CA2352477A1 - Compositions comprising angiostatin and kringle 5 region of plasminogen and methods of use thereof - Google Patents

Abstract

The present invention relates to one or more compositions that inhibit the proliferation and/or migration of endothelial cells. The compositions contai n a kringle 5 region of plasminogen and/or Angiostatin. If two separate compositions are used, one containing a kringle 5 region of plasminogen, or a biologically active portion thereof, and the other containing Angiostatin, t he compositions may be administered sequentially or simultaneously. The compositions are useful to treat angiogenic associated disorders.

Description

WO 00131244 PCT/US99/2?243 PLASMINOGEN AND METHODS OF USE THEREOF
Field of the Invention The present invention relates to compositions useful to inhibit proliferation and/or migration of endothelial cells. The compositions comprise a combination of angiostatin protein plus the kringle-5 region of plasnainogen. The compositions of the present invention are capable of inhibiting angiogenesis related diseases and modulating angiogenic processes.
to Background of the Invention As used herein, the term "angiogenesis" means the generation of new blood vessels into tissue or organ. Under nonrnal physiological conditions, humans or animals undergo angiogenesis only in very restricted situations. For example, angiogenesis is normally observed in wound healing, fetal and embryonic 15 development and formation of the corpus luteum, endometrium and placenta.
The team "endothelium" means a thin layer of flat endothelial cells that lines serous cavities, lymph vessels, and blood vessels.
Endothelial cells and pericytes, surrounded by a basement membrane, form capillary blood vessels. Angiogenesis begins with the erosion of the basement 2o membrane by enzymes released by endothelial cells and leukocytes. The endothelial cells, which line the lumen of blood vessels, then protrude through the basement membrane. Angiogenic stimulants induce the endol:helial cells to migrate through the eroded basement membrane. The migrating cells form a "sprout" off the parent blood vessel, where the endothelial cells undergo mitosis and proliferate. The endothelial 25 sprouts merge with each other to form capillary loops, creating new blood vessels.
Pathological angiogenesis occurs in a number of disease states, for example tumor metastasis and abnormal growth by endothelial cells, and suppos-ts the pathological damage seen in these conditions. The diverse pathological disease states in which abnormal angiogenesis is present have been grouped together as "angiogenic 3o dependent" or "angiogenic associated" disorders. F'or a review of angiogenesis and its relation to tumor growth, see PCT publication W'U 95/29242 and references cited therein, hereby incorporated by reference in its entirety.

Angiogenesis is tightly regulated by both positive and negative signals.
Angiogenic stimulators, such as fibroblast growth factor (FGF), and vascular endothelial growth factor (VEGF), are potent mitogc.ns for endothelial cell proliferation and strong chemoattractants for endothelial cell migration.
These positive regulators can promote neovascularization l:o sustain the expansion of both primary and metastatic tumors (Cwoss, J. L. et. al., ('.I 993) J. Natl. Cancer Iycst.
8S(2):121-i31; Kim, K.J. et. al., (1993) Nature 362(6243):841-844). Among the negative regulators described to date, Angiostatin ranks as one of the most effective endogenous inhibitors of angiogenesis (O'Reilly, Mf. S. et. al., (1994}
Cel179:315-to 328; O'Reilly, M: S. et. al., (1996) Nat.. Meal. 2:689-692; Wu, Z. et. al., (1997) Biochem. Biophys. Res. Commun. 236:651-654). Angiostatin comprises an internal fragment of plasrninogen and consists of four tx-iple--looped kringle domains constrained by three disulfide bonds. Angiostatin was shown to inhibit endothelial cell proliferation in vitro and to suppress growth factor-:induced angiogenesis in vivo is (O'Reilly (1994), supra). Inhibition of angiogenesis by treatment with angiostatin results in significant suppression of tumor growth in both murine and human tumor models (O'Reilly {1994); O'Reilly (1996); Wu (1997), supra).
Angiostatin has been described as a potent angiogenesis inhibitor that could markedly suppress the growth of a variety of tumors, including carcinomas of lung, 20 prostate, colon, and breast (Cao, Y. et. al., {1998) J. Clin. Invest.
101(S): IOSS-1063;
O'Reilly, et. al ., ( 1996) supra; O'Rei Ily, et. aL, ( 1994) supra).
The individual kringle domains of angiostatin have distinct anti-proliferative and anti-migratory activities toward endothelial cells (Cao, Y. et. al., ( 1996) J. Biol.
Chem. 271:29461-29467; Ji, W.R. et. al., ( 1998) FASEB Jrnl {in press)). It has been 2S documented that the first three kringles of angiostatin exhibit potent inhibitory activities on endothelial cell proliferation whereas lcringle 4 has a marginal effect. It was also shown that the intact kringle structure is essential for the anti-proliferative activities of angiostatin.
The kringle S of human plasminogen displays high structural similarity and 3o about S0% sequence identity to the four kringles of angiostatin. Kringle S
was reported to inhibit bFGF-elicited endothelial cell growth in a dose-dependent manner (Ji, W.R. et. al., (1998) Biochem Biophys Res Commun. 247(2):414-4.19; Cao, Y.
et.
al., (1997) J. Biol. Chem. 272:22924-22928). These data suggest that lmingle S, like angiostatin, may have potent anti-angiogenic activities.

