EP1161258A1 - Compositions and methods of use of ldl-like receptor ligands for the treatment of cancer and angiogenic-based disease - Google Patents

Abstract

Compositions and methods effective in inhibiting abnormal or undesirable cell proliferation, particularly endothelial cell proliferation and angiogenesis related to neovascularization and tumor growth are provided. The compositions comprise naturally occuring or synthetic protein, peptide, or protein fragment capable of binding to low density lipoprotein (or low density lipoprotein-like) receptors. The compositions may be administred using a pharmaceutically acceptable carrier. The methods involve administering to a human or animal the compositions described herein in a dosage sufficient to inhibit cell proliferation, particularly endothelial cell proliferation. The methods are useful for treating diseases and processes, such as cancer, mediated by undesired and uncontrolled cell proliferation particularly by inhibiting angiogenesis. Administration of the compositions of the present invention to a human or animal having prevascularized metastasized tumors is useful for preventing the growth or expansion of such tumors.

Description

COMPOSITIONS AND METHODS OF USE OF LDL-LIKE RECEPTOR LLGANDS FOR THE TREATMENT OF

CANCER AND ANGIOGENIC-BASED DISEASE

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims pπoπty to Unites States Patent Application Seπal No. 09/270,982, filed March 17, 1999.

FIELD OF THE INVENTION

The present invention relates to compositions and methods for the inhibition of cellular proliferation. More particularly, the present invention relates to the use of proteins or peptides, and/or fragments thereof, that bind to lipoprotem receptors, particularly low density lipoprotem receptors, and inhibit angiogenesis and angiogenesis-related diseases.

BACKGROUND OF THE INVENTION

Cellular proliferation is a normal ongoing process in all living organisms and is one that involves numerous factors and signals that are delicately balanced to maintain regular cellular cycles The general process of cell division is one that consists of two sequential processes: nuclear division (mitosis), and cytoplasmic division (cytokinesis). Because organisms are continually growing and replacing cells, cellular proliferation is a central process that is vital to the normal functioning of almost all biological processes Whether or not mammalian cells will grow and divide is determined by a vaπety of feedback control mechanisms, which include the availability of space in which a cell can grow, and the secretion of specific stimulatory and inhibitory factors in the immediate environment

When normal cellular proliferation is disturbed or somehow disrupted, the results can affect an array of biological functions. Disruption of proliferation could be due to a myπad of factors such as the absence or overabundance of vaπous signaling chemicals or presence of altered environments Some disorders characteπzed by abnormal cellular proliferation include cancer, abnormal development of embryos, improper formation of the corpus luteum, difficulty in wound healing as well as malfunctioning of inflammatory and immune responses.

Cancer is characteπzed by abnormal cellular proliferation. Cancer cells exhibit a number of properties that make them dangerous to the host, often including an ability to mvade other tissues and to induce capillary ingrowth, which assures that the proliferating cancer cells have an adequate supply of blood. One of the defining features of cancer cells is that they respond abnormally to control mechanisms that regulate the division of normal cells and continue to divide in a relatively uncontrolled fashion until they kill the host.

Angiogenesis and angiogenesis related diseases are closely affected by cellular proliferation. As used herein, the term "angiogenesis" means the generation of new blood vessels into a tissue or organ. Under normal physiological conditions, humans or animals undergo angiogenesis only in very specific restricted situations. For example, angiogenesis is normally observed in wound healing, fetal and embryonal development and formation of the corpus luteum, endometπum and placenta. The term "endothehum" is defined herein as a thin layer of flat cells that lines serous cavities, lymph vessels, and blood vessels. These cells are defined herein as "endothelial cells". The term "endothelial inhibiting activity" means the capability of a molecule to inhibit angiogenesis in general The inhibition of endothelial cell proliferation also results m an inhibition of angiogenesis.

Both controlled and uncontrolled angiogenesis are thought to proceed in a similar manner. Endothelial cells and peπcytes, surrounded by a basement membrane, form capillary blood vessels. Angiogenesis begins with the erosion of the basement membrane by enzymes released by endothelial cells and leukocytes The endothelial cells, which line the lumen of blood vessels, then protrude through the basement membrane Angiogenic stimulants induce the endothelial cells to migrate through the eroded basement membrane. The migrating cells form a "sprout" off the parent blood vessel, where the endothelial cells undergo mitosis and proliferate. The endothelial sprouts merge with each other to form capillary loops, creating the new blood vessel.

