor cells (Matrigel) to serve as a model angiogenic stimulus (Kleinman, H., 1986; Pas ⁇ aniti, A., 1992) .
  • Subcutaneou ⁇ Matrigel implant ⁇ in C57BL6/J mice were evaluated 14 days after inoculation for vessel formation, cellular infiltration and hemoglobin content.
  • 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 rEMAP II Plasma clearance and tissue deposition of infused rEMAP II.
  • the resorption phase of rEMAP II handling m vivo wa ⁇ best described as a first order proces ⁇ .
  • the elimination pha ⁇ e following IP administration fit to a monoexponential decline, and the re ⁇ orption and elimination half-lives were 50.1 ⁇ 0.1 and 102 ⁇ 6 mm, respectively.
  • the precipitability of the tracer in trichloroacetic acid (20%) was greater in the tumor compared with other tissues, consistent with a relative accumulation of apparently intact rEMAP II in tumor tissue.
  • mice receiving active rEMAP II showed a striking reduction tumor volume (Fig.
  • rEMAP H-mduced area ⁇ of pykno ⁇ is had a general perivascular distribution, though pyknotic cells often extended beyond the vasculature.
  • Fig %F-G a site with several microvessels is visualized by staining for thrombomodulin, and evaluation of an adjacent section demonstrates DNA fragmentation using the TUNEL assay.
  • rEMAP II treatment was begun 72 hr prior to resection of the primary tumor and was continued through the end of the experiment (See Fig ⁇ . 8A-E) .
  • Animals receiving rEMAP II 1000 ng IP every 12 hrs) showed significantly fewer and smaller surface nodules, compared with vehicle by gross inspection and histologic study. Consistent with these data, rEMAP II-treated animals demonstrated 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 ti ⁇ ue hypoxia/hypoxemia (Olive, P., 1992; Kalra, R., 1994) , it wa ⁇ assessed whether rEMAP II might display enhanced activity under oxygen deprivation.
  • Neovascularization is a critical regulator of the growth of both primary and metastatic neoplasm ⁇ (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
  • VEGF vascular endothelial growth factor
  • Maciag, T. , 1984 acidic fibroblast growth factor
  • bFGF basic fibroblast growth factor
  • angiogenin Fett, J., 1985; King, T., 1991, Olson, K. , 1994
  • a switch in phenotype from pancreatic adenoma to malignancy was closely tied to expression of angiogenic mediators
  • 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 admini ⁇ tration of an agent ⁇ uch 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 suppressed 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 mto bFGF-contammg implants was blocked by rEMAP II.
  • cytokines such as transforming growth factor- ⁇ or TNF-a have been found to induce vascular ingrowth in angiogenesis models (Leibovich, S., 1987; Fraker-Schroder, M , 1987; Mad ⁇ , J., 1992) .
  • 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, thu ⁇ cau ⁇ ing tumor injury initially clo ⁇ e to the va ⁇ culature, 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, i ⁇ 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 suppres ⁇ ed.
  • the predominate affect appeared to be induction of apoptosis, especially in the actively dividing cell population.
  • hypoxia could potentially sensitize endothelium to EMAP II by ⁇ everal mechanisms, including arrest of cells at the Gl/S mterface (Shreeniwas, R., 1991) or increased sensitivity to subsequent encounters with oxidizing ⁇ timuli.
  • Gl/S mterface Shreeniwas, R., 1991
  • oxidizing ⁇ timuli oxidizing ⁇ timuli.
  • pilot studies sugge ⁇ t that EMAP II has an important effect on cellular redox status as addition of N-acetylcysteine blocks EMAP H-mediated endothelial apoptosis.
  • Analysis of mechanisms 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-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 expression of other mediators which control the local angiogenic balance, including enhanced activity of pathways regulating production of angio ⁇ tatic peptides, such as angiostatin or thrombospondin, and/or might suppress expression of pro-angiogenic factors in the tumor bed
  • EMAP II might elicit endothelial production of mediators which directly impair tumor cell viability (as mentioned above) .
  • 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 expression of Vascular Endothelial Growth Factor (VEGF) was therefore carried out.