wo 00/3124a PCTNS99/272a3 Summary of the Invention In accordance with the present invention, compositions are provided comprising an Angiostatin molecule plus a kringie 5 molecule. Applicants herein provide evidence that a combination of Angiostatin plus a kringle 5 molecule exhibit a synergistic effect, and shows superior results over Angiostatin, the kningle S region alone, or kringles 1-5 of plasminogen.
The present invention provides methods and compositions for treating diseases and processes mediated by undesired arid uncontrolled angiogenesis by administering 1 o to a human or animal a composition comprising Anl;iostatin and the kringle 5 region of plasminogen. The present invention is particularly useful for treating, or for repressing the growth of, tumors. Administration of compositions of the present invention to a human or animal with prevascularized metastasized tumors will prevent the growth or expansion of those tumors.
is The methods of the present invention encompass the use of a single composition comprising Angiostatin and the kringle: 5 region of plasminogen.
Additionally, the methods of the present invention encompass the use of a composition comprising Angiostatin and a composition comprising the kringle 5 region ~of plasminogen, said compositions administered simultaneously or 2o sequentially.
All references cited herein, whether supra or it f~a, are hweby incorporated by reference in their entirety.
Brief Description of the Figures 25 Figure 1 is a graph showing the additive anti-endothelial cell migratory activities of kringle 5 of human plasminogen in combination with Angiostatin.
Figure lA shows additive inhibition of HCTVEC cell migration by kringle 5 of human plasminogen in combination with Angiostatin; Figure 1B shows additive inhibition of BCE cell migration by kringle 5 of human plasminagen in combination with 3o Angiastatin. (Abbreviations: KS = kringle 5; AST == Angiostatin; bFGF =
basic fibroblast growth factor).