Persistent, unregulated angiogenesis occurs in a multiplicity of disease states, tumor metastasis and abnormal growth by endothelial cells and supports the pathological damage seen in these conditions. The diverse pathological disease states in which unregulated angiogenesis is present have been grouped together as angiogenic-dependent, angiogenic- associated, or angiogenic-related diseases. These diseases are a result of abnormal or undesirable cell proliferation, particularly endothelial cell proliferation

The hypothesis that tumor growth is angiogenesis- dependent was first proposed in 1971 by Judah Folkman (N. Engl. Jour.

Med. 285:1182 1186, 1971). In its simplest terms the hypothesis proposes that once tumor "take" has occurred, every increase in tumor cell population must be preceded by an increase in new capillaπes converging on the tumor. Tumor "take" is currently understood to indicate a prevascular phase of tumor growth in which a population of tumor cells occupying a few cubic millimeters volume and not exceeding a few million cells, survives on existing host microvessels Expansion of tumor volume beyond this phase requires the induction of new capillary blood vessels For example, pulmonary micrometastases m the early prevascular phase m mice would be undetectable except by high power microscopy on histological sections. Further indirect evidence supporting the concept that tumor growth is angiogenesis dependent is found in U S Patent Νos 5,639,725, 5,629,327, 5,792,845, 5,733,876, and 5,854,205, all of which are incorporated herein by reference. Thus, it is clear that cellular proliferation, particularly endothelial cell proliferation, and most particularly angiogenesis, plays a major role m the metastasis of a cancer If this abnormal or undesirable proliferation activity could be repressed, inhibited, or eliminated, then the tumor, although present, would not grow In the disease state, prevention of abnormal or undesirable cellular proliferation and angiogenesis could avert the damage caused by the invasion of the new microvascular system Therapies directed at control of the cellular pro ferative processes could lead to the abrogation or mitigation of these diseases

What is needed therefore are compositions and methods which can inhibit abnormal oi undesirable cellular proliferation, especially the growth of blood vessels into tumors The compositions should be able to overcome the activity of endogenous growth factors m premetastatic tumors and prevent the formation of the capillaries in the tumors thereby inhibiting the development of disease and the growth of tumors The compositions should also be able to modulate the formation of capillaπes in angiogenic processes, such as wound healing and reproduction Finally, the compositions and methods for inhibiting cellular proliferation should preferably be non-toxic and produce few side effects. SUMMARY OF THE INVENTION

Compositions and methods are provided that are effective m inhibiting abnormal or undesirable cell proliferation, particularly endothelial cell proliferation and angiogenesis related to neovasculaπzation and tumor growth. The compositions compπse a naturally occurring or synthetic protein, peptide, or protein fragment containing all, or an active portion of low density lipoprotem receptor (LDLR) binding ligand, optionally combined m a pharmaceutically acceptable earner. Representative gands useful for the present invention compπse proteins or peptides and fragments thereof belonging to the following categoπes: protemases (i.e. tissue -type plasmmogen activator (tPA)), proteinase inhibitors (i.e. serpins, TFPI), proteinase/inhibitor complexes (urokinase-type plasmmogen activator/plasmmogen activator inhibitor 1 (uPA/PAI), thrombin/antithrombm), poprotems (i.e. apo poprotem B, apo poprotem J or clusteπn), and matπx proteins (i.e. thrombospondin). In addition, specific proteins or peptides compπsing representative hgands useful for the present invention include' alpha2- macroglobulm, beta-amyloid precursor protein (APP), proteinase nexm II (PN2), proteinase nexm I (PN1), pro-uPA, antithrombm III, lipoprotem lipase, lactofeπn, PAI-1, horse leukocyte elastase inhibitor, protein C inhibitor, Cl -inhibitor, alpha2~antιplasmιn, alpha 1 -proteinase inhibitor, and alphal-antichymotryps , hepaπn cofactor II.

