JP2002539174A - Compositions and methods using LDL-like receptor ligands for the treatment of cancer and angiogenesis-related diseases - Google Patents

Abstract

  (57) [Summary] Provided are compositions and methods that are effective in inhibiting abnormal and unwanted cell proliferation, particularly endothelial cell proliferation, and angiogenesis associated with neovascularization and tumor growth. The compositions include natural or synthetic proteins, peptides or protein fragments, which can bind to low density lipoprotein (or low density lipoprotein-like) receptors. The composition can be administered using a pharmaceutically acceptable carrier. The method includes administering a composition described herein to a human or animal in an amount sufficient to inhibit cell proliferation, particularly endothelial cell proliferation. This method is useful for treating diseases and advanced processes, such as cancer, resulting from unwanted and unregulated cell growth, particularly by inhibiting angiogenesis. Administering the present composition to a human or animal having a tumor with metastasis due to pro-angiogenesis is useful in preventing the growth and spread of such a tumor.

Description

DETAILED DESCRIPTION OF THE INVENTION

(Related Application) The priority of the present application is US application Ser. No. 09 / 270,982 (filing date: March 1999)
17).

FIELD OF THE INVENTION [0002] The present invention relates to compositions and methods for inhibiting cell proliferation. More particularly, the present invention relates to the use of proteins or peptides, and / or fragments thereof, which bind to lipoprotein receptors, especially low density lipoprotein receptors, to produce angiogenesis and angiogenesis-related diseases. Suppress.

BACKGROUND OF THE INVENTION Cell proliferation is a process that is constantly occurring in all living organisms, and involves many factors and signals that balance the delicate balance of normal cells. The cycle has been maintained. The general process of cell division consists of two sequential processes, the nuclear division (mitosis) and the cytoplasm division (cytokinesis). As organisms constantly grow and rotate cells, cell proliferation is a core process that is essential for the functioning of almost all biological processes. Governing whether mammalian cells grow or divide is governed by a variety of feedback-regulatory mechanisms, including the space available for cells to grow, and specific stimuli and deterrents in a given environment. Secretion of factors.

[0004] When disruption or some inhibition of normal cell growth occurs, various biological functions are affected as a result. Inhibition of growth can be due to a myriad of factors, such as the deficiency or excess of various signaling chemicals, or changes in the environment. Characteristic disorders of abnormal cell proliferation include cancer, abnormal development of the embryo, inappropriate formation of the corpus luteum, difficulty in wound healing, and dysfunction in infection and immune response.

[0005] Cancer is characterized by abnormal cell growth. Cancer cells have a number of characteristics that are dangerous to the host, often allowing them to invade other tissues and cause ingrowth in capillaries, resulting in proliferating cancer cells. A sufficient blood supply is provided. One of the defining characteristics of cancer cells is that they respond abnormally to the regulatory mechanisms that control normal cell division, continue dividing almost dysregulated, and eventually kill the host. is there.

[0006] Angiogenesis and angiogenesis-related diseases are closely linked to cell proliferation. As used herein, the term “angiogenesis” means the formation of new blood vessels into a tissue or organ. Under normal physiological conditions, angiogenesis occurs in humans or animals only in very specific and limited conditions. For example, angiogenesis is commonly found in wound healing, fetal and embryo development,
This is the case in the formation of the corpus luteum, endometrium and placenta. The term "endothelium" is defined herein as a thin layer of flat cells lining the serous cavities, lymph vessels and blood vessels. These cells are defined herein as "endothelial cells.""
The term "endothelial inhibitory activity" generally refers to the ability of a molecule to inhibit angiogenesis. As a result of suppressing endothelial cell proliferation, angiogenesis is also suppressed.

[0007] Angiogenesis is thought to proceed in a similar manner, with or without regulation. Endothelial cells and pericytes are enveloped by the basement membrane and form capillaries. Angiogenesis begins with erosion of the basement membrane by enzymes released from endothelial cells and leukocytes. The endothelial cells lining the lumen of the blood vessel then bulge through the basement membrane. Endothelial cells migrate through the eroded basement membrane as stimulated by angiogenic stimuli. The migrated cells separate from the original blood vessels to form "buds" where the endothelial cells mitose and proliferate. Endothelial cell sprouts connect with each other and form capillary loops, creating new blood vessels.

[0008] Sustained and unregulated angiogenesis is observed in the case of overlapping disease states, metastasis of tumors and abnormal growth of endothelial cells, resulting in the pathological damage observed in these conditions. The various pathological disease states in the presence of unregulated angiogenesis are angiogenetic-dependent, angiogenic-associated (ang).
iogenic-associate and angiogenesis-related (angiage)
nic-related). These diseases are the result of abnormal or unwanted cell proliferation, especially endothelial cell proliferation.

[0009] The hypothesis that tumor growth is vascular-dependent was established in 1971 by Judah Fol.
Kman (N. Engl. Jour. Med. 28
5: 1182-1186,1971). To put it simply, in this hypothesis,
It has been proposed that once a tumor "take" has occurred, new capillaries are concentrated on top of the tumor, always prior to expansion of the tumor cell population. This "acquisition" of tumors is now understood to refer to the prevascular phase in tumor growth, where the population of tumor cells occupies a volume of a few cubic millimeters and the number of cells is in the millions. And live in the host's microvessels. In order for the tumor volume to expand beyond this phase, the induction of new capillaries is required. For example, in mice, micrometastasis in the lung in the early pre-vascular phase is not found unless histological sections are examined under high power microscopy. Indirect evidence supporting that tumor growth is vascular dependent is disclosed in US Pat.
9,725, 5,629,327, 5,792,845, and 5
Nos., 733,876 and 5,854,205, all of which are incorporated herein by reference.

What is clear from the above is that cell proliferation, particularly endothelial cell proliferation, and most importantly, angiogenesis plays a major role in cancer metastasis.
If this abnormal or undesirable proliferative activity could be suppressed, suppressed or eliminated, the tumor would not grow, if at all. this
In disease states, damage from erosion of new microvascular pathways can be avoided by interfering with abnormal or unwanted cell proliferation and angiogenic processes. Therapies for controlling the cell growth process will be able to control or mitigate these diseases.

Therefore, what is needed here is abnormal or undesired cell proliferation,
In particular, a composition and a method capable of suppressing the growth of blood vessels into tumors. The composition must be capable of inhibiting the activity of endogenous growth factors in the pre-metastatic tumor and preventing the formation of capillaries inside the tumor, thereby preventing disease progression and tumor growth. The composition must also be able to regulate the formation of capillaries in the angiogenic process, for example in the case of wound healing and regeneration. Finally, compositions and methods for inhibiting cell growth are non-toxic,
Preferably, there are few side effects.

SUMMARY OF THE INVENTION [0012] Compositions and methods are provided that are effective in inhibiting abnormal and undesirable cell proliferation, particularly endothelial cell proliferation, and angiogenesis associated with neovascularization and tumor growth. The composition comprises a low density lipoprotein receptor (LDLR) bound to a natural or synthetic protein, peptide, or protein fragment or ligand thereof.
And optionally associated with a pharmaceutically acceptable carrier.