Detailed Description of the lmvention Angiostatin has been described as a potent angiogenesis inhibitor that can markedly suppress the growth of a variety of tumors, including carcinomas of lung, prostate, colon, and breast. Angiostatin and the sequences of mouse, human, rhesus monkey, porcine and bovine plasminogen are given iin U.S. Patent No.
5,639,725, the disclosure of which is incorporated herein by reference in its entirety.
The kringle 5 of human plasminogen displays high structural similarity and about 50% sequence identity to the four k~ingles of angiostatin. Kringle 5 was reported to inhibit bFGF-elicited endothelial cell growth in a dose-dependent manner.
to These data suggest that kringle 5, like angiostatin, may have potent anti-angiogenic activities.
The present invention for the first time presents evidence that combination therapy, involving the administration of Angiostatin and a kringle 5 fi~agment of plasminogen, exhibits superior angiogenesis inhibition over Angiostatin alone, kringle 15 5 alone, and a kringle 1-5 region of plasminogen.
Compositions are provided comprising a kringle 5 region of plasminogen and Angiostatin. The source plasminogen (i.e., the plasminogen from which the kringle 5 region andlor the Angiostatin is derived) may be from the same species (e.g., human) or from different species (e.g., a kringle 5 region from murine plasminogen and 2o Angiostatin comprising kringles I-4 from human pl~usminogen). A kringle 5 region from plasminogen, or biologically active portion thereof, and said Angiostatin, may be deuived, for example, fi~om murine plasminogen, lhuman plasrninogen, Rhesus plasminogen, porcine plasrninogen, canine plasminogen, or bovine plasminogen.
Preferably, the kringle 5 region and the Angiostatin we derived from the same 25 species.
Additionally, the kringle 5 fragment and the Angiostatin may be administered in the same composition, or they may be administered in separate compositions.
If separate compositions are used, the compositions may be administered simultaneously or sequentially.
so Also encompassed within the scope of the present invention are variations of the kringle 5 fi~agrnent, including biologically active fragments, and biologically active analogs involving amino acid deletions, additions and/or substitutions.
"Biologically active fragment" includes fi-agments of the kringle 5 region that maintain the same biological activity of the kringle :S region. "Biologically active analogs" includes variations of the kringle 5 region that do not materially alter the biological activity (i.e., anti-angiogenic activity) of the I~;ringle S
fragment. Included within the scope of the invention are changes made to txie kringle 5 fragment that increase anti-angiogenic activity.
Preferred analogs include a kr~ingle 5 fragment and Angiostatin molecule whose sequences differ from the wild-type sequence by one or more conservative amino acid substitutions or by one or more non-conservative amino acid substitutions, deletions or insertions which do not abolish the biological activity of the molecules.
Conservative substitutions typically include the substitu~.tion of one amino acid for to another with similes characteristics, e.g., substitutions within the following groups:
valine, glycine; glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. Other canservative amino acid substitutions c;an be taken from the table below.
Table 1 Conservative amino acid replacements For Amino Acid Code Replace with any of Alanine A D-Ala, Gly, beta-Ala, lL-Cys, D-Cys Arginine R D-Arg, Lys, D-Lys, homo-Arg, D-homo-Arg, Met, Ile, D-M et, D-I1 e, Orn, D-Orn Asparagine N D-Asn, Asp, D-Asp, Glu, D-Glu, Gln, D-Gln Aspartic Acid D D-Asp, D-Asn, Asn, Glu, D-Glu, Gln, D-Gln Cysteine C D-Cys, S-Me-Cys, Met, D-Met, Thr, D-Thr Glutamine Q D-Gln, Asn, D-Asn, Glu, D-Glu, Asp, D-Asp Glutamic Acid E D-Glu, D-Asp, Asp, Asn, D-Asn, Gln, D-Gln Glycine G Ala, D-Ala, Pro, D-Pro, f3-Ala, Acp Isoleucine I D-Ile, Val, D-Val, Leu, D-Leu, Met, D-Met Leucine L D-Leu, Val, D-Val, Met, D-Met Lysine K D-Lys, Arg, D-Arg, horno-Arg, D-homo-Arg, Met, D-Met, 11 e, D-I1 e, Osn, D-Orn Methionine M D-Met, S-Me-Cys, Ile, D-Ile, Leu, D-Leu, Val, D-Val Phenylalanine F D-Phe, Tyr, D-Thr, L-lDopa, His, D-His, Tip, D-Trp, Trans-3,4, or S-phenylproline, cis-3,4, or 5-phenylproline Proline P D-Pro, L-1-thioazolidine-4-carboxylic acid, D- or L-1-oxazolidine-4-cwboxylic acid Serine S D-Ser, Thr, D-Thr, allo-Thr, Met, D-Met, Met(O), D-Met(O), L-Cys, D-Cys;

