JP2002531385A - Compositions comprising the kringle 5 region of angiostatin and plasminogen and methods of using the same - Google Patents

Abstract

(57) SUMMARY The present invention relates to one or more compositions that inhibit the growth and / or migration of endothelial cells. The composition contains the kringle 5 region of plasminogen and / or angiostatin. If one uses the kringle 5 region of plasminogen or a biologically active portion thereof and the other uses two independent compositions containing angiostatin, the compositions can be administered sequentially or simultaneously. The compositions are useful for treating angiogenesis-related disorders.

Description

DETAILED DESCRIPTION OF THE INVENTION

TECHNICAL FIELD The present invention relates to compositions useful for inhibiting endothelial cell proliferation and / or migration. This composition is a kringle 5 of angiostatin protein and plasminogen.
Includes combinations with regions. The compositions of the present invention can inhibit angiogenesis-related diseases and modulate angiogenic processes.

BACKGROUND OF THE INVENTION [0002] As used herein, the term "angiogenesis" refers to the generation of new blood vessels in a tissue or organ. Under normal physiological conditions, humans or animals only undergo angiogenesis under very limited circumstances. For example, angiogenesis is normally observed during wound healing, fetal and embryonic development and formation of the corpus luteum, endometrium and placenta. The term "endothelium" refers to a thin layer of flat endothelial cells lining serous cavities, lymph vessels and blood vessels.

[0003] Endothelial cells and pericytes surrounded by a basement membrane form capillaries. Angiogenesis begins with erosion of the basement membrane by enzymes released by endothelial cells and leukocytes. The endothelial cells lining the lumen of the vessel then protrude through the basement membrane. Angiogenic stimulators cause endothelial cells to migrate across the eroded basement membrane. The migrating cells form "sprouts" that sprout from the parent blood vessel, where the endothelial cells undergo mitosis and proliferation. Endothelial sprouts coalesce with each other to form a capillary loop and create new blood vessels.

[0004] Pathological angiogenesis occurs in many disease states, such as tumor metastasis and abnormal proliferation by endothelial cells, and supports the pathological damage seen in those states. The various pathological disease states in which abnormal angiogenesis is observed are "angiogenesis dependent" or "
It has been classified into one group as "angiogenesis-related" disorders. For an overview of angiogenesis and the relationship of angiogenesis to tumor growth, see PCT Publication No. WO 95/29242 and the references cited therein, which are incorporated herein by reference in their entirety. Is).

[0005] Angiogenesis is tightly regulated by both positive and negative signals. Angiogenesis stimulating factors such as fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) are potent mitogens for endothelial cell proliferation and potent chemoattractants for endothelial cell migration. These positive regulators can promote neovascularization and sustain the growth of both primary and metastatic tumors (Gross, JL et al. (1993).
J. Natl. Cancer Inst. 85 (2): 121-131; Kirn, KJ et al. (1993) Nature 362 (624).
3): 841-844). Of the negative regulators described to date, Angiostatin is one of the most potent endogenous inhibitors of angiogenesis (O
'Reilly, MS et al. (1994) Cell 79: 315-328; O'Reilly, MS et al. (1996) Nat. Med. 2
: 689-692; Wu, Z. et al. (1997) Biochem. Biophys. Res. Commun. 236: 651-654). Angiostatin contains an internal fragment of plasminogen and consists of four triple loop kringle domains constrained by three disulfide bonds. Angiostatin has been shown to inhibit endothelial cell proliferation in vitro and suppress growth factor-induced angiogenesis in vivo (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'Reil)
ly (1996); Wu (1997) supra).

[0006] Angiostatin has been described as an extremely potent angiogenesis inhibitor that can significantly inhibit the growth of various tumors, including lung, prostate, colon and breast cancer (
(1998) J. Clin. Invest. 101 (5): 1055-1063; O'Reilly, et al. (1996) supra; O'Reilly et al. (1994) supra).

The individual cling domains of angiostatin have different antiproliferative and antimigratory activities on endothelial cells (Cao, Y. et al. (1996) J. Biol. Chem. 271: 29461-29467;
Ji, WR et al. (1998) FASEB Jrnl (printing). It has been reported that the first three kringles of angiostatin show potent inhibitory activity on endothelial cell proliferation, whereas kringle 4 has only a minor effect. It has also been shown that complete kringle structure is essential for the antiproliferative activity of angiostatin.