  • rEMAP II had a striking effect on C6 gliomas grown subcutaneou ⁇ ly in nude mice, cau ⁇ ing a ⁇ ix-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. Ultrastructural study of tumor vasculature from animal ⁇ treated with rEMAP II showed intravascular accumulation of platelets and fibrin, as well a ⁇ finding ⁇ consistent with apoptosis of the endothelium.
  • 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) .
  • neoplastic cells In the central nervous system, in which vasculature is 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 site ⁇ of local necrosis (and presumably, hypoxia) , enhancing neoves ⁇ el formation by attracting endothelium which ha ⁇ been shown to selectively express the VEGF receptor Flk-1 (Plate, K , 1992; Shweiki, D. 1992; Wemdel, K. , 1994, Plate, K. , 1994; Samoto, K , 1995) .
  • VEGF has emerged as an angioge c factor involved in physiologic and pathophysiologic vascular response ⁇ (Houck, K., 1991; Keck, P., 1989) .
  • This polypeptide was initially characterized based on its ability to increase vascular permeability when injected subcutaneou ⁇ ly into guinea pig ⁇
  • VEGF ha ⁇ been show to have other properties associated with inflammatory mediators vitro, including induction of the procoagulant tis ⁇ ue factor on endothelial cells and mononuclear phagocytes (Clauss, M. , 1990) . Although the relevance of these findings to the biology of VEGF m vivo ha ⁇ not been clarified, it ha ⁇ been ⁇ peculated that thi ⁇ could account for pathologic findings the vasculature of gliomas, including evidence of vascular leakage and local thrombi. The role of VEGF a ⁇ a central angiogemc mediator has been demonstrated more directly.
  • VEGF vasculogenesi ⁇
  • 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 tumor ⁇ elicited thrombohemorrhage and sensitized tumor vasculature to subsequent systemic infusion of tumor necrosis factor.
  • Treatment of mice bearing Lewis Lung Carcinomas or B16 melanomas with low concentrations of EMAP II administered systemically for several weeks resulted in tumor regression and a pathologic picture of patchy apoptosis apparently radiating from tumor vasculature (Schwarz, M. , 1995) .
  • C6 glioma cells (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 de ⁇ cribed (Gumkow ⁇ ki, F., 1987) .
  • Human umbilical vein endothelial cells were grown and characterized as described (Kao, J., 1992) . DNA fragmentation was evaluated by agarose gel electrophoresis (xBorczyca et al . , 1993- ask dave p.) .
  • Radioligand binding studies employed 125 I-rEMAP II and cultured C6 glioma or endothelial cells.
  • rEMAP II was prepared from E. coli transformed with a plasmid containing the coding sequece for mature murine EMAP II, and was purified by a modification of our previous procedure (Kao, J.
  • the protocol for binding included washing cultured endothelium (2xl0 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 exces ⁇ of unlabelled rEMAP II.
  • Well ⁇ were incubated for 2 hr ⁇ at 4 ° C, unbound material wa ⁇ removed by ⁇ ix rapid washes (for a total of 6 sec/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 Research) + 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 gram ⁇ ; Charle ⁇ River) by a modification of method ⁇ de ⁇ cribed in the literature (San- Galli, F., 1989; Bernstein, J., 1990) .
  • Intratumoral administration involved positive pre ⁇ sure microinfu ⁇ ion 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 animals in each treatment group. At the end of the experiment, animal ⁇ were ⁇ acrificed by humane euthana ⁇ ia, the cranium was opened, the brain removed, incubated in formalin (4%) at 4°C for 72 hrs, 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
  • each of the groups was 12-14 mm 3 .
  • 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 the indicated times.
  • tissue was treated with proteinase K (1 ⁇ g/ml) , and incubated with d ⁇ gox ⁇ genm-11-dUTP, klenow, and dNTPs overnight Nitroblue tetrazolium and alkaline phosphatase were used to reveal the digoxigenin labelled DNA fragments.
  • 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 patient ⁇ (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, has been shown to effectively deliver therapeutic agents within the central nervous sy ⁇ tem without elevating intracranial pre ⁇ ure.