Threonine T D-Thr, Ser, D-Ser, allo-Thr, Met, D-Met, Met(O), D-Met(O), Val, D-Val Tyrosine Y D-Tyr, Phe, D-Phe, L-:Dopa, His, D-His Valine ~ V ~ D-Val, Leu, D-Leu, Ile, D-Ile, Met, D-Met 4 PCTlUS99127243 Other analogs within the invention are those with modifications which increase protein or peptide stability; such analogs m;~y contain, for example, one or more non-peptide bonds (which replace the peptide bonds) in the protein or peptide sequence. Also included are analogs that include re:>idues other than naturally occurring L-amino acids, e.g., D-amino acids or non-naturally occuuring or synthetic amino acids, e.g., fi or y amino acids.
The kringle 5 regions and Angiostatin useful in the present invention may be obtained by synthetic means, i.e., chemical synthesis of the polypeptide from its 1 o component amino acids, by methods known to those; of ordinary skill in the aat. For example, the solid phase procedure described in Houghton et al., Proc.
Natl.'Acad.
Sci. 82, 5131-5135 (1985) rnay be employed. The polypeptides may be obtained by production in prokaryotic or euka~yotic host cells expressing a DNA sequence coding for all or pant of the desired fragment, or by in vitro translation of the mRNA encoded 15 by a DNA sequence coding for the desired peptide. Techniques for the production of a kringle 5 fragment and Angiostatin by these means ai-e known in the an, and are described herein.
The kringle S region and Angiostatin produced in this manner may then be isolated and purified to some degree using vaa-ious protein purification techniques.
2o For example, chromatographic procedures such as ion exchange chromatography, gel filtration chromatography and immunoaffinity chromatography may be employed.
If the polypeptides of the invention (i.e., a kringle 5 fragment and Angiostatin) aa~e made through recombinant techniques, the DNA, sequences useful to prepare the polypeptides of the invention can be obtained using various methods well known to 25 those of ordinary skill in the art. The expression vectors can be partly or wholly synthesized chemically and/or paz-tly or wholly prepared through genetic engineering techniques. Fragments can be sequentially ligated (via appropriate temninal restz~iction sites or complementary terminal sequences) so as to form the col~~ect linear sequence of nucleotides.
3o Expression vehicles of the invention for production of the anti-angiogenic polypeptides of the invention include plasrnids or other vectors. In general, such vectors contain control sequences that allow expression in various types of hosts.
Suitable expression vectors containing the desired coding and contl-ol sequences may WO OOI31244 PCT/US99/2'7243 be constructed using standard recombinant DNA techniques known in the as~t, many of which are described in Sambrook et al. (1989), Molecular Cloning: A
Laboratory Manual, 2nd edition, Cold Spuing Harbor Laboratory, Cald Spring Harbor, NY.
An expression vector as contemplated by the; present invention is capable of directing the replication of the vector in bacteria, ye~~st, insect, andlor mammalian cells. One class of vectors utilizes yeast DNA elements that provide autonomously replicating origins such as the yeast 2~. element or A,RS 1 sequence which yield extrachromosornal plasmids. A second class of vectors relies upon the integration of the desired gene sequences into the host cell chromosome. The vectors may also to incorporate a bacterial origin of replication. Suitable bacterial origins of replication include, for example, the ColEl, pSC101 and MI3 origins of replication.
Expression vectors useful of the present invc,ntion typically contain a promoter located 5' to (i.e., upstream of) the DNA sequence to be expressed, and a transcription termination sequence located 3' to (i.e., downstream ofj the sequence to be expressed.
15 Suitable promoters include, for example, the yeast ADHI promoter. The promoter sequence may also be inducible, to allow modulation of expression {e.g., by the presence or absence of nutrients or other inducers in the growth medium).
Examples include the yeast GALI, CUPl, and MET35 promoters. Suitable termination sequences include, for example, the yeast CYCI teumination and polyadenylation 2o sequences.
The expression vectors may also include other regulatory sequences for optimal expression of the desired product. Such sequences include secretoiy leader sequences, which provide for secretion of the expressed product or direct membrane localization, and restriction enzyme recognition sequences, which provide sites for 25 cleavage by restriction endonucleases. All of these materials are known in the ay-t and most a~-e commercially available.
A suitable expression vector may also include marking sequences, which allow phenotypic detection and/or selection of tt-ansformed yeast or bacterial cells.
Such a marker may provide prototrophy to an auxooophic host (e.g., amino acid 30 biosynthetic genes), biocide resistance or supersens~itivity (e.g., antiobiotic resistance) or a phenotypically detectable signal (e.g,, fluorescence). The selectable marker gene can be either directly linked to the DNA gene sequences to be expressed, or introduced into the same cell by transformation. Examples of yeast selectable markers include Basidium pullularrs AURI-C gene, the S. cerevisiae URA3 or genes and the like. Examples of bacterial selectable markers include the ampicillin resistance gene. A preferred vector is pYESII, containing the ColEl and 2p.
origins of replication, the yeast URA3 and bacterial ampR genes, and the yeast GALI
promoter sequence (Inviti~ogen).
s In a further alternative, the constructs may be introduced into a cell by transformation in conjunction with a gene allowing for selection where the construct will become integrated into the host genome or persist episomally. Usually, the constZ-uct will be part of a vector having homologous sequences for integration or a replication system recognized by the host cell.
1o The compositions of the present invention, comprising a kringle 5 fragment of plasminogen (or a biologically active fragment or analog thereof) and Angiostatin, are useful to treat angiogenic associated disorders. The present invention includes the method of tz-eating an angiogenic-associated disorder with an effective amount of a composition comprising a kringle 5 fragment and Angiostatin. As described above, a 1s single composition comprising a lcringle 5 fragment and Angiostatin may be used, or sepwate compositions (a first comprising a kringle 5 fiagment and a second comprising Angiostatin) may be administered simultaneously or sequentially.
The angiogenesis mediated diseases include, but are not limited to, solid tumors, blood bom tumors such as leukemias; tumor metastasis; benign tumors, for 2o example, hemangiomas, acoustic acuromas, neurofibrornas, trachomas, and pyogenic granulomas; rheumatoid arthritis; psoriasis; ocular angiogenic diseases, for example, diabetic retinopathy, retinopathy of prematurity, macular degenet-ation, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis; Osler-Webber Syndrome; myocardial angiogenesis; plaque neovascularization; telangiectasia;
25 hemophiliac joints; angiofibroma; and wound granulation. The compositions of the present invention are useful in treatment of disease of excessive or abnormal stimulation of endothelial cells. These disorders include, but are not limited to, intestinal adhesions, atherosclerosis, scleroderma, and hypeW-ophic scars, i.e., keloids.
The compositions can also be used as birth control agents by preventing 30 vascularization required for embryo implantation.
The compositions and methods of the present invention may be used in combination with other compositions and procedures for the treatment of angiogenic associated disorders. For example, a tumor may be treated conventionally with surgery, radiation or chemotherapy, and then compositions comprising a kiingle S