Representative ligands useful for the present invention include proteins or peptides and fragments thereof belonging to the following categories: That is, proteinases (ie, tissue plasminogen activator (tPA)), proteinase inhibitors (ie, serpin, TFPI), proteinase /
Inhibitor complexes (urokinase plasminogen activator / plasminogen activator inhibitor 1 (uPA / PAI), thrombin / antithrombin), lipoproteins (ie, apolipoprotein B, apolipoprotein J or clusterin), and Substrate proteins (ie, thrombospondin) and the like. Still further, specific proteins or peptides containing representative ligands useful for the present invention include: That is, α2-macroglobulin, beta-amyloid precursor protein (APP), proteinase nexin II (PN2), proteinase nexin I (PN1), pro-uPA, antithrombin III, lipoprotein lipase, lactoferrin, PAI-1 , Equine leukocyte elastase inhibitor, protein C inhibitor, C1 inhibitor, α2-antiplasmin, α1-proteinase inhibitor, and α1-antichymotrypsin, heparin cofactor II and the like.

Preferred LDLR ligands include proteins belonging to the serpin (serine protease inhibitor) protein family having Kunitz-type or non-Kunitz-type domains, such as tissue factor pathway coagulation inhibitor 1 (
TFPI-1), tissue factor pathway coagulation inhibitor 2 (TFPI-2), antithrombin III, amyloid protein precursor (APP), β-amyloid precursor protein, collagen VI, and bovine pancreatic trypsin inhibitor (BP
TI).

[0015] The protein, peptide or fragment thereof preferably comprises all or an active part of the protein described above. As used herein, "
The term "active moiety" refers to that portion of a protein that suppresses cancer, preferably by inhibiting abnormal or undesired growth of the cancer. The present invention also includes analogs, peptides, or protein fragments, or combinations thereof, of the above-described proteins that inhibit abnormal or undesirable cell growth. The protein, peptide, or protein fragment is LD
Most preferably, it is a protein, peptide or protein fragment of the ligand that binds to LR.

Without wishing to be bound by theory, by inhibiting endothelial cell proliferation, the methods and compositions described herein inhibit tumor growth by inhibiting tumor angiogenesis. It is thought to be useful in suppressing metastasis.

The methods provided herein for treating diseases and processes arising from undesired and uncontrolled cell proliferation, such as cancer, include methods for inhibiting cell proliferation, particularly endothelial cell proliferation. Administering a composition described herein to a human or animal at a sufficient dose is included. This method is particularly useful for treating or suppressing tumor growth, particularly by inhibiting angiogenesis. Administering the present compositions to humans or animals having tumors with metastases due to pro-angiogenesis is useful to prevent the growth or spread of such tumors.

Accordingly, it is an object of the present invention to provide methods and compositions for treating diseases or processes that are caused by abnormal or undesirable cell proliferation.

It is another object of the present invention to provide methods and compositions for treating or inhibiting cancer growth.

Yet another object of the present invention is to provide methods and compositions for the treatment of cancer with minimal side effects.

[0021] Another object of the present invention is to provide methods and compositions for treating diseases and progressive processes resulting from angiogenesis.

Yet another object of the present invention is to provide methods and compositions that inhibit the growth of cells, especially endothelial cells, including using proteins, peptides, active fragments and analogs thereof. .

Another object of the present invention is a method and composition for treating diseases and progressive processes caused by angiogenesis by administering an anti-angiogenic compound comprising a low density lipoprotein receptor ligand. It is to provide.

Another object of the present invention is a method and composition for treating diseases and progressive processes caused by angiogenesis by administering an anti-angiogenic compound comprising a low density lipoprotein receptor ligand. Wherein the ligands include proteinases, proteinase inhibitors, lipoproteins and substrate proteins.

[0025] Another object of the present invention is a method and composition for treating diseases and progressive processes caused by angiogenesis, comprising administering an anti-angiogenic compound comprising a low density lipoprotein receptor ligand. Wherein the ligands include antithrombin III and amyloid protein precursor.

It is a further object of the present invention to reduce cancer and inhibit tumor growth in humans or animals with cancer by administering an anti-angiogenic compound comprising a low density lipoprotein receptor ligand. Wherein the ligand comprises a proteinase, a proteinase inhibitor, a lipoprotein and a substrate protein.

Yet another object of the present invention is to provide a compound for anti-angiogenesis comprising a low density lipoprotein receptor ligand, wherein the ligand is a proteinase, a proteinase inhibitor, a lipoprotein and a substrate protein. As a combination with a pharmaceutically acceptable carrier.

Yet another object of the present invention is to provide a compound for anti-angiogenesis comprising a low density lipoprotein receptor ligand, wherein the ligand comprises a proteinase, a proteinase inhibitor, a lipoprotein and a substrate. Protein
It is included in combination with a pharmaceutically acceptable carrier, which can be administered by intramuscular, intravenous, transdermal, oral or subcutaneous injection.

Yet another object of the present invention is to provide compositions and methods for treating diseases and progressive processes resulting from angiogenesis, including hemangiomas, solid tumors , Blood-borne tumors
or), leukemia, metastasis, telangiectasia, psoriasis, edema sclerosis, suppurative granuloma, myocardial neovascularization, Crohn's disease, plaque neovascular proliferation, arteriovenous insufficiency, corneal disease,
Rubeosis, neovascular glaucoma, diabetic retinopathy, post lens fibroplasia, arthritis, diabetic neovascular growth, macular degeneration, wound healing, peptic ulcer, Helicobacter-related illness, fracture, keloid, angiogenesis, hematopoiesis, ovulation Menstruation, placenta formation, and cat scratching fever, but are not limited thereto.

The above and other objects, features and advantages of the present invention will become apparent with reference to the disclosure of embodiments described below and the appended claims.

DETAILED DESCRIPTION The following is a description of the best currently conceivable embodiment for practicing the present invention. The description has been made in order to explain the general principles of the invention and should not be taken as limiting. The entire text of the references referred to herein is hereby incorporated herein by reference, including but not limited to US patent application Ser. No. 09 / 270,982 (199).
U.S. patent application Ser. No. 09 / 130,273 (filed Mar. 17, 1998).
And US Patent No. 5,981,471 (issued November 9, 1999).

[0032] Compositions and methods are provided for treating diseases and processes resulting from or associated with abnormal or undesirable cell proliferation. The composition includes a low density lipoprotein (or low density lipoprotein-like) receptor (LD
LR), including isolated or natural proteins or peptides, or fragments of proteins, including all or an active portion of LR). These compositions may optionally include a pharmaceutically acceptable carrier, for delivery to humans or animals.