Prefeπed LDLR hgands compπse proteins belonging to the protein family of serpins (seπne protease inhibitors) those containing

Kunitz or non-Kunitz domains such as tissue factor pathway inhibitor 1 (TFPI-1), tissue factor pathway inhibitor 2 (TFPI-2), antithrombm III, amyloid protein precursor (APP); beta-amyloid precursor protein, collagen VI, and bovme pancreatic tryps inhibitor (BPTI) Preferably, the protein, peptide or fragment thereof, contains all or an active portion of the above identified proteins The term "active portion", as used herein, means a portion of a protein that inhibits cancer, preferably by inhibiting abnormal or undesirable cell proliferation. Also included in the present invention are homologs, peptides, or protein fragments, or combinations thereof of the above-identified proteins, that inhibit abnormal or undesirable cell proliferation. Most preferably, the protein, peptide, or protein fragment is a protein, peptide or protein fragment of an LDLR binding ligand. Though not wishing to be bound by the following theory, it is believed that by inhibiting endothelial cell proliferation, the methods and compositions descπbed herein are useful for inhibiting tumor growth and metastasis by blocking tumor vasculaπzation. The methods provided herein for treating diseases and processes mediated by undesired and uncontrolled cell proliferation, such as cancer, involve administeπng to a human or animal the composition descπbed herein in a dosage sufficient to inhibit cell proliferation, particularly endothelial cell proliferation The methods are especially useful for treating or repressing the growth of tumors, particularly by inhibiting angiogenesis Administration of the compositions to a human or animal having prevasculaπzed metastasized tumors is useful for preventing the growth or expansion of such tumors

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

It is another object of the present invention to provide methods and compositions for treating or repressing the growth of a cancer It is yet another object of the present invention to provide methods and compositions for therapy of cancer that has minimal side effects

It is another object of the present invention to provide methods and compositions for treating diseases and processes that are mediated by angiogenesis

Yet another object of the present invention is to provide methods and compositions compπsing the use of pioteins, peptides, active fragments and homologs thereof, that inhibit cell proliferation, particularly endothelial cell proliferation Another object of the present invention is to provide methods and compositions for treating diseases and processes that are mediated by angiogenesis by administrating antiangiogemc compounds compπsing low density lipoprotem receptor hgands

It is another object of the present invention to provide methods and compositions for treating diseases and processes that are mediated by angiogenesis by administrating antiangiogemc compounds compπsing low density lipoprotem receptor hgands, wherein the hgands compπse protemases, proteinase inhibitors, hpoprotems and matπx proteins

It is another object of the present invention to provide methods and compositions for treating diseases and processes that are mediated by angiogenesis compπsing administration of antiangiogemc compounds compπsing low density lipoprotem receptor hgands wherein the hgands compπse antithrombm III and amyloid protein precursor

It is a further object of the present invention to provide methods and compositions for reducing cancer and inhibiting tumor growth m a human or animal having cancer by administrating antiangiogemc compounds compπsing low density lipoprotem receptor gands, wherein the hgands compπse protemases, proteinase inhibitors, hpoprotems and matπx proteins

It is still another object of the present invention to provide antiangiogemc compositions compπsing low density lipoprotem receptor hgands, wherein the hgands compπse protemases, proteinase inhibitors, hpoprotems and matπx proteins in combination with pharmaceutically acceptable earners

Yet another object of the present invention is to provide antiangiogemc compositions compπsmg low density lipoprotem receptor hgands, wherein the hgands compπse protemases, proteinase inhibitors, hpoprotems and matnx proteins optionally combined with pharmaceutically acceptable earners that may be administered intramuscularly, intravenously, transdermally, orally, or subcutaneously It is yet another object of the present invention to provide compositions and methods for treating diseases and processes that are mediated by angiogenesis including, but not limited to, hemangioma, solid tumors, blood borne tumors, leukemia, metastasis, telangiectasia, psoriasis, scleroderma, pyogemc gianuloma, myocardial angiogenesis, Crohn's disease, plaque neovasculaπzation, arterio venous malformations, corneal diseases, rubeosis, neovascular glaucoma, diabetic retmopathy, retrolental fibroplasia, arthritis, diabetic neovasculaπzation, macular degeneration, wound healmg, peptic ulcer, Hehcobacter related diseases, fractures, keloids, vasculogenesis, hematopoiesis, ovulation, menstruation, placentation, and cat scratch fever These and other objects, features and advantages of the present invention will become apparent after a review of the following detailed descπption of the disclosed embodiment and the appended claims

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a graph showing the partial neutralization of TFPI activity by RAP