WO 00!31244 PCTNS99/2'7243 region and Angiostatin may be subsequently administered to the patient to extend the dormancy of micrometastases and to stabilize any residual primary tumor.
The present invention also provides pharmaceutical (i.e., therapeutic) compositions comprising a kringle 5 region of plasrr~inogen (or a biologically active fragment or analog thereof) and Angiostatin, optionally in combination with at least one additional active compound, and any pharmaceutically acceptable carrier, adjuvant or vehicle. "Additional active compounds''' encompasses, but is not limited to, an agent or agents selected from the group consisting of an irnmunosuppressant, an anti-cancer agent, an anti-viral agent, an anti-inflammato;y agent, an anti-fungal 1o agent, an antibiotic, or an anti-vascular hypeiproliferation compound.
The term "pharmaceutically acceptable cawier, adjuvant or vehicle" refers to a cazTier, adjuvant or vehicle that may be administered to a subject, incorporated into a composition of the present invention, and which does not destroy the pharmacological activity thereof. Pharmaceutically acceptable carriers, adjuvants and vehicles that 15 may be used in the pharmaceutical compositions of the present invention include, but are not limited to, the following: ion exchangers, alumina, aluminum stearate, lecithin, self emulsifying drug delivery systems ("SEDDS") such as d(-tocopherol polyethyleneglycol 1000 succinate, surfactants used in pharmaceutical dosage forms such as Tweens or other similar polymeric delivery ;matrices, serum proteins such as 2o human serum albumin, buffer substances such as phosphates, giycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyroHdone, cellulose-based substances, 25 polyethylene glycol, sodium carboxyrnethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.
Cyclodextrins such as a-, (3- and y-cyclodextrin, or chemically modified derivatives such as hydroxyalkylcyclodextrins, including 2- and 3-hydroxypropyl-(3-cyclodextrins, or other solubilized derivatives may also be used to enhance delivery of 3o the compositions of the present invention.
The compositions of the present invention rr~ay contain other therapeutic agents as descuibed below, and may be formulated, :For example, by employing conventional solid or liquid vehicles or diluents, as well as pharmaceutical additives of a type appropriate to the mode of desired administration (for example, excipients, binders, preseZVatives, stabilizers, flavors, etc.) according to techniques such as those well known in the aart of phaz~naceutical foimularion.
The compositions of the present invention rr~ay be administered by any suitable means, for example, orally, such as in the fcn~rn of tablets, capsules, granules or powders; sublingually; buccally; parenterally, such as by subcutaneous, intravenous, inta~amuscular, or intrastemal injection or infusion techniques (e.g., as sterile injectable aqueous or non-aqueous solutions or suspensions}; nasally such as by inhalation spray; topically, such as in the form of a cream or ointment; or rectally such as in the form of suppositories; in dosage unit fornnulations containing non-toxic, l0 pharmaceutically acceptable vehicles or diluents. T'he present compositions may, for example, be administered in a form suitable for immediate release or extended release. Immediate release or extended release may be achieved by the use of suitable pharmaceutical compositions comprising a ki-ingle 5 fragment and Angiostatin, or, particularly in the case of extended release, by the use of devices such as subcutaneous implants or osmotic pumps. The present compositions may also be administered liposomally.
Exemplary compositions for oral administration include suspensions which may contain, for example, microciystalline cellulose for imparting bulk, alginic acid or sodium alginate as a suspending agent, methylcellulose as a viscosity enhances, arid 2o sweeteners or flavoring agents such as those known in the aa~t; and immediate release tablets which may contain, for example, microczyst~alline cellulose, dicalcium phosphate, starch, magnesium stearate andlor lactose and/or other excipients, binders, extenders, disintegrants, diluents and lubricants such as those known in the art. The present compounds may also be delivered through t:he oral cavity by sublingual andlor 2s buccal administr ation. Molded tablets, compressed tablets or freeze-dried tablets are exemplary forms which may be used. Exemplary compositions include those formulating the present compositions with fast dissolving diluents such as rnannitol, lactose, sucrose andlor cyclodextrins. Also included in such fomnulations may be high molecular weight excipients such as celluloses (avicel) or polyethylene glycols 30 {PEG}. Such foumulations may also include an excipient to aid mucosal adhesion such as hydroxy propyl cellulose {HPC), hydroxy propyl methyl cellulose (HPMC), sodium carboxy methyl cellulose (SCMC), malefic ;anhydride copolymer {e.g., Gantrez), and agents to control release such as polyaciylic copolymer (e.g., Carbopol 934). Lubricants, glidants, flavors, coloring agents .and stabilizers may also be added for ease of fabrication and use.
Exemplazy compositions for nasal aerosol or inhalation administration include solutions in saline which may contain, for example, benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, andlor other solubilizing or dispersing agents such as those knovvn in the art.
Exemplary compositions for parenteral administration include injectable solutions or suspensions which may contain, for example, suitable non-toxic;
parenterally acceptable diluents or solvents, such as mannitol, 1,3-butanediol, water, io Ringer's solution, an isotonic sodium chloride solution, or other suitable dispersing or wetting and suspending agents, including synthetic mono- or diglycerides, and fatty acids, including oleic acid. The team "parenteral" as used herein includes subcutaneous, inti~acutaneous, intravenous, inti-amuscular, inti~aanicular, intt~aaz-terial, intrasynovial, inh-asternal, int<-athecal, intt-alesional and inti-acranial injection or 15 infusion techniques.
Exemplary compositions for rectal administration include suppositories which may contain, for example, a suitable non-irritating c;xcipient, such as cocoa butter, synthetic glyceride esters or polyethylene glycols, which are solid at ordinary temperatures, but liquify and/or dissolve in the rectal cavity to release the active 2o compounds (i.e., the kringle 5 fragment and Angiostatin).
Exemplary compositions for topical administration include a topical carrier such as Plastibase (mineral oil gelled with polyethylene).
The effective amount of a compound of the present invention may be determined by one of ordinary skill in the art, and includes exemplary dosage amounts 25 for an adult human of from about 0.1 to 500 mg/kg of body weight of active compound per day, which may be administered in a single dose or in the form of individual divided doses, such as from 1 to 5 times per day. It will be understood that the specific dose level and fi~equency of dosage for any particular subject may be varied and will depend upon a variety of factors including the activity of the specific 3o compound employed, the metabolic stability and length of action of that compound, the species, age, body weight, general health, sex and diet of the subject, the mode and time of administration, rate of excretion and clearance, drug combination, and severity of the particular condition. Preferred subjects for treatment include animals, most preferably mammalian species such as humans, and domestic animals such as dogs, cats and the like, subject to angiogenic associated disorders.
The compositions of the present invention may be employed alone or in combination with other suitable therapeutic agents useful in the treatment of s angiogenic associated disorders, such as angiogenesis inhibitors other than those of the present invention, antiinflammatories, antiproliferatives, chemotherapeutic agents, immunosuppressants, and antimicrobials.
Other therapeutic agents, when employed in combination with the compositions of the present invention,-may be used, for example, in those amounts to indicated in the Physicians' Desk Reference {PDR) or as otherwise determined by one of ordinary skill in the art.
The following examples ar-e further illustrative of the present invention.
These examples are not intended to limit the scope of the present invention,, and provide further understanding of the invention.
Example l: Gene construction, expression, and purification of recombinant human angiostatin The human angiostatin cDNA was amplified from a human plasminogen cDNA template {American Type Culture Collection, Rockville, MD) by standard 2o polymerase chain reaction (PCR) with the foilowinl; two primers:

5'-GCGGATCCATGAAAGTGTATCTCTCAGAGTGCAAG (forward primer for residue 98-458); and 5'-GCGGATCCTCACTATTCTGTTCCTCiAGCATTTTTTCAG (reverse primer for residue 98-458).
The amplified cDNA fragment was Iigated into the BamHI site of the pMelBacA vector (InVitrogen, San Diego, CA). The angiostatin cDNA plasmid was then co-transfected into S~ cells with viral BaculoCroldTM DNA (PharMingen, San Diego, CA). Briefly, lx 106 Sf~ cells were seeded in a T25 tissue culture flask and incubated at 27°C with 1 ml of the transfection solution containing 2 ~g of transfer 3o vector DNA, 0.5 pg of BaculoGoldTM DNA, and 6 ~.l of Cellfectin (Gibco BRL, Gaithersburg, MD). The transfection solution was removed 4 hours post-transfection and replaced with 3 m1 of Sf900II medium (Gibco BRL, Gaithersburg, MD). Four days after incubation, the viral supernatant was harvested and individual clones were identified by limiting dilution. The clone with the lhighest protein expression, as determined by SDS-PAGE/Coomassie blue staining, was amplified in Sf9 cells for protein production. High Five insect cells (InVitrogen, San Diego, CA} (1.5 x 10~
cells/rnl) were infected with approximately 1 x I 0' viral panicleslml of the recombinant virus. After 48 houm, the culture supernatant was collected by centrifugation at 5,000 x g for 30 minutes. The supernatant was then applied to a lysine-Sepharose column and angiostatin protein eluted with s-aminocaproic acid as previously described (Wu, Z., et. al., (1997) Biochem. Biophys. Res. Commun.
236:651-654).
Example 2: Production of recombinant knngle 5 of human plasminogen The cDNA for K4-5 was amplified from HPg cDNA template by PCR as previously described {Menhart, N., et. al., {1993) Biochemistry 32:8799-8806). The amplified cDNA was inserted between the Avr Il and the Not I sites of the pPIC9K vector (InVitrogen, San Diego, CA). The transfer plasmid, pPIC9K[K4-KS of HPg], was then linearized with restriction endonuclease Sac I and transformed into the strain of Pichia pasto~is by electi~oporation. Isolation of high yield clones and the following high bio-mass fermentation were performed as previously described (NiIsen, S. L., et. al., (1997) Biotech. ar~cl Applied Biochem. 25:63-74). The protein of human plasminogen was purified from the fermentation medium by lysine 2o affinity chromatography as described (Chang, Y., et. al., (1997) Biochemistry 36(25):7652-7663). The purified products were extensively dialyzed against water;
lyophilized, and digested with elastase at room temperature for 16 hours in 0.1 M
phosphate/15 mM s-aminocaproic acid (pH 7.8) at a protein: elastase ratio of 1:250 (w/w). The digest was then reapplied to a lysine-Sepharose chromatography column z5 and the kringle 5 fragment collected in the flow-through. The kringle S
protein was extensively dialyzed against water and lyophilized.
Example 3: Anti-Endothelial Cell Proliferative/Mil;ratory Activity of Kringle 5 in Combination With Angiostatin 3o Two primary endothelial cell types, human umbilical vein endothelial cell (HLTVEC) and bovine capillary endothelial {BCE) cell, were chosen foa- the Boyden-chamber based migration assay. Briefly, HUVEC cells were obtained commercially (Clonetics, San Diego, CA). Bovine capillary endothelial cells were harvested fi-orn bovine adrenal glands as previously described (Fol~kman, J. et. al., (1979}
Proc. Natl.