As used herein, the term “active moiety” refers to the moiety required to bind LDLR,
Defined as the antiproliferative portion of the protein. The active moiety has the ability to inhibit cell growth, such as endothelial cell growth, by in vivo or in vitro assays or well known methods. Preferred as the LDLR ligand composition of the present invention include proteins belonging to the following categories. That is, proteinases (ie, tissue plasminogen activator (tPA)), proteinase inhibitors (ie, serpin, TFPI), proteinase / inhibitor complexes (urokinase plasminogen activator / plasminogen activator inhibitor 1 (uPA / PAI), thrombin / antithrombin), lipoproteins (ie, apolipoprotein B), and substrate proteins (ie, thrombospondin). The most preferred proteins, peptides or protein fragments are proteins, peptides or protein fragments containing representative ligands, including but not limited to the following: That is, α2-macroglobulin, β-amyloid precursor protein (APP), proteinase nexin II (PN2), proteinase nexin I (PN1), pro-uPA, lipoprotein lipase, lactoferrin, PAI-1, horse leukocyte elastase Inhibitors, protein C inhibitors, C1-inhibitors, α2-antiplasmin, α1-proteinase inhibitors, α1-antichymotrypsin, heparin cofactor II, and antithrombin III.

As mentioned above, the compositions of the present invention may optionally include a pharmaceutical carrier.
As used herein, the term "carrier" includes delivery mechanisms well known to those skilled in the art, for example, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), and other adjuvants. However, the present invention is not limited to these. The low-density lipoprotein receptor ligand composition of the present invention further comprises an adjuvant, which is known and used in a pharmaceutical composition of the prior art.
It is to be understood that preservatives, diluents, emulsifiers, stabilizers and other ingredients may be included. Any of the currently known adjuvant systems can be used in the compositions of the present invention. Illustrative examples of such adjuvants are Freund's incomplete adjuvant, Freund's complete adjuvant, polydisperse β- (1,4) -linked acetylated mannan (“Acemannan”), TiterMAX®. (Polyoxyethylene-polyoxypropylene copolymer adjuvant, Cytrix Corporation (CytRx Corpo)
ration, Norcross, Georgia), Chiron Corp.
Aguila Biopharm, a modified lipid adjuvant from the company, Emeryville, California).
adjuvant, inactivated Bordetella pertussis (Aceuticals, Worcester, Mass.).
ertussis), lipopolysaccharide (LPS) from Gram-negative bacteria, large polymerizable anions such as dextran sulfate, alum, inorganic gels such as aluminum hydroxide or aluminum phosphate, egg albumin, flagellin, thyroglobulin,
Examples include, but are not limited to, serum albumin of all species, gamma globulin of all species, and polymers of D- and / or L-amino acids.

Alternatively, for continuous administration, the gene for a protein, peptide or protein fragment, having all or an active portion of the protein, can be incorporated into the vector using gene therapy techniques. The vector may be administered in an excipient that has specificity for a target site, such as a tumor.

According to the method of the present invention, a composition described herein comprising a protein, peptide or protein fragment with all or an active portion of an LDLR ligand is optionally combined with a pharmaceutically acceptable carrier. For inhibiting undesired cell proliferation, particularly endothelial cell proliferation, or angiogenesis or angiogenesis-related diseases such as cancer, in humans or animals in which undesired cell proliferation is observed. Administer enough. Definitions "a", "an" and "the" mean one or more as used herein and include plural unless the context makes sense.

The term “peptide” is a chain of amino acids (typically L-amino acids), the α-carbon of which has a carboxyl group at the α-carbon of one amino acid and an amino group at the α-carbon of another amino acid. Are linked by a peptide bond formed by a condensation reaction between There is a free amino group at the terminal amino acid at one end of the chain (ie, the amino terminus) and a free carboxyl group at the terminal amino acid at the other end of the chain (ie, the carboxy terminus). . Thus, the term “amino terminal” (abbreviated N-terminal) refers to the free α-amino group of an amino acid at the amino terminus of a peptide, or to the α-amino acid of an amino acid anywhere within the peptide. Refers to a group (an imino group if participating in a peptide bond). Similarly, the term "carboxy terminal" (abbreviated C-terminal) refers to the free carboxyl group on an amino acid at the carboxy terminal of a peptide, or the carboxyl group of an amino acid anywhere within the peptide. pointing.

Typically, the amino acids making up the peptide are numbered sequentially, starting with the amino terminal and increasing in the direction of the carboxy terminal of the peptide.
Thus, when one amino acid is said to "follow" another, that amino acid will be closer to the carboxy terminus of the peptide than the previous amino acid.

As used herein, the term “residue” refers to an amino acid (D or L) incorporated into a peptide by an amide bond. Thus, the amino acid may be a natural amino acid, or, unless otherwise limited, a known natural amino acid homolog (ie, amino acid mimetics) that exhibits a similar function to the natural amino acid. May also be included. Further, amide bond mimetics include modification of the peptide backbone, as is well known to those skilled in the art.

As used herein, “consisting essence
The phrase "entry of)" excludes any element that substantially alters the essential properties of the peptide to which the term applies. Thus, a description of a peptide "consisting essentially of" excludes any amino acid substitutions, additions or deletions that substantially alter the biological activity of the peptide.

Further, as will be appreciated by the skilled artisan, as noted above, one or a small number of amino acids (typically 5% or less, more typically Conservatively modified changes if individual substitutions, deletions or additions, such as alterations, additions or deletions (less than 1%), result in the replacement of the amino acids with chemically similar amino acids. is there. The table for conservative substitutions shows functionally similar amino acids and is well known to those skilled in the art. Each of the following six groups are amino acids that can be conservatively substituted for one another. 1) alanine (A), serine (S), threonine (T); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V);
And 6) phenylalanine (F), tyrosine (Y), tryptophan (W).

The phrase “isolated” or “biologically pure” refers to a substance that is substantially or essentially free of compounds that, in nature, usually accompany it. Thus, the peptides described herein are free of substances normally associated with their natural environment. Typically, the isolated antiproliferative peptides described herein are at least about 80% pure, usually at least about 90%, preferably at least about 90%, as measured by band intensity on a silver stained gel. Has at least about 95% purity.

As is well known to those skilled in the art, the purity or homogeneity of a protein can be measured in a number of ways, for example, subjecting a protein sample to electrophoresis on a polyacrylamide gel, followed by staining. To visualize. Depending on the purpose, high resolution is required, and HPLC or a method similar thereto is used.

If the length of the antiproliferative peptide is relatively short (ie, less than about 50 amino acid units), it is often synthesized using standard chemical peptide synthesis techniques.

In solid phase synthesis, the C-terminal amino acid of the sequence is attached to an insoluble support and then the remaining amino acids of the sequence are added sequentially, which is preferred for the chemical synthesis of the antiproliferative peptides described herein. Is the way. Techniques for solid phase synthesis are well known to those skilled in the art.

[0046] Alternatively, the antiproliferative peptides described herein are synthesized by recombinant nucleic acid synthesis. Generally, in this method, a nucleic acid sequence encoding a peptide is created,
The nucleic acid is introduced into an expression cassette under the control of a specific promoter, the peptide is expressed in a host, the expressed peptide or polypeptide is isolated, and if necessary,
Regenerate the peptide. Techniques sufficient to cause a technician to perform such operations are described in the literature.