Figure 2 is a dose response graph showing cell proliferation activity in the presence of vaπous amounts of TFPI and RAP and is a representative of four expenments Error bars represent standard deviation

Figure 3 is a graph showing the dose-dependent inhibitory effect of clusteπn on bFGF-induced endothelial cell proliferation

DETAILED DESCRIPTION

The following descπption includes the best presently contemplated mode of caπymg out the invention This descπption is made for the purpose of illustrating the general pnnciples of the inventions and should not be taken in a limiting sense The entire text of the references mentioned herein are hereby incorporated in their entireties by reference, including U S Patent Application Senal No 09/270,982, filed March 17, 1999, U S Patent Application Senal No 09/130,273, filed August 6 1998, and United States Patent No 5,981,471, issued November 9, 1999

Compositions and methods for the treatment of diseases and processes that are mediated by, or associated with, abnormal or undesirable cellular proliferation are provided The compositions comprise isolated naturally occurπng or synthetic protein, peptide, or protein fragment, containing all or an active portion of hgands that bind low density lipoprotem (or low density poprotein-hke) receptors (LDLR) For delivery to a human or animal, the compositions may optionally compπse a pharmaceutically acceptable earner

The term "active portion" is defined herein as the antiprohferative portion of the protein necessary for binding LDLR The active portion has the ability to inhibit cell proliferation such as endothelial cell proliferation by in vivo or in vitro assays or other known techniques Preferably, the LDLR ligand compositions of the present invention compπse proteins belonging to the categoπes of protemases (i.e. tissue- type plasmmogen activator (tPA)), proteinase inhibitors (i.e serpins, TFPI), protemase/inhibitor complexes (urokmase-type plasmmogen activator/plasminogen activator inhibitor 1 (uPA/PAI), thrombm/antithrombin), hpoprotems (i.e. apohpoprotem B), and matπx proteins (i.e. thrombospondin). Most preferably, the protein, peptide, or protein fragment is a protein, peptide or protein fragment compnsmg representative hgands including, but not limited to, alpha2-macroglobuhn, beta-amyloid precursor protein (APP), proteinase nexm II (PN2), proteinase nexm I (PN1), pro-uPA, lipoprotem lipase, lactofeπn, PAI-1, horse leukocyte elastase inhibitor, protein C inhibitor, Cl -inhibitor, alpha2- antiplasmin, alpha 1 -proteinase inhibitor, alphal-antichymotrypsm, hepaπn cofactor II, and antithrombm III.

As noted above, the compositions of the present invention may be optionally combined with a pharmaceutical earner. The term

"earner" as used herein compπses delivery mechanisms known to those skilled in the art including, but not limited to, keyhole limpet hemocyamn (KLH), bovme serum albumin (BSA) and other adjuvants It is to be understood that the low density lipoprotem receptor ligand compositions of the present invention can further compπse adjuvants, preservatives, diluents, emulsifiers, stabilizers, and other components that are known and used for pharmaceutical compositions of the pnor art Any adjuvant system known in the art can be used for the compositions of the present invention Such adjuvants include, but are not limited to, Freund's incomplete adjuvant, Freund's complete adjuvant, polydispersed β-(l,4) linked acetylated mannan ("Acemannan"), TlTERMAX® (polyoxyethylene- polyoxypropylene copolymer adjuvants from CytRx Corporation (Norcross, Georgia), modified pid adjuvants from Chiron Corporation (Emeryville, California), saponin denvative adjuvants from Aguila Biopharmaceuticals (Worcester, Massachusetts), killed Bordetella pertussis, the hpopolysacchande (LPS) of gram-negative bactena, large polymenc anions such as dextran sulfate, and inorganic gels such as alum, aluminum hydroxide, or aluminum phosphate, ovalbumin, flagellin; thyroglobuhn, serum albumin of any species; gamma globulin of any species; and polymers of D- and/or L- amino acids.

Alternatively, the gene for the protein, peptide, or protein fragment, containing all or an active portion of the protein, may be delivered in a vector for continuous administration using gene therapy techniques. The vector may be administered in a vehicle having specificity for a target site, such as a tumor.

In accordance with the methods of the present invention, the compositions descπbed herein, containing a protein, peptide, or protein fragment including all or an active portion of an LDLR ligand, optionally combined in a pharmaceutically acceptable earner, is administered to a human or animal exhibiting undesirable cell proliferation in an amount sufficient to inhibit the undesirable cell proliferation, particularly endothelial cell proliferation, angiogenesis or an angiogenesis-related disease, such as cancer.