The human plasminogen kringle 5 shows high structural similarity and about 50% sequence identity to the four kringles of angiostatin. Kringle 5 is bFGF
Dose-dependently inhibits endothelial cell proliferation induced by (Ji, WR)
(1998) Biochem Biophys Res Commun. 247 (2): 414-419; Cao, Y. et al. (1997) J. Biol. Chem. 272: 22924-22928). These data suggest that kringle 5 may have potent anti-angiogenic activity as well as angiostatin.

SUMMARY OF THE INVENTION In accordance with the present invention, there is provided a composition comprising an angiostatin molecule and five kringle molecules. Applicants now provide evidence that the combination of angiostatin and the kringle 5 molecule exhibits a synergistic effect, with superior results over the angiostatin, kringle 5 region alone, or the plasminogen kringle 1-5. .

[0010] The present invention is directed to a method of treating diseases and processes mediated by unwanted uncontrolled angiogenesis by administering to a human or animal a composition comprising angiostatin and the kringle 5 region of plasminogen. A composition of The invention is particularly useful for treating tumors or for suppressing tumor growth.
If the composition of the present invention is administered to a human or animal having a pre-angiogenic metastatic tumor,
The growth or proliferation of those tumors will be prevented.

[0011] The method of the invention involves the use of a single composition comprising angiostatin and the kringle 5 region of plasminogen. The method of the present invention also provides a composition comprising angiostatin, which is administered simultaneously or sequentially, and a kringle 5 of plasminogen.
Also encompasses use with compositions that include a region.

[0012] All documents cited herein, both those mentioned above and those mentioned below, are incorporated herein by reference in their entirety.

DETAILED DESCRIPTION Angiostatin is described as a very potent angiogenesis inhibitor that can significantly inhibit the growth of various tumors, including lung, prostate, colon and breast cancer.
The sequences of angiostatin and murine, human, rhesus, pig and bovine plasminogen are described in U.S. Patent No. 5,639,725, the disclosure of which is incorporated herein by reference in its entirety.

[0014] Kringle 5, a human plasminogen, shows high structural similarity and about 50% sequence identity to the four kringles of angiostatin. Kringle 5 is bFGF
Was reported to inhibit endothelial cell proliferation induced by Dose in a dose-dependent manner. These data suggest that kringle 5 may have potent anti-angiogenic activity as well as angiostatin.

The present invention provides evidence that the combination therapy of administering angiostatin and the kringle 5 fragment of plasminogen exhibits superior angiogenesis inhibition over angiostatin alone, kringle 5 alone, and the kringle 1-5 region of plasminogen. It is presented for the first time.

There is provided a composition comprising the kringle 5 region of plasminogen and angiostatin. The original plasminogen (ie, the kringle 5 region and / or the plasminogen from which angiostatin is derived) may be from the same species (eg, human) or different species (eg, the kringle 5 region from murine plasminogen and human plasminogen). Angiostatin containing kringle 1-4). The kringle 5 region of plasminogen or a biologically active part thereof and the angiostatin are, for example, murine plasminogen, human plasminogen,
It can be derived from rhesus monkey plasminogen, porcine plasminogen, dog plasminogen or bovine plasminogen. Preferably, the kringle 5 region and the angiostatin are from the same species.

The kringle 5 fragment and angiostatin can be administered as the same composition, or they can be administered as separate compositions. If separate compositions are used, they can be administered simultaneously or sequentially.

Mutants of the kringle 5 fragment, such as, for example, biologically active fragments and biologically active analogs containing amino acid deletions, additions and / or substitutions are also within the scope of the invention. "Biologically active fragments" include fragments of the kringle 5 region that retain the same biological activity as the kringle 5 region. "Biologically active analogs" include variants of the kringle 5 region that do not significantly alter the biological activity (ie, anti-angiogenic activity) of the kringle 5 fragment. Modifications made to the kringle 5 fragment that increase anti-angiogenic activity are within the scope of the present invention.