After expression, the recombinant peptide can be purified by standard methods. These methods include ammonium sulfate fractionation, affinity columns, column chromatography, gel electrophoresis, and the like. A substantially pure composition is preferably about 50-95% homogeneous, but for use as a therapeutic agent, most preferably more than 80-95% homogeneous.

[0048] One skilled in the art will appreciate that after chemical synthesis, biological expression or purification, the antiproliferative peptide will adopt a conformation substantially different from the native conformation of the component peptides. Know that there is sex. Often, it is necessary to denature and reduce the antiproliferative peptide and then refold the peptide to the desired conformation. Those skilled in the art know how to regenerate proteins after reduction and denaturation.

As used herein, the term “biological activity” refers to compounds obtained from, or reacting with, a biological system, or the functionality, reaction, It refers to the functionality, reactivity, and specificity of a compound that mimics sex and specificity. Suitable examples of biologically active compounds include enzymes, antibodies, antigens and proteins.

The term “body fluid” as used herein refers to saliva, gingival secretions, cerebrospinal fluid, gastrointestinal fluid, mucus, genitourinary secretions, synovial fluid, blood, serum, plasma, urine Cyst fluid, lymph fluid, ascites fluid, pleural effusion, interstitial fluid, intracellular fluid, ocular fluid, semen, mammary secretions, and vitreous humor, and nasal secretions, but are not limited thereto.

Characteristics of Low Density Lipoprotein Receptor As used herein, the term “low density lipoprotein receptor (LDLR)” includes low density lipoprotein (LDL), low density lipoprotein receptor related Protein (LRP), very low density lipoprotein receptor (VLDLR), α ₂
Macroglobulin receptor / low-density lipoprotein receptor-related protein (α ₂ M
R / LRP), gp330 / megalin Apolipoprotein E receptor 2 (apo
ER2) and the receptor LR8B family of proteins.

The LDL receptor family of proteins is characterized by a transmembrane protein composed of about four members (LDL, LRP, VLDLR and gp330 / megalin) (Strrickland et al., FASEB 9:
890-898 (1995). All of which are incorporated herein by reference). These proteins exhibit similar structural patterns and exhibit related functions, such as mediating plasma membrane penetration and cytolysis of various ligands. A common structural feature of the LDL receptor family is the presence of one or more repeats of the NPXY sequence in the cytoplasmic domain, which results in the accumulation of the receptor in clathrin-coated pits of cells. Can be. On the other hand, the extracellular domain contains many copies of the epidermal growth factor-like domain, which allows the separation of the receptor ligand within the endosome.

Binding of Ligands to Low Density Lipoprotein Receptors According to a novel discovery in the present invention, the anti-angiogenic or anti-proliferative activity of compounds such as TFPI requires LDL receptor expressed on the surface of endothelial cells. It was shown that a body-like protein was mediated. As shown in Example 2, the antiproliferative activity of TFPI was reduced in the presence of LDL (LDLR ligand). Furthermore, VL
The present inventors have also shown that the presence of DLR antibodies reduces the antiproliferative activity of TFPI, further confirming the role of LDLR.

Ligands known to penetrate the plasma membrane via the LDL receptor protein family include members of the following categories: That is,
Proteinases (eg, tPA), proteinase inhibitors (eg, serpin, TFPI), proteinase / inhibitor complexes (uPA / PA)
I, thrombin / antithrombin), lipoproteins (eg, apolipoprotein B), and substrate proteins (eg, thrombospondin). Preferred LDLR ligands include α ₂ -macroglobulin, β-amyloid precursor protein, proteinase nexin II, proteinase nexin I, pro-uPA, lipoprotein lipase, lactoferrin, PAI-1, horse leukocyte elastase inhibitors, protein C inhibitor, C1- inhibitor, alpha ₂ - antiplasmin, alpha 1-proteinase inhibitor, alpha 1-antichymotrypsin, heparin cofactor II, tissue plasminogen activator, antithrombin III, tissue factor pathway coagulation inhibitor, apolipoprotein B, apolipoprotein J, clusterin, thrombospondin, and active fragments thereof.

In particular, the serpin superfamily (Serine proteinase inhibitor) having a domain of Kunitz or non-Kunitz
Ligands belonging to are preferred for the methods and compositions of the present invention. Serpins are a family of structurally similar proteins known to irreversibly inhibit a wide range of serine proteases. Their tertiary structure is flexible, containing three β sheets (designated A, B and C), several α-helices (named A to I), and highly variable residues. It consists of a sexual reactive loop. The reactive site is in the reactive loop, approximately 30 to 40 amino acids from the carboxy terminus of the protein, and contains peptide bonds that mimic the normal substrate of the target protease. Upon binding to the protease, the conformation of the serpins changes significantly, whereby the active loop is incorporated into β-sheet A. Serpin family ligands include:
Tissue factor pathway coagulation inhibitor 1 (TFPI-1) and tissue factor pathway coagulation inhibitor 2 (TFPI-2), antithrombin III, amyloid protein precursor (APP), amyloid beta precursor protein, collagen VI, bovine pancreatic trypsin inhibitor ( BPTI), α ₁ -proteinase inhibitor, α ₁ -antichymotrypsin, α ₂ -antiplasmin, heparin cofactor II,
Protein C inhibitor (PCI), proteinase nexin-1 (PN-1)
), And plasminogen activator inhibitors (PAI-1 and P
AI-2).

[0056] Representative proteins of protease inhibitors that bind to LDLR include TFPI. TFPI is a glycoprotein having a molecular weight of about 32-45.
kDa. TFPI is composed of approximately 276 amino acids, followed by an acidic amino terminus, followed by three Kunitz-type protease inhibitor domains called Kunitz-1, Kunitz-2 and Kunitz-3, and a basic carboxyl-terminal region. There is a structure that there is.

TFPI, also known to those skilled in the art as a lipoprotein-binding coagulation inhibitor, is a protease inhibitor that plays an important role in regulating tissue factor-induced blood coagulation. Generally, TFPI is found in plasma, platelets, and on endothelium. Typically, after the bleeding has stopped,
TFPI is three times the concentration normally found in plasma. TF in blood vessels
PIs exist in several forms, which are well known to those skilled in the art. The majority of plasma TFPIs have a molecular weight of 34-41 kDa, but other higher molecular weight types are also present. TFPI is synthesized in endothelial cells and then exocytosed on the cell surface to produce heparin sulfate proteoglycan (HSP
G) and remain there (Narita et al., Journal. Biol. Chemistry 270 (42): 248).
00-24804 (1995)).