Definitions

The terms "a", "an" and "the" as used herein are defined to mean one or more and include the plural unless the context is mappropπate. The term "peptides," are chains of ammo acids (typically L- ammo acids) whose alpha carbons are linked through peptide bonds formed by a condensation reaction between the carboxyl group of the alpha carbon of one ammo acid and the amino group of the alpha carbon of another ammo acid. The terminal ammo acid at one end of the chain {i.e., the ammo terminal) has a free amino group, while the terminal amino acid at the other end of the chain (. e , the carboxy terminal) has a free carboxyl group As such, the term "ammo terminus" (abbreviated N-termmus) refers to the free alpha-ammo group on the ammo acid at the amino terminal of the peptide, or to the alpha-amino group (imino group when participating in a peptide bond) of an amino acid at any other location withm the peptide

Similarly, the term "carboxy terminus" (abbreviated C-termmus) refers to the free carboxyl group on the amino acid at the carboxy terminus of a peptide, or to the carboxyl group of an amino acid at any other location within the peptide Typically, the ammo acids making up a peptide are numbered in order, starting at the amino terminal and increasing in the direction toward the carboxy terminal of the peptide Thus, when one amino acid is said to "follow" another, that amino acid is positioned closer to the carboxy terminal of the peptide than the preceding amino acid The term "residue" is used herein to refer to an ammo acid

(D or L) that is incorporated into a peptide by an amide bond As such, the ammo acid may be a naturally occurπng amino acid or, unless otherwise limited, may encompass known analogs of natural am o acids that function in a manner similar to the naturally occurring amino acids (i.e. , amino acid mimetics). Moreover, an amide bond mimetic includes peptide backbone modifications well known to those skilled in the art.

The phrase "consisting essentially of" is used herein to exclude any elements that would substantially alter the essential properties of the peptides to which the phrase refers. Thus, the descπption of a peptide "consisting essentially of . . ." excludes any amino acid substitutions, additions, or deletions that would substantially alter the biological activity of that peptide. Furthermore, one of skill will recognize that, as mentioned above, individual substitutions, deletions or additions which alter, add or delete a single amino acid or a small percentage of amino acids (typically less than 5%, more typically less than 1%) in an encoded sequence are conservatively modified vanations where the alterations result in the substitution of an ammo acid with a chemically similar amino acid.

Conservative substitution tables providing functionally similar amino acids are well known in the art. The following six groups each contain ammo acids that are conservative substitutions for one another: 1) Alan e (A), Seπne (S), Threomne (T), 2) Aspartic acid (D), Glutamic acid (E),

3) Asparagine (N), Glutamine (Q);

4) Argmine (R), Lys e (K),

5) Isoleucine (I), Leucme (L), Methiomne (M), Va ne (V); and

6) Phenylalanine (F), Tyrosme (Y), Tryptophan (W). The phrases "isolated" oi "biologically pure" refer to matenal which is substantially or essentially free from components which normally accompany it as found in its native state Thus, the peptides descnbed herein do not contain matenals normally associated with their in situ environment Typically, the isolated, antipro ferative peptides described herein are at least about 80% pure, usually at least about 90%, and preferably at least about 95% as measured by band intensity on a silver stained gel.

Protein puπty or homogeneity may be indicated by a number of methods well known the art, such as polyacrylamide gel electrophoresis of a protein sample, followed by visualization upon staining. For certain purposes high resolution will be needed and HPLC or a similar means for puπfication utilized. When the antiprohferative peptides are relatively short m length (i e , less than about 50 am o acids), they are often synthesized using standard chemical peptide synthesis techniques

Solid phase synthesis in which the C-termmal amino acid of the sequence is attached to an insoluble support followed by sequential addition of the remaining ammo acids in the sequence is a preferred method for the chemical synthesis of the antiprohferative peptides descπbed herein Techniques for solid phase synthesis are known to those skilled m the art Alternatively, the antiprohferative peptides descnbed herein are synthesized using recombinant nucleic acid methodology Generally, this involves creating a nucleic acid sequence that encodes the peptide, placing the nucleic acid in an expression cassette under the control of a particular promoter, expressing the peptide in a host, isolating the expressed peptide or polypeptide and, if required, renatunng the peptide Techniques sufficient to guide one of skill through such procedures are found in the literature

Once expressed, recombinant peptides can be puπfied according to standard procedures, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like Substantially pure compositions of about 50 to 95% homogeneity are prefeπed, and 80 to 95% or greater homogeneity are most preferred for use as therapeutic agents

One of skill in the art will recognize that after chemical synthesis, biological expression or puπfication, the antiprohferative peptides may possess a conformation substantially different than the native conformations of the constituent peptides In this case, it is often necessary to denature and reduce the antiprohferative peptide and then to cause the peptide to re-fold into the preferred conformation Methods of reducing and denatunng proteins and inducing le-folding are well known to those of skill in the art

As employed herein, the phrase "biological activity" refers to the functionality, reactivity, and specificity of compounds that are derived from biological systems or those compounds that are reactive to them, or other compounds that mimic the functionality, reactivity, and specificity of these compounds Examples of suitable biologically active compounds include enzymes, antibodies, antigens and proteins

The term "bodily fluid," as used herein, includes, but is not limited to, saliva, gmgival secretions, cerebrospinal fluid, gastrointestinal fluid, mucous, urogemtal secretions, synovial fluid, blood, serum, plasma, uπne, cystic fluid, lymph fluid, ascites, pleural effusion, interstitial fluid, mtracellular fluid, ocular fluids, seminal fluid, mammary secretions, and vitreal fluid, and nasal secretions.

Low Density Lipoprotein Receptor Characteristics

As used herein, the term "low density lipoprotem receptor (LDLR)" includes a family of proteins compπsing low density lipoprotem (LDL), low density lipoprotem receptor-related protein (LRP), very low density lipoprotem receptor (VLDLR), α₂macroglobuhn receptor/low - density- poprotein-receptor-related protein ( ₂MR LRP), gp330/megahn apohpoprotem E receptor 2 (apoER2) and receptor LR8B.

The LDL-receptor family of proteins is characteπzed by transmembrane proteins compπsing approximatey four members: LDL, LRP, VLDLR, and gp330/megalm (see Stπckland et al. FASEB 9:890-898

(1995) which is incorporated herein in its entirety) These proteins exhibit similar structural patterns and perform related functions such as the mediation of endocytosis and cellular degradation of a diverse vaπety of hgands One common structural aspect of the LDL-receptor family is the presence, in the cytoplasmic domain, of one or more repeats of an NPXY sequence responsible for targeting the receptor to clathπn-coated pits of the cells, while the extracellular domain contains multiple copies of the epidermal growth factor-like domain responsible for the release of the receptor hgands within the endosomes.

Ligands Binding Low Density Lipoprotem Receptors

The novel findings of the present inventors demonstrate that the antiangiogemc, or antiprohferative, activity of compounds such as TFPI is mediated by a LDL receptor-like protein expressed on the surface of endothelial cells As shown in Example 2, the antiprohferative activity of

TFPI was reduced m the presence of LDL (an LDLR ligand). In addition, further support for the role of LDLR is provided wherein the inventors demonstrated that antiprohferative activity of TFPI was reduced in the presence of VLDLR antibodies. Ligands that are known to be endocytosed via the LDL receptor family of proteins compπse members of the following categoπes: protemases (e.g., tPA), proteinase inhibitors (e.g., serpins, TFPI), protemase/mhibitor complexes (uPA/PAI, thrombm/antithrombin), hpoprotems (e.g. apohpoprotem B), and matπx proteins (e.g., thrombospondin) Preferred LDLR ligands compnse oc₂-macroglobulm, beta-amyloid precursor protein, proteinase nexin II, proteinase nexin I, pro-uPA, lipoprotem lipase, lactofenn, PAI-1, horse leukocyte elastase inhibitor, protein C inhibitor, Cl-mhibitor, α₂-antιplasmιn, alphal- proteinase inhibitor, alphal-antichymotrypsm, hepaπn cofactor II, tissue- type plasmmogen activator, antithrombm III, tissue factor pathway inhibitor, apohpoprotem B, apohpoprotem J, clusteπn, thrombospondin, and active fragments thereof.

In particular, ligands belonging to the serpm superfamily (senne proteinase inhibitors) having either Kunitz or non-Kunitz domains 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 