Preferred analogs include one or more conservative amino acid substitutions or one or more non-conservative amino acid substitutions, deletions or insertions with the wild-type sequence that do not abolish the biological activity of the molecule. Kringle 5 fragments and the angiostatin molecule with the differentiating sequences are included. Conservative substitutions typically involve the replacement of one amino acid by another of like characteristics, for example, substitution within each of the following groups: valine, glycine; glycine, alanine; valine, isoleucine. , Leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; phenylalanine, tyrosine. Other conservative amino acid substitutions can be selected from the following table.

[Table 1] Conservative amino acid substitution

Other analogs encompassed by the present invention are those with modifications that increase the stability of the protein or peptide. For example, such analogs may contain one or more non-peptide bonds (instead of peptide bonds) in the protein or peptide sequence. Also included are analogs containing residues other than naturally occurring L-amino acids, such as D-amino acids or non-natural or synthetic amino acids (eg, β or γ-amino acids).

The kringle 5 region and angiostatin useful in the present invention can be synthesized by synthetic means.
That is, it can be obtained by chemically synthesizing the polypeptide from the constituent amino acids by a method known to those skilled in the art. For example, Houghton et al., Proc. Natl. Ac
ad. Sci. 82, 5131-5135 (1985). The polypeptide is obtained by production in a prokaryotic or eukaryotic host cell expressing a DNA sequence encoding all or part of the desired fragment, or by in vitro translation of mRNA encoded by the DNA sequence encoding the desired peptide. Can be. Techniques for producing kringle 5 fragments and angiostatin by these means are known in the art and are described herein.

The kringle 5 region and angiostatin thus produced can then be isolated and purified to some extent using various protein purification techniques. For example, chromatographic methods such as ion exchange chromatography, gel filtration chromatography, and immunoaffinity chromatography can be used.

[0023] Polypeptides of the Invention (ie Kringle 5 Fragments and Angiostatin)
When is produced by recombinant techniques, DNA sequences useful for the production of the polypeptide of the present invention can be obtained using various methods well known to those skilled in the art. An expression vector can be partially or completely chemically synthesized and / or can be partially or completely produced by genetic engineering techniques. Each fragment can be joined sequentially (via appropriate terminal restriction sites or complementary terminal sequences) to form the correct primary sequence of nucleotides.

The expression vehicle of the invention for producing an anti-angiogenic polypeptide of the invention includes a plasmid or other vector. Generally, such vectors contain control sequences that allow expression in various types of hosts. Suitable expression vectors containing the desired coding and control sequences are prepared using standard recombinant DNA techniques known in the art.
Technology and many such standard recombinant DNA techniques are described in Sambrook et al.
989) Molecular Cloning: A Laboratory Manual (2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

An expression vector contemplated by the present invention can direct the replication of the vector in bacterial, yeast, insect and / or mammalian cells. One type of vector is yeast D, which provides an autonomous origin of replication, such as a yeast 2μ sequence or an ARS1 sequence that results in an extrachromosomal plasmid
Use NA sequence. Another type of vector relies on the integration of the desired gene sequence into the host cell chromosome. The vector may include a bacterial origin of replication. Suitable bacterial origins of replication include, for example, Col El, pSC101 and M13 origins of replication.

[0026] Useful expression vectors of the present invention typically include a promoter located 5 '(ie, upstream) to the DNA sequence to be expressed and a promoter located 3' (ie, downstream) to the sequence to be expressed. And a transcription termination sequence. Suitable promoters include, for example, the yeast ADH1 promoter. The promoter sequence may be inducible to allow for regulation of expression (eg, by the presence or absence of nutrients or other inducers in the growth medium). Examples include the yeast GAL1 , CUP1 and MET25 promoters. Suitable termination sequences include, for example, yeast CYC1 termination and polyadenylation sequences.

The expression vector may contain other regulatory sequences to optimize expression of the desired product. Examples of such a sequence include a secretory leader sequence that prepares for secretion of an expression product or indicates membrane localization, a restriction enzyme recognition sequence that provides a cleavage site by a restriction endonuclease, and the like. All of these materials are known in the art and most are commercially available.