Other proteins that include a Kunitz-type domain and that can be used in the compositions of the invention include amyloid precursor protein (APP), collagen VI, and bovine pancreatic trypsin inhibitor (BPTI). APP contains a Kunitz-type protease inhibitor domain, which exists in at least four different forms, but as different numbers of amino acids from approximately 700-800 residues (Niwano et al., J. Lab. Clin. Med.,
125 (2): 215-256 (1995)). Collagen VI is found on the vessel wall, has a triple helical structure, and has a Kunitz-type protease inhibitor domain (Kehrel, Semin. Thromb.
. Hemost. 21 (2): 123-9 (1995)). Bovine pancreatic trypsin inhibitor (BPTI) is a 58-residue protein with three disulfide bonds and belongs to the Kunitz family of serine proteinase inhibitors (Moss et al., J. Gen. Physiol. 108 (6):
473-84 (1996)).

In another embodiment, a preferred category of LDLR binding ligands is ligand conjugates. Although the ligand complex contains the LDL receptor-like protein, typically, the individual components of the complex are not self-contained. For example, plasminogen activator inhibitor 1 (PAI-1
) And urokinase plasminogen activator (uPA) alone do not bind to LRP. However, when each adds to form a complex, the conformation of PAI-1 changes, and the uPA / PAI-1 complex binds to LRP using the PAI-1 component. Such ligand complexes include:
Like thrombin / antithrombin and uPA / antithrombin,
Protease / serpin complexes are included. Further limiting the scope of the present invention and the ligand complexes contained therein, uPA / PAI-1 and tPA / P
AI-1, uPA / PN-1 (proteinase nexin-1), elastase /
alpha ₁ proteinase inhibitor, trypsin / alpha ₁ proteinase inhibitor, thrombin / heparin cofactor II, and thrombin PAI-1, uPA /
PCI (protein C inhibitor), uPA / antithrombin III, uP
A / C1-inhibitor, low molecular weight uPA / PAI-1, low molecular weight uPA / PC
I, tPA / PCI, thrombin / PCI, thrombin / PN-1 and the like. The serine proteinase / serpin complex can be obtained, for example, using the E.
ur. J. Biochem. 248: 270-281 (1997).

The LDLR ligand of the present invention includes body fluids (including serum, urine, ascites, etc.)
(Not limited to these), or can be synthesized by chemical or biological methods, such as cell culture, recombinant gene expression, peptide synthesis, and the like. Recombinant techniques include the polymerase chain reaction (PCR
), Gene amplification from RNA using reverse transcriptase / PCR, and the like. LDLR ligand is a known protein extraction method,
In particular, W. F. Novotny et al. Biol. Chem. , 264:
18832-18837 (1989).

Peptides or Protein Fragments Peptides or protein fragments containing LDLR binding ligands can be made from the above-described proteins, and their anti-proliferative or anti-angiogenic activity is determined using techniques and methods well known to those skilled in the art. You can find out. For example, full-length recombinant T
FPI (rTFPI) can be made using a baculovirus gene expression system. Full-length proteins can be cleaved into individual domains, and are described, for example, in Enjoji et al. (Biochemistry 34: 5725-).
5735 (1995)). In the method of Enjoji et al., RTFPI is treated with a digestive enzyme, human neutrophil elastase, and the digest is purified using a heparin column.
Human neutrophil elastase cleaves TFPI at Leu ⁸⁹ into two fragments, one of which is Kunitz-1 and the other is Kunitz-2 and Kunitz-type.
Type 3 is included. For further fragmentation, see Balian et al. (Bioche
It is preferable to treat a fragment containing Kunitz-2 type and Kunitz-3 type (Kunitz-2 / Kunitz-3) with the digestible compound hydroxylamine using the method of Mistry 11: 3798-3806 (1972). The digest is purified using a heparin column. Hydroxylamine cleaves a fragment containing the Kunitz-2 type and a Kunitz-3 type into two fragments, and separates the fragment containing the Kunitz-3 type domain from the fragment containing the Kunitz-2 type domain.

As an alternative, there is a method in which the whole or a large fragment of the protein exhibiting antiproliferative activity is digested while removing amino acids one at a time to obtain a fragment. Each successively shortened fragment is tested for antiproliferative activity. Similarly, a method of synthesizing fragments with varying lengths and testing the antiproliferative activity is also possible. The exact number and attributes of amino acids in the protein required to confer antiproliferative activity, using routine digestion, synthesis and screening tools well known to those skilled in the art, while increasing or decreasing the length of the fragment. And the sequence will be determined by those skilled in the art.

The antiproliferative activity is assessed by testing the fragment's activity in suppressing the growth of new vascular cells, referred to herein as inhibiting angiogenesis, in situ. A preferred assay is the chicken embryo chorioallantoic membrane (CAM) assay, which is described by Crum et al., Science 230: 1375 (1985);
And U.S. Pat. No. 5,001,116. The CAM test is briefly summarized as follows. Embryos of fertilized chickens are dehulled on day 3 or 4 and discs of methylcellulose containing the fragment of interest (dis)
c) is implanted in the chorioallantoic membrane. The embryos are examined 48 hours later and if a clear avascular area appears around the methylcellulose disc, the diameter of that area is measured. The larger the diameter of this region, the greater the antiproliferative activity. Another preferred assay is the HUVEC assay described in Example 2.

The active fragment is preferably a fragment containing a part of the ligand necessary for binding to LDLR. Specifically, ligands having either a Kunitz-type domain or a non-Kunitz-type domain and belonging to the family of protease inhibitors are preferred. Serpin ligands include, but are not limited to: That is, tissue factor pathway coagulation inhibitor 1 (TFPI-1), tissue factor pathway coagulation inhibitor 2 (TFPI-2), antithrombin II
I, amyloid protein precursor (APP), amyloid beta precursor protein, collagen VI, bovine pancreatic trypsin inhibitor (BPTI), α ₁ -proteinase inhibitor, α1-antichymotrypsin, α2-antiplasmin, heparin cofactor II, protein C Inhibitors (PCI), proteinase nexin-1 (PN-1), and plasminogen activator inhibitors (PAI-1 and PAI-2).

As noted above, it will be appreciated by the skilled artisan that one or a small number of amino acids (typically less than 5%, more typically less than Individual substitutions, deletions or additions that alter, add or delete (less than 1%) conservatively modified changes if the change would result in the replacement of an amino acid with a chemically similar amino acid. is there. Tables for conservative substitutions indicating functionally similar amino acids are well known to those skilled in the art. Thus, proteins having conservatively altered changes relative to the claimed protein are also included in the invention, provided that the chemical reactivity of the protein is not significantly different from the claimed protein. You.

Formulation The natural or synthetic protein, peptide or protein fragment containing all or the active portion of the LDLR ligand can be prepared using known techniques in a physiologically acceptable formulation, such as a pharmaceutically acceptable carrier. Can be prepared. For example, a protein, peptide or protein fragment can be in the form of a therapeutic composition in combination with a pharmaceutically acceptable excipient.

Alternatively, the gene for a natural or synthetic protein, peptide or protein fragment, including all or the active portion of the LDLR ligand, can be incorporated into a vector and administered continuously by gene therapy techniques. The vector may be administered in an excipient that has specificity for a target site, such as a tumor.