Acad. Sci. USA 76:5217-5221). BCE cells were maintained in DMEM in the presence of 10% bovine calf serum, 1% antibiotics, and 3 nglrnl of bFGF (PeproTech, Rockhill, NJ).
To evaluate endothelial cell migration, a Boyden Chamber-based assay was performed. First, polycarbanate membranes with 8 l.~m pore sizes (Neuro Probe Inc., Cabin John, MD) were coated with 100 ~tg/ml of collagen type I following manufacturer's instructions (Becton Dickinson, Bedford, MA). BCE cells between passage 10 to 14, or I-IWEC cells between passage I to 3, were harvested with 0.05%
tiypsin solution, washed, resuspended to a density of 75,000 cells/ml in DMEM
to containing 10% bovine calf serum and 10 ng/ml of bFGF, and incubated at 37°C for 30 minutes. During cell incubation, various concentrations of kningle 5, Angiostatin, or a combination of kringle 5 and Angiostatin samples were loaded into the lower chambers. The collagen-coated membrane filter ways placed on top of the lower chamber and the top chamber then attached. After the 30 minute incubation, endothelial cells were loaded into the top chamber and incubated at 37"C for 4 hours.
The chemotaxis chamber was then dismantled and the filter membrane removed.
The non-migrated cells were scraped off the upper surface of the membrane with cotton swabs three times. After rinsing with PBS, the membrane was fixed with 10%
buffered formalin for 45 minutes and then stained with Gill No. 2 hematoxyiin overnight (J.B. Baker, Phillipsburg, NJ). The membrane was then rinsed with PBS
and mounted with Cytoseal (Stephens Scientific, Riverdale, NJ). Each sample was tested in quach-uplicate and a representative field in each well was counted at 100x magnification to determine the number of migrated cells.
Table 2 below summarizes the inhibitory activities of kningle 5 ("KS"), Angiostatin ("AST"), KS+AST combination, andkongle I-5 ("Kl-5") of pl asminogen.
Table 2 Protein lCso (nM) ICso (nM) (HUVEC),............................................._..._.....................
................~BCE)..............................................._..........
....