Suitable expression vectors are used for phenotypic detection of transformed yeast or bacterial cells.
And / or may include a marking sequence to allow selection. Such a mer
Kerr provides auxotrophic hosts with prototrophy (eg, amino acid biosynthesis genes)
Biocide resistance or biocide hypersensitivity (eg, antibiotic resistance) or phenotypically
Or provide a detectable signal (eg, fluorescence). Selectable marker gene
Can be directly linked to the DNA gene sequence to be expressed, or
It can be introduced into cells. Examples of yeast selectable markers include:Basidium pullu lans AUR1-C gene,S. cerevisiae URA3OrLEU2There are genes. Bacterial
An example of a selectable marker is an ampicillin resistance gene. Preferred vectors are Col E1 and 2μ origin of replication, yeastURA3And bacteria amp^RGenes and yeastGAL1Step
PYESII (Invitrogen) containing the promoter sequence.

As another alternative, the constructs can be introduced into cells by transformation, with genes that allow for selection when the constructs are integrated into the host genome or maintained as episomes. Usually, the construct will be part of a replication system recognized by a vector or host cell having homologous sequences for integration.

[0030] Compositions of the invention comprising the kringle 5 fragment of plasminogen (or a biologically active fragment or analog thereof) and angiostatin are useful for treating angiogenesis-related disorders. The present invention includes a method of treating an angiogenesis-related disorder with an effective amount of a composition comprising a Kringle 5 fragment and angiostatin. As mentioned above, kringle 5
A single composition comprising the fragments and angiostatin can be used, or separate compositions (one containing the kringle 5 fragment and the other containing angiostatin) can be administered simultaneously or sequentially.

[0031] Angiogenesis mediated diseases include, for example, solid tumors, hematological tumors such as leukemia; tumor metastases; benign tumors such as hemangiomas, acoustic acuromas, neurofibromas, trachoma and suppurative granuloma; Rheumatoid arthritis; Psoriasis; Intraocular neovascular diseases such as diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retro lens fibroplasia, Rubeosis; Ausler-Weber (Osl
er-Webber) syndrome; myocardial neovascularization; plaque neovascularization; telangiectasia; hemophilia arthropathy; angiofibroma; wound granulation. The compositions of the present invention are useful for treating diseases in which endothelial cells are excessively or abnormally stimulated.
These disorders include, but are not limited to, for example, intestinal adhesions, atherosclerosis, scleroderma, and hyperplastic scars (ie, keloids). The composition can also be used as a birth control drug by preventing angiogenesis required for implantation of embryos.

The compositions and methods of the present invention can be used in conjunction with other compositions and techniques for treating angiogenesis-related disorders. For example, a tumor may be treated conventionally with surgery, radiation or chemotherapy and then administered to the patient with a composition comprising kringle 5 regions and angiostatin, thereby extending the dormancy of the micrometastases and leaving the remaining primary tumor Can be stabilized.

The present invention relates to a pharmaceutically acceptable carrier comprising the kringle 5 region of plasminogen (or a biologically active fragment or analogue thereof) and angiostatin, optionally together with at least one additional active compound, Drugs containing adjuvants or vehicles (
That is, therapeutic) compositions are also provided. The "additional active compound" includes, for example, one or more drugs selected from the group consisting of immunosuppressants, anticancer drugs, antiviral drugs, anti-inflammatory drugs, antifungal drugs, antibiotics or anti-vascular hyperproliferative compounds. Included, but not limited to.

The term “pharmaceutically acceptable carrier, adjuvant or vehicle” refers to a carrier, adjuvant that can be incorporated into a composition of the present invention and administered to a subject without destroying the pharmacological activity of the composition of the present invention. Refers to an agent or medium. Pharmaceutically acceptable carriers, adjuvants and vehicles that can 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-alpha-tocopherol polyethylene glycol 1000 succinate, surfactants or other similar polymer delivery matrices such as Tween used in pharmaceutical dosage forms, human serum albumin, etc. Serum proteins, buffer substances such as phosphate, glycine, 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 salt, colloidal silica, magnesium trisilicate, Polyvinylpyrrolidone, cellulosic substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylate, wax, polyethylene-polyoxypropylene block polymer, polyethylene glycol and wool fat. Chemically modified derivatives such as cyclodextrins such as α-, β- and γ-cyclodextrin and hydroxyalkyl cyclodextrins including 2- and 3-hydroxypropyl-β-cyclodextrin or other solubilized derivatives are also provided by the compositions of the present invention. Can be used to improve the delivery of

The compositions of the present invention may contain other therapeutic agents, as described below, and also include, for example, conventional solid or liquid vehicles or diluents and pharmaceutical additives of a type suitable for the desired mode of administration. (Eg, excipients, binders, preservatives, stabilizers, flavors, and the like) and can be formulated according to techniques well known in the pharmaceutical arts.