  • Animals receiving rEMAP II by the IT/IP routes showed the greatest suppression of tumor growth; at a dose of 100 ng
  • IP 10 ng (IT) /l ⁇ g (IP)
  • IP 100 ng
  • rEMAP II IP/IT
  • rEMAP II was 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) ; thi ⁇ method allowed a comparison of each animal with itself.
  • Histologic appearance of rEMAP II-treated C6 gliomas showed small tumors with evidence of pyknotic changes and apoptosis throughout the lesions, compared with larger tumors in vehicle-treated controls which di ⁇ played homogeneou ⁇ central areas and apoptotic/necrotic changes limited to the periphery.
  • VEGF- ediated vascular ingrowth into Matrigel implants effect of rEMAP II.
  • Matrigel is a complex mixture of basement membrane proteins, a ⁇ well a ⁇ other cell products, from Engelbreth-Holm-Swarm (EHS) tumor cells (Kleinman, H. , 1986; Pas ⁇ aniti, A., 1992) .
  • EHS Engelbreth-Holm-Swarm
  • Addition of an exogenou ⁇ growth factor, such as basic fibroblast growth factor, to Matrigel has been shown to provide a model for assessment of vessel ingrowth (Kleinman, H., 1986; Passaniti, A., 1992) .
  • This model was employed by mixing Matrigel with recombinant human VEGF and subcutaneously implanting the mixture into mice.
  • Ultrastruetural properties of tumor vasculature effect of rEMAP II. Tumors harvested after 3, 6 or 9 days of EMAP II treatment were noticeably different from controls. Macroscopically, reddish pinpoint areas were observed, presumably the result of red blood cell stasis, extravsation and thrombus formation. This impression was confirmed by microscopic studies showing platelet thrombi and red cell stasis, especiallly in large (40 ⁇ m dimabeter) venular vessels. Consistent with the presence of fibrin, ultrastructural ⁇ tudie ⁇ showed a 21 nm periodicity of the fibrin strand ⁇ . Vasculature in both control and EMAP
  • EMAP II has several properties which are consi ⁇ tent with the hypothesis that it ⁇ pecifically 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, such as basic fibroblast growth factor (Stan, A. , 1995) .
  • EMAP II exerts its affects on tumors from the pathologic picture in treated tumors bed of tumors treated with EMAP II
  • a mechamsm 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 cells, such as cytotoxic agents or anti-sense to Insulin-like Growth Factor, the latter having been shown to suppress glioma growth (Res coff, M., 1994) .
  • agents directly targetting neoplastic cells such as cytotoxic agents or anti-sense to Insulin-like Growth Factor

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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US60/003,898 1995-09-18

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US5885798A (en) * 1996-08-28 1999-03-23 Incyte Pharmaceuticals, Inc. DNA encoding a monocyte activating cytokine
JP2002542145A (ja) 1998-11-13 2002-12-10 チルドレンズ・ホスピタル・オヴ・ロス・アンジェルス 血管成長を促進する方法
AUPQ879500A0 (en) * 2000-07-14 2000-08-10 Meditech Research Limited Hyaluronan as cytotoxic agent, drug presensitizer and chemo-sensitizer in the treatment of disease
USD692527S1 (en) 2012-03-12 2013-10-29 Kohler Co. Shower faceplate
US9468939B2 (en) 2012-03-12 2016-10-18 Kohler Co. Faceplate for shower device
WO2014126796A2 (fr) * 2013-02-13 2014-08-21 Indiana University Research & Technology Corporation Méthodes de diagnostic, de traitement et de surveillance de la rétinopathie diabétique
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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US5019556A (en) * 1987-04-14 1991-05-28 President And Fellows Of Harvard College Inhibitors of angiogenin
US5086164A (en) * 1989-01-10 1992-02-04 Repligen Corporation Novel methods and compositions for treatment of angiogenic diseases
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
US5641867A (en) * 1993-09-29 1997-06-24 The Trustees Of Columbia University In The City Of New York Antibody which specifically binds to endothelial-monocyte activating polypeptide II

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