.................................... 489.1 .........._........................_..._.:........._.......................

AST 650.2 K5 100.0 89.4 K5 + AST 9.3 8.8 ...~,."~,., K1-' w,~"~""~.."~,., 8~~ "~",.,N." 33.7-~.~"w .,~.,~"..""~.,~.,.",.

For HUVEC migration, the ICso's (protein concentrations at 50% of inhibition) of angiostatin and kringle 5 are approximately 0.651ZM and 0.10 p.M, respectively (Figure lA & Table 2). A combination of these two agents produced a significant increase of the anti-migratory activity wiith an ICso of about 9.3 nM. This one-log increase in anti-endothelial cell activity implies that kringle S and angiostatin may inhibit angiogenesis via different yet coordinated pathways. For BCE cell migration, the ICsc~'s of angiostatin and lcringle 5 were approximately 0.49 p.M and 0.09 ~.M, respectively {Figure 1B & Table 2). A combination of these two proteins resulted in a nearly one log increase in. the anti-migratory activity (ICso =
8.8 nM).
to This synergism suggests that kringle S and angiostatin may function in cooperation to block angiogenesis. As a reference, the recombinant kringle I-5 protein was generated in the Bacculovirus expression system. It is shown to potently inhibit ITEJVEC and BCE cell migration in a dose-dependent manner. The anti-migratory activities of lcringle I -5 are close to those of angiost,atin and kringle 5 combinations for HUVEC (ICso of kringle 1-5 = 8 nM), but is approximately 4-fold less potent for BCE migration (ICso = 33.7 nM for BCE). This shows that the combination of a kringle 5 fragment of plasminogen plus Angiostatin shows superior activity even over a ki-ingle I-5 fragment of plasminogen. Ki-ingle 5 of human pIasminogen may thus be used in combination with Angiostatin to potentiate ias anti-angiogenic functions.
Although the present invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims.

Claims (14)

We claim:

1. A therapeutic composition for inhibiting endothelial cell proliferation and/or migration comprising a kringle 5 region from plasminogen, or biologically active portion thereof, and angiostatin, and a pharmaceutically acceptable carrier.

2. The therapeutic composition of claim 1 wherein said kringle 5 region from plasminogen, or biologically active portion thereof, and said angiostatin are derived from the same or different species.

3. The therapeutic composition of claim 1 wherein said kringle 5 region from plasminogen, or biologically active portion thereof, and said angiostatin are derived from murine plasminogen, human plasminogen, Rhesus plasminogen, porcine plasminogen, canine plasminogen, or bovine plasminogen.

4. A method of inhibiting endothelial cell proliferation and/or migration comprising administering to an endothelial cell a proliferation and/or migration inhibiting amount of a composition comprising a kringle 5 region from plasminogen, or a biologically active potion thereof, and angiostatin.

5. The method of claim 4 wherein said kringle 5 region from plasminogen, or biologically active portion thereof, and said angiostatin are derived from the same or different species.

6. The method of claim 4 wherein said kringle 5 region from plasminogen, or biologically active portion thereof, and said angiostatin are derived from murine plasminogen, human plasminogen, Rhesus plasminogen, porcine plasminogen, canine plasminogen, or bovine plasminogen.

7. A method of treating a mammal with an angiogenic associated disorder comprising administering to said mammal a treatment effective amount of a composition comprising a kringle 5 region from plasminogen, or a biologically active portion thereof, and angiostatin.

8. The method of claim 7 wherein said kringle 5 region from plasminogen, or biologically active portion thereof, and said angiostatin are derived from the same or different species.

9. The method of claim 7 wherein said kringle 5 region from plasminogen, or biologically active portion thereof, and said angiostatin are derived from murine plasminogen, human plasminogen, Rhesus plasminogen, porcine plasminogen, canine plasminogen, or bovine plasminogen.

10. A method of treating a mammal with an angiogenic associated disorder comprising administering to said mammal a treatment effective amount of a combination of a first and a second composition, said first composition comprising a kringle 5 region from plasminogen, or a biologically active portion thereof, said second composition comprising angiostatin.

11. The method of claim 10 wherein said first composition and said second composition are administered simultaneously.

12. The method of claim 10 wherein said first composition and said second composition are administered sequentially.

13. The method of claim 10 wherein said kringle 5 region from plasminogen, or biologically active portion thereof, and said. angiostatin are derived from the same or different species.

14. The method of claim 10 wherein said kringle 5 region from plasminogen, or biologically active portion thereof, and said angiostatin are derived from murine plasminogen, human plasminogen, Rhesus plasminogen, porcine plasminogen, canine plasminogen, or bovine plasminogen.