The compositions of the present invention may be administered by any suitable means, for example, orally, such as in tablets, capsules, granules or powders, or by sublingual administration, or by buccal administration, or subcutaneously, intravenously Parenteral, such as by intramuscular or intrasternal injection or infusion (eg, as a sterile injectable aqueous or non-aqueous solution or suspension), or nasally, such as by inhalation spray, or in the form of creams or ointments. Topically, or rectally in the form of a suppository or the like, as a unit dosage formulation containing a non-toxic pharmaceutically acceptable vehicle or diluent. The composition can be administered, for example, in a form suitable for immediate release or extended release. Immediate or prolonged release can be achieved by the use of a suitable pharmaceutical composition comprising the kringle 5 fragment and angiostatin, or, especially for prolonged release, by the use of a device such as a subcutaneous implant or an osmotic pump. . The composition can also be administered using liposomes.

Representative compositions for oral administration include, for example, microcrystalline cellulose to increase bulk, alginic acid or sodium alginate as suspending agent, methylcellulose as thickening agent and known in the art. Suspensions which may contain such sweetening or flavoring agents and the like, for example as microcrystalline cellulose, dicalcium phosphate, starch, magnesium stearate and / or lactose and / or as known in the art. Immediate release tablets which may contain other excipients, binders, extenders, disintegrants, diluents and lubricants. The compounds can also be delivered through the oral cavity by sublingual and / or buccal administration. Molded tablets, compressed tablets or lyophilized tablets are exemplary dosage forms that can be used. Representative compositions include those formulated with a fast-dissolving diluent, such as mannitol, lactose, sucrose, and / or cyclodextrin. Such formulations may also contain high molecular weight excipients, such as cellulose (Avicel) or polyethylene glycol (PEG). Such formulations may also assist in mucosal adhesion, for example, hydroxypropylcellulose (HPC), hydroxypropylmethylcellulose (HPMC), sodium carboxymethylcellulose (SCMC), maleic anhydride copolymers (eg, Gant
excipients such as rez) and agents that control release, such as, for example, polyacrylic copolymers (eg, Carbopol 934). Lubricants, glidants, flavors, colorings and stabilizers may be added for ease of manufacture and use.

Representative compositions for nasal aerosol or inhalation administration include, for example, benzyl alcohol or other suitable preservatives, absorption enhancers to enhance bioavailability and / or are known in the art. Saline solutions which may contain other stabilizers or dispersants as described above.

Representative compositions for parenteral administration include, for example, suitable non-toxic parenterally acceptable diluents or solvents such as mannitol, 1,3-butanediol, water,
Ringer's solution, isotonic saline solution or other suitable dispersing or wetting and suspending agent (
Injectable solutions or suspensions, which may contain synthetic mono- or diglycerides) as well as fatty acids such as oleic acid. The term "parenteral" as used herein refers to subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrabursal, intrasternal, intrathecal, intralesional and intracranial injection or infusion Technology.

Representative compositions for rectal administration include suitable nonirritating agents which are solid at room temperature but liquefy and / or dissolve in the rectal cavity to release the active compounds (ie, kringle 5 fragments and angiostatin). Excipients include suppositories which may contain, for example, cocoa butter, synthetic glyceride esters or polyethylene glycol.

Exemplary compositions for topical application 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 may be about 0.
It contains a typical dosage for adult humans of 1-500 mg of active compound, which can be administered in a single dose or in divided doses, for example, 1 to 5 times a day. The specific dose level and frequency of dosing for a particular subject can be varied and include the activity of the compound used, the metabolic stability and length of action of the compound, the species, age,
It will be appreciated that it will depend on a variety of factors including body weight, general health, sex and diet, mode and time of administration, rate of excretion and clearance, concomitant medications and the severity of the condition. Preferred subjects for treatment include animals prone to angiogenesis-related disorders, most preferably mammalian species, for example humans and domestic animals such as dogs, cats and the like.