[0068] These compositions may be in the form of a solid, liquid, aerosol, or the like. Examples of solid compositions include tablets, creams and implantable dosage units. Tablets can be administered orally. The therapeutic cream is administered topically. Implantable dosage units can be administered locally, for example, at the site of a tumor, or can be implanted, for example, subcutaneously, to provide a sustained release of the therapeutic composition. Examples of the liquid composition include a prescription as a subcutaneous, intravenous, or intraarterial injection and a prescription for topical and intraocular administration. Aerosol formulations include inhaler formulations for administration to the lung.

[0069] The compositions can be administered using standard routes of administration. Generally, they are administered by topical, oral, enteral, nasal or parenteral (eg, intravenous, subcutaneous, intramuscular injection) routes. In addition, the composition can be included in a sustained release matrix, such as a biodegradable polymer, which is implanted near the site of desired delivery, for example, at the site of a tumor. The methods include single administration, repeated administration at predetermined time intervals, and sustained release administration for a predetermined period.

The matrix for sustained release used herein is a matrix usually obtained from a polymer material, which can be degraded by hydrolysis or dissolution by an enzyme or an acid-base. Once inserted into the body, it is enzymes and body fluids that act on this matrix. The matrix for sustained release is preferably selected from biocompatible materials, such as liposomes, polylactide (polylactide acid), polyglycolide (polymer of glycolic acid), and polylactide-co-glycolide (lactic acid and glycol). Acid copolymers), polyanhydrides,
Poly (ortho) ester, polypeptide, hyaluronic acid, collagen, chondroitin sulfate, carboxylic acid, fatty acid, phospholipid, polysaccharide, nucleic acid, polyamino acid, amino acid such as phenylalanine, tyrosine, isoleucine, polynucleotide,
Polyvinyl propylene, polyvinyl pyrrolidone and silicon. Preferred as biodegradable matrices are either polylactide, polyglycolide or polylactide-co-glycolide (copolymer of lactic acid and glycolic acid).

The dose of the composition will depend on the condition to be treated, the particular composition used, and clinical factors such as weight and patient condition, and the route of administration.

Further, the term “effective amount” refers to inhibiting the growth of unwanted cells, particularly endothelial cells, when administered to a human or animal, resulting in reduced cancer or spread and growth of the cancer. It refers to the amount of the composition as controlled. The effective amount is
It can be easily determined by those skilled in the art according to ordinary methods.

For example, an antiproliferative composition according to the present invention can be administered parenterally or orally in a range of about 1.0 μg to 1.0 mg per patient, provided that the range is It has no limiting meaning. The amount of antiproliferative composition required to produce the proper effect will, in fact, vary from patient to patient and will depend on the strength of the composition being administered, the condition being treated and the responsiveness of the individual patient. I do. As such, the dosage to be administered to an individual patient will be determined based on routine trials and the training and experience of those skilled in the art.

The composition may be administered in combination with other compositions or means for treating the disease. For example, to treat unwanted cell proliferation, as before,
Surgery, radiation therapy, chemotherapy may be combined with the administration of this composition,
And subsequently, additional doses may be administered to the patient to stabilize and completely suppress any remaining unwanted cell growth.

LDL Receptor Ligand Antibodies Also included in the invention are LDLR ligand antibodies, which are used for therapeutic as well as diagnostic purposes. The antibodies provided herein are monoclonal or polyclonal antibodies having binding specificity for an LDLR ligand.
As the antibody, a monoclonal antibody having higher specificity for a ligand is preferable. This antibody has very low or no cross-reactivity to other proteins and peptides. This antibody is a proteinase, a proteinase inhibitor,
Preferably, it is specific for ligands including serpins, proteinase / inhibitor complexes, thrombin / antithrombin, lipoproteins and substrate proteins.

[0076] Monoclonal antibodies are made by immunizing animals, such as mice and rabbits, with whole or immunogenic portions of an LDLR ligand, such as antithrombin III. Spleen cells are removed from the immunized animal and the sensitized spleen cells are transferred to a myeloma cell line, such as mouse SP2 / O myeloma cells (ATCC
, Manassas, Virginia). To induce cell fusion, polyethylene glycol is added. Hybridomas can be chemically sorted by culturing the cells in a selective medium containing hypoxanthine, aminopterin and thymidine.

[0077] The hybridomas are then screened for the ability to produce monoclonal antibodies against the LDLR ligand. Hybridoma-producing antibodies that bind to the LDLR ligand are cloned, expanded, and stored frozen for future production. Hybridomas producing monoclonal antibodies having the IgG isotype, more preferably the IgG1 isotype are preferred.

In the same manner as above, a polyclonal antibody is produced by immunizing an animal such as a mouse or rabbit with an LDLR ligand. Serum is then collected from the animal and antibodies in serum are screened for binding to an LDLR ligand, preferably an antigen reactive with the monoclonal antibody described above.

[0079] Monoclonal or polyclonal antibodies, or both, can also be directly labeled with a detectable label to identify and quantify LDLR ligands in biological or environmental samples, as described below. Will be described.
Labels for use in immunoassays are generally well known to those of skill in the art and include enzymes, radioisotopes, fluorescence, luminescence, chromogenic materials, eg, colored materials such as gold colloidal gold or latex beads. In an immunoassay, the antibody may be bound to a solid phase in order to separate the antibody-antigen complex from the untreated component. Illustrative solid phase materials include, but are not limited to, microtiter plates, test tubes, magnetic powder, plastic particles, glass particles, slides, and the like. Methods for binding an antibody to a solid phase are well known to those skilled in the art.

Alternatively, the antibody may be a labeled substance having affinity for immunoglobulins,
Indirect labeling can also be performed, for example, by reacting with protein A or G, or a second antibody. The antibody can be conjugated to a second substance, and detection can be performed using a labeled third substance having an affinity for the second substance conjugated to the antibody. For example, an antibody can be conjugated to biotin, and the antibody-biotin conjugate detected using labeled avidin or streptavidin. Similarly, an antibody can be conjugated to a hapten, and the antibody-hapten conjugate can be detected using a labeled anti-hapten antibody. These and other methods for using antibody conjugates for labeling and assays are well known to those skilled in the art.

The present invention provides sensitive immunoassays using one or more of the antibodies described above. In various samples, particularly biological samples such as human or animal biological fluids, this immunoassay is useful for detecting the presence and amount of LDLR ligand. The sample can be taken from any source, as long as the LDLR ligand is present. For example, these samples include, but are not limited to, blood, saliva, semen, tears, urine, and the like.

The antibody-antigen complex formed in the immunoassay of the present invention can be detected using an immunoassay known to those skilled in the art, for example, a sandwich immunoassay or a competitive immunoassay. The antibody-antigen complex is contacted with an antibody similar to that used to capture the antigen, but labeled with a detectable label. Preferred labeling methods are listed: chemiluminescent labeling methods such as ruthenium and aequorin, bioluminescent labeling methods such as luciferase, fluorescent labeling methods such as FITC, and such as alkaline phosphatase, β-galactosidase and horseradish peroxidase. There is an enzymatic labeling method.