The compositions of the present invention can be used alone or in any other suitable therapeutic agent useful in the treatment of angiogenesis-related diseases, such as angiogenesis inhibitors other than the angiogenesis inhibitors of the invention, anti-inflammatory agents, and antiproliferative agents It can be used in combination with drugs, chemotherapeutics, immunosuppressants and antimicrobial agents.

When used in combination with the compositions of the present invention, the other therapeutic agent can be used in the amounts given in the Handbook of Pharmaceuticals for Pharmaceuticals (PDR) or in amounts determined by those skilled in the art by other methods.

The following examples further illustrate the present invention. These examples are not intended to limit the scope of the invention, but rather to provide a better understanding of the invention.

Examples Example 1: Gene construction, expression and purification of recombinant human angiostatin Human plasminogen cDNA template (American Type Culture Collection)
Human Angiostatin cDNA was amplified by standard polymerase chain reaction (PCR) from Rockville, Md. Using the following two primers: 5'-GCGGATCCATGAAAGTGTATCTCTCAGAGTGCAAG (forward primer for residues 98-458) And 5'-GCGGATCCTCACTATTCTGTTCCTGAGCATTTTTTCAG (reverse primer for residues 98-458).

[0047] The amplified cDNA fragment was transferred to a pMelBacA vector (InVitrogen, San Francisco, CA).
Diego) at the BamHI site. Next, this angiostatin cDNA plasmid
BaculoGold virus into Sf9 cells^TM DNA (PharMingen, Sande, CA)
(Jiego). Briefly, 1 × 10⁶Sf9 fine
The cells are inoculated into a T25 tissue culture flask and 2 μg of the transfer vector DNA, 0.5 μg of BaculoGo
ld^TM DNA and 6 μl cellfectin (Gibco BRL, Gaithers, MD)
Incubate at 27 ° C with 1 ml of transfection solution containing
I did it. Remove transfection solution 4 hours after transfection
Replace with 3 ml Sf900II medium (Gibco BRL, Gaithersburg, MD)
Was. After 4 days of culture, viral supernatants are collected and individual clones are
Identified. Protein expression levels determined by SDS-PAGE / Coomassie blue staining were
Higher clones were amplified in Sf9 cells for protein production. High Five insect cells (
InVitrogen, San Diego, CA (1.5 x 10)⁶Cells / ml), about 1 × 10 ⁷ Infected with virus particles / ml recombinant virus. After 48 hours, 30 minutes at 5,000 x g
The culture supernatant was collected by centrifugation during. Next, the supernatant is added to lysine-sepharose.
On a column and as previously described (Wu, Z. et al. (1997)Biochem.Biophys.Res.Commun. Two
36: 651-654), dissolve angiostatin protein using ε-aminocaproic acid
Issued.

Example 2 Production of Recombinant Kringle 5 of Human Plasminogen As described previously (Menhart, N. et al. (1993) Biochemistry 32: 8799-8806), HPg cDN
K4-5 cDNA was amplified from the A template by PCR. Convert the amplified cDNA to pPIC9K
The vector (InVitrogen, San Diego, CA) was inserted between the AvrII and NotI sites. Next, the introduced plasmid pPIC9K [K4-K5 of HPg] was linearized with the restriction endonuclease SacI and electroporated into Pichia pastoris.
Of KM71 strain. Isolation of high yield clones followed by high biomass fermentation was performed as previously described (Nilsen, SL et al. (1997) Biotech . And Applied Biochem . 25: 63-74). The K4-K5 protein of human plasminogen was purified from the fermentation medium by lysine affinity chromatography as described previously (Chang, Y. et al. (1997) Biochemistry 36 (25): 7652-7663). The purified product is dialyzed extensively against water, lyophilized and 1: 250 (w / w) protein: elastase in 0.1 M phosphate / 15 mM ε-aminocaproic acid (pH 7.8) at room temperature. Elastase digestion by ratio. Next, the digest was applied to a lysine-Sepharose chromatography column again, and the kringle 5 fragment in the flow-through solution was collected. The kringle 5 protein was dialyzed extensively against water and lyophilized.

Example 3 Anti-Endothelial Cell Proliferation of Kringle 5 in Combination with Angiostatin /
Migration activity Two major endothelial cell types, human umbilical vein endothelial cells (HUVEC) and bovine capillary endothelium, were selected for the Boyden chamber-based migration assay. Briefly, HUVEC cells were purchased commercially (Clontics, San Diego, CA). Bovine capillary endothelial cells were collected from bovine adrenal gland as previously described (Folkman, J. et al.
(1979) Proc. Natl. Acad. Sci. USA 76: 5217-5221). BCE cells were maintained in DMEM in the presence of 10% calf serum, 1% antibiotic and 3 ng / ml bFGF (PeproTech, Rock Hill, NJ).

A Boyden chamber based assay was performed to assess endothelial cell migration. First, an 8 μm pore size polycarbonate membrane (Neuro Probe Inc., Cabin John, MD) was coated with 100 μg / ml type I collagen according to the manufacturer's instructions (Becton Dickinson, Bedford, Mass.). BCE cells at passages 10-14 or HUVEC cells at passages 1-3 using a 0.05% trypsin solution are collected, washed, and washed with DMEM containing 10% calf serum and 10 mg / ml bFGF. To 75,00
Resuspended at a density of 0 cells / ml and incubated at 37 ° C for 30 minutes. Various concentrations of Kringle 5, angiostatin, or a combination of Kringle 5 and angiostatin samples were added to the lower chamber during cell incubation. The collagen-coated membrane filter was placed on the lower chamber before the upper chamber was fitted. After incubation for 30 minutes, endothelial cells were added to the upper chamber and incubated at 37 ° C for 4 hours. Next, the chemotaxis chamber was disassembled, and the filter membrane was taken out. Non-migrating cells were scraped off the top of the membrane three times with a cotton swab. After rinsing with PBS, membranes were fixed in 10% buffered formalin for 45 minutes, and then stained overnight with Gill's hematoxylin # 2 (JBBaker, Phillipsburg, NJ). The membrane is then rinsed with PBS and Cytoseal (Stephens Sc
ientific, Riverdale, NJ). Each sample was tested in quadruplicate and the number of migrating cells was determined by counting the representative field of each well at 100 × magnification.

Table 2 below shows Kringle 5 (“K5”), Angiostatin (“AST”), K5 + A
The ST combinations and the inhibitory activity of plasminogen on Kringle 1-5 ("K1-5") are summarized.

[Table 2]

For HUVEC migration, the IC ₅₀ (protein concentration at 50% inhibition) for angiostatin and kringle 5 are approximately 0.65 μM and 0.10 μM, respectively (FIG. 1A and Table 2).
. Combination of these two drugs significantly increases anti-migratory activity with an IC ₅₀ of about 9.3 nM
Became. This 1 log increase in anti-endothelial cell activity implies that kringle 5 and angiostatin can inhibit angiogenesis by different but cooperative pathways. Respect BCE cell migration, IC ₅₀ of angiostatin and kringle were respectively about 0.49μM and 0.09μM (FIG. 1B and Table 2). Combining these two proteins increased anti-migratory activity by almost 1 log (IC ₅₀ = 8.8 nM).
This synergy suggests that kringle 5 and angiostatin may cooperate to block angiogenesis. As a reference example, recombinant kringle 1
The -5 protein was produced in a baculovirus expression system. This is HUVEC and BCE
It was found that cell migration was strongly inhibited in a dose-dependent manner. The anti-migratory activity of kringle 1-5 is close to that of the combination of angiostatin and kringle 5 for HUVEC (IC _{50 of} kringle 1-5 = 8 nM), but about 4 times less potent for BCE migration. Yes (IC _{50 for} BCE = 33.7 nM). This indicates that the combination of the kringle 5 fragment of plasminogen and angiostatin shows superior activity even more than the kringle 1-5 fragment of plasminogen. Thus, the use of human plasminogen kringle 5 in combination with angiostatin can enhance its anti-angiogenic function.

While the 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 modifications and changes may be made within the scope of the appended claims.

[Brief description of the drawings]

FIG. 1 is a graph showing the additional anti-endothelial cell migration activity of human plasminogen kringle 5 in combination with angiostatin. FIG. 1A shows the additional inhibition of HUVEC cell migration by human plasminogen kringle 5 in combination with angiostatin. FIG. 1B shows the additional inhibition of BCE cell migration by kringle 5 of human plasminogen in combination with angiostatin. (Abbreviation: K5 = Kringle 5, AST = Angiostatin, bFGF = Basic fibroblast growth factor)

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 17/00 A61P 27/02 17/06 29/00 101 27/02 35/00 29/00 101 43 / 00 121 35/00 A61K 37/02 43/00 121 37/47 // C12N 15/09 ZNA C12N 15/00 ZNAA (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI , FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN , TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM) AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, EE, ES, FI, GB, GD , GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, UZ, VN , YU, ZA, ZW (72) Inventor Mohammand E. Condri, 1818 Yearing Drive, New Hope, PA 18938 (72) Inventor Pamela A Trail USA 19067 Yardley, PA, USA No. 1419 F term (reference) 4B024 AA01 BA21 CA04 DA02 EA02 EA04 FA02 GA11 HA01 4C084 AA02 AA03 BA01 BA02 BA05 BA08 BA09 BA10 BA23 BA24 BA25 BA41 BA42 CA01 CA04 CA05 CA17 CA49 CA53 CA56 CA59 DC50 MA02 MA13 MA17 MA35 MA31 MA31 MA43 MA52 MA57 MA59 MA60 MA63 MA66 MA67 NA05 NA14 ZA332 ZA392 ZA442 ZA892 ZB152 ZB262 ZC802

Claims (14)

[Claims] 1. A therapeutic composition for inhibiting the proliferation and / or migration of endothelial cells, comprising the kringle 5 region of plasminogen or a biologically active portion thereof and angiostatin and a pharmaceutically acceptable carrier. 2. The therapeutic composition of claim 1, wherein said kringle 5 region of plasminogen or a biologically active portion thereof and said angiostatin are from the same or different species. 3. The therapeutic composition of claim 1, wherein said kringle 5 region of plasminogen or a biologically active portion thereof and said angiostatin are derived from murine plasminogen, human plasminogen, rhesus monkey plasminogen, porcine plasminogen, canine plasminogen or bovine plasminogen. object. 4. The method according to claim 1, wherein the growth and / or migration of the composition comprising the kringle 5 region of plasminogen or a biologically active part thereof and angiostatin is administered to the endothelial cell. How to inhibit. 5. The method of claim 4, wherein said kringle 5 region of plasminogen or a biologically active portion thereof and said angiostatin are from the same or different species. 6. The method of claim 4, wherein said kringle 5 region of plasminogen or a biologically active portion thereof and said angiostatin are derived from murine plasminogen, human plasminogen, rhesus monkey plasminogen, porcine plasminogen, canine plasminogen or bovine plasminogen. 7. A method of treating a mammal having an angiogenesis-related disorder, comprising administering to the mammal a therapeutically effective amount of a composition comprising kringle 5 region of plasminogen or a biologically active portion thereof and angiostatin. A method consisting of: 8. The method of claim 7, wherein said kringle 5 region of plasminogen or a biologically active portion thereof and said angiostatin are from the same or different species. 9. The method of claim 7, wherein said kringle 5 region of plasminogen or a biologically active portion thereof and said angiostatin are derived from murine plasminogen, human plasminogen, rhesus monkey plasminogen, porcine plasminogen, canine plasminogen or bovine plasminogen. 10. A method of treating a mammal having an angiogenesis-related disorder, comprising combining a first composition comprising the kringle 5 region of plasminogen or a biologically active portion thereof with a second composition comprising angiostatin. Administering to the mammal an effective amount of the above. 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 of plasminogen or a biologically active portion thereof and said angiostatin are from the same or different species. 14. The method of claim 10, wherein said kringle 5 region of plasminogen or a biologically active portion thereof and said angiostatin are derived from murine plasminogen, human plasminogen, rhesus monkey plasminogen, porcine plasminogen, canine plasminogen or bovine plasminogen.