Next, the labeled complex is detected using a detection method or device specialized for detecting the label used. Incubation of one or more soluble antigens with magnetic beads coated with non-specific antibodies in the same assay will determine the background value of the sample analyzed in the assay.

Diseases and Conditions to be Treated The methods and compositions described herein are useful for treating human and animal diseases and processes arising from abnormal or undesirable cell proliferation. Examples of such diseases include hemangiomas, solid tumors, leukemias, metastases, telangiectasia, psoriasis, edema sclerosis, purulent granulomas, myocardial neovascularization, plaque neovascular growth, tubular collaterals ( coronary collaterals), ischemic limb angiogenesis
, Corneal disease, rubeosis, neovascular glaucoma, diabetic retinopathy, post-lens fibroplasia, arthritis, diabetic neovascular growth, macular degeneration, wound healing, peptic ulcer, fracture, keloid, angiogenesis, hematopoiesis, ovulation , Menstruation, placenta formation, etc., but are not limited thereto. The methods and compositions inhibit angiogenesis,
It is particularly useful for diseases and disorders related to angiogenesis.

The methods and compositions described herein are particularly useful for treating cancer, arthritis, macular degeneration, and diabetic retinopathy. Administering the present composition to a human or animal having a tumor with metastasis due to pro-angiogenesis is useful in preventing the growth and spread of such a tumor.

The compositions and methods of the present invention are further illustrated by the following non-limiting examples, which should not be construed as limiting the scope of the invention in any way. On the contrary, after reading this description, various other embodiments, modifications and variations thereof may be suggested to one skilled in the art without departing from the spirit of the invention and / or the appended claims. It should be clearly understood that measures may be taken for equivalents.

Example 1 Analysis of Human Umbilical Vein Vascular Endothelial Cells Human umbilical vein vascular endothelial cells (HUVEC) and their media (EGM and EBM) were purchased from Clonetics (San Diego, Calif.). HUVEC were cultured in EGM to saturation in growth in the usual manner.
Cells were trypsinized and EBM1OOμ supplemented with 2% serum and antibiotics.
Plated in 96-well plates at a rate of 5,000 cells per liter. Cells were allowed to attach to the plate for at least 2 hours. Then 10ng / m
1 bFGF and various concentrations of anti-angiogenic drugs were added to the wells. Cells were cultured for 48 hours at 37 ° C. in 5% CO ₂ gas. Cell proliferation was determined using the uridine incorporation method (Boelinger Mannheim Corporation, Indianapolis, IN).

Example 2 Endothelial Cell Proliferation Test in the Presence of TFPI and LDL A proliferation test well known to those skilled in the art of using human umbilical vein vascular endothelial cells HUVEC includes tissue factor pathway coagulation inhibitor (TFPI) and Performed using low density lipoprotein (LDL). (LDL is an LDLR ligand known to promote endothelial cell growth.) Materials and Methods Materials for this experiment include HUVEC and media for their growth,
"Endothelial cell basal medium" (EBM) and "endothelial cell growth medium" (EGM) (Cl
Onetics, San Diego, CA). Also, full-length TFPI (American Diagnostics Inc., Greenwich, CT) was used. In addition, a cell proliferation ELISA BrdU
(Boehringer Mannheim Corporation, Indianapolis, IN), bFGF (R & D, Minneapolis, MN)
, And LDL (PerImmune, Inc., Rockville, MD) were also used.

For the growth test, the HU was routinely grown to saturation in EGM medium.
Culturing the VEC was included. Cells were treated with trypsin and treated with EBM medium 1
Plated in 96-well plates at a rate of 5000 cells per 00 μl. Cells were allowed to attach to the plate for at least 2 hours. Next, 10
ng / ml bFGF and various concentrations of full-length TFPI were added to the wells. Cells were grown for 48 hours, after which cell proliferation was measured using a standard uridine incorporation method.

Results In the absence of LDL, 150 nM TFPI inhibited bFGF-induced HUVEC proliferation by 50%. Under the same experimental conditions, but with LDL present at 100 μg / ml, the same concentration of TFPI inhibited HUVEC proliferation by 30%. This T
The partial decrease in FPI activity demonstrates that LDLR-like proteins affect the antiproliferative effect of TFPI. Without wishing to be bound by subsequent theory, the involvement of the receptor by LDL results in a partial internalization of the receptor that prevents its binding to TFPI and induction of mitotic arrest signals.

Example 3 Endothelial Cell Proliferation Assay in the Presence of TFPI and VLDLR Antibodies In previous experiments performed by the inventors, 550 nM TFPI was converted to HUVEC bFGF
It has been shown to inhibit induced proliferation by up to 90%. HUVEC, 3
When incubated in the presence of 1 μm VLDLR antibody (American Red Cross (Rockville, Md.)) For 1 hour at 7 ° C., the same concentration of TFP resulted in HUV
Inhibited bFGF-induced proliferation of EC by 50%. This partial decrease in TFPI activity demonstrates, at least in part, that VLDLR affects the antiproliferative properties of TFPI.

Example 4 Proliferation Test in the Presence of Receptor Binding Protein The proliferation test described in Examples 1 and 2
) Was conducted to evaluate the effect. RAP is a 39 kDa protein that binds with high affinity to members of the LDL receptor system protein, and antagonizes the former ligand binding property.

[0093] 275 nM TFPI inhibited bFGF-induced proliferation of HUVEC by 100%. Under the same conditions, in the presence of 100 nM RAP (Rockville, MD) the same concentration of TFPI inhibited bFGF-stimulated HUVEC replication by almost 50%. Again, partial neutralization of TFPI activity by RAP demonstrates that LDLR-like proteins are involved in the transduction of TFPI antimitotic signals. The results of this experiment are illustrated in FIG.

Example 5 Endothelial Cell Migration Test in the Presence of TFPI and RAP The proliferation test described in Examples 1 and 2 was performed to evaluate the effect of RAP on endothelial cell proliferation. RAP is a 39 kDa protein that binds with high affinity to LDL receptor proteins, and antagonizes the ligand binding properties of the former protein.

TFPI inhibits bFGF-induced HUVEC migration in a dose-dependent manner. The presence of 500 nM RAP in the culture medium neutralized the anti-migratory activity of TFPI. The result of this experiment is illustrated in FIG.

Example 6 Endothelial Cell Proliferation Test in the Presence of Clusterin Clusterin, apolipoprotein J, is a multifunctional 80 kDa protein implicated in the terminal complement pathway. Clusterin is synthesized by endothelial cells to produce gp330 / megalin receptor and LRP (Zlo).
Kovic et al., Proc. Natl. Acad. Sci. USA 1996 9
3 (9), 4229-4234 and Morales et al., 1996 Biol. R
eprod. 55 (3), 676-683).

Proliferation studies performed as described in Examples 1 and 2 above demonstrate that clusterin inhibits bFGF-induced endothelial cell proliferation in a dose-dependent manner. The result of this experiment is shown visually in FIG.

Of course, the foregoing is only relevant in the preferred embodiment of the invention, and numerous modifications or alterations may depart from the spirit and scope of the invention as set forth in the appended claims. Without that, it must be understood that what can be done therein.

[Brief description of the drawings]

FIG. 1 is a graph when the activity of TFPI is partially neutralized by RAP.

FIG. 2 is a dose response curve showing cell growth activity in the presence of various amounts of TFPI and RAP, representing four examples. The error line represents the standard deviation.

FIG. 3 is a graph showing the dose-dependent inhibitory effect of clusterin on bFGF-induced endothelial cell proliferation.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 27/02 A61K 37/48 35/00 37/64 (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, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, DZ, 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, TZ, UA, UG, UZ, VN, YU, ZA, ZWF term (reference) 4C084 AA01 AA02 AA03 BA36 BA44 DA39 DA40 DC02 DC03 DC21 DC32 DC34 DC35 DC36 DC41 NA14 ZA332 ZA962 ZB112 ZB262 ZC412

Claims (20)

[Claims] Claims: 1. A method of treating a human or animal having undesired cell proliferation, comprising administering to said human or animal a composition comprising an isolated protein or peptide, or an antiproliferative fragment thereof. A method wherein the protein or peptide has the ability to bind to low density lipoprotein receptor, and the effective amount is an amount sufficient to inhibit unwanted cell growth. 2. The low-density lipoprotein receptor comprises low-density lipoprotein, low-density lipoprotein receptor-related protein, ultra-low-density lipoprotein receptor, gp330 / megalin, and an active fragment thereof. The described method. 3. The isolated protein or peptide includes proteinases, proteinase inhibitors, serpins, proteinase / inhibitor complexes, thrombin / antithrombin, lipoproteins, interstitial proteins and active fragments thereof. The method described in. 4. The isolated protein or peptide comprises α2-macroglobulin, β-amyloid precursor protein, proteinase nexin I, proteinase nexin II, pro-uPA, lipoprotein lipase, lactoferrin, PAI-1 , Equine leukocyte elastase inhibitor, protein C inhibitor, C1-inhibitor, α2-antiplasmin, α1-proteinase inhibitor, α1-antichymotrypsin, heparin cofactor II, tissue plasminogen activator, antithrombin III, tissue factor pathway coagulation inhibitor , Apolipoprotein B, apolipoprotein J, clusterin, thrombospondin, tissue factor pathway coagulation inhibitor 1, tissue factor pathway coagulation inhibitor 2, amyloid protein Precursor, amyloid beta precursor protein, collagen VI, bovine pancreatic trypsin inhibitor, and methods of claim 1 selected from the group consisting of active fragments thereof. 5. The method of claim 3, wherein said proteinase / inhibitor complex comprises urokinase plasminogen activator / plasminogen activator inhibitor 1 complex or thrombin / antithrombin complex. 6. The low-density lipoprotein receptor comprises a low-density lipoprotein receptor-related protein, and the isolated protein or peptide, or an antiproliferative fragment thereof, comprises α2-macroglobulin. The method of claim 1. 7. The low-density lipoprotein receptor comprises a low-density lipoprotein receptor-related protein, and the isolated protein or peptide, or an antiproliferative fragment thereof, comprises antithrombin III. Item 1. The method according to Item 1. 8. The method of claim 1, wherein said composition further comprises a pharmaceutically acceptable excipient, carrier or sustained release matrix. 9. The method of claim 1, wherein said undesired cell growth is undesired endothelial cell growth. 10. The method of claim 9, wherein said inhibiting endothelial cell proliferation is inhibiting neovascularization. 11. The method of claim 1, wherein said undesired cell proliferation is a disease associated with angiogenesis. 12. The method according to claim 4, wherein the angiogenesis-related disease is a disease selected from the group consisting of cancer, arthritis, macular degeneration, and diabetic retinopathy. 13. A method for treating undesired angiogenesis in a human or animal, comprising administering to a human or animal having undesired angiogenesis a proteinase, a proteinase inhibitor, a serpin, a proteinase / inhibitor complex, thrombin / antithrombin, A method of treatment comprising administering a composition comprising an effective amount of an anti-angiogenic compound comprising a lipoprotein and a substrate protein. 14. The angiogenesis-inhibiting compound is α2-macroglobulin, β
Amyloid precursor protein, proteinase nexin I, proteinase nexin II, pro-uPA, lipoprotein lipase, lactoferrin, PAI
-1, horse leukocyte elastase inhibitor, protein C inhibitor, C1
Inhibitors, α2-antiplasmin, α1-proteinase inhibitor, α1-antichymotrypsin, heparin cofactor II, tissue plasminogen activator, antithrombin III, tissue factor pathway coagulation inhibitor, apolipoprotein B, apolipoprotein J, clusterin, Thrombospondin, tissue factor pathway coagulation inhibitor 1, tissue factor pathway coagulation inhibitor 2, amyloid protein precursor, amyloid beta precursor protein, collagen VI,
14. The method according to claim 13, wherein the method is selected from the group consisting of bovine pancreatic trypsin inhibitor, and active fragments thereof. 15. The method according to claim 14, further comprising a pharmaceutically acceptable excipient, carrier or sustained release matrix. 16. The method of claim 16, wherein the undesired angiogenesis is cancer, arthritis, macular degeneration,
15. The method of claim 14, which is associated with angiogenesis-related diseases, including diabetic retinopathy. 17. A composition for treating unwanted angiogenesis in a human or animal comprising an effective amount of an anti-angiogenic compound, wherein the composition comprises a ligand that binds to a low density lipoprotein receptor. 18. The method of claim 17, wherein the low density lipoprotein receptor comprises low density lipoprotein, low density lipoprotein receptor-related protein, very low density lipoprotein receptor, gp330 / megalin and active fragments thereof. A composition as described. 19. The composition according to claim 17, wherein said ligands include proteinases, proteinase inhibitors, serpins, proteinase / inhibitor complexes, thrombin / antithrombin, lipoproteins, substrate proteins and active fragments thereof. 20. The ligands include α2-macroglobulin, β-amyloid precursor protein, proteinase nexin I, and proteinase nexin II.
, Pro-uPA, lipoprotein lipase, lactoferrin, PAI-1, equine leukocyte elastase inhibitor, protein C inhibitor, C1-inhibitor, α2-antiplasmin, α1-proteinase inhibitor, α1-antichymotrypsin, heparin cofactor II, tissue plus Minogen activator, antithrombin III, tissue factor pathway coagulation inhibitor, apolipoprotein B, apolipoprotein J, clusterin, thrombospondin, tissue factor pathway coagulation inhibitor 1, tissue factor pathway coagulation inhibitor 2, amyloid protein precursor, amyloid beta 18. The composition according to claim 17, wherein the composition is selected from the group consisting of precursor protein, collagen VI, bovine pancreatic trypsin inhibitor, and active fragments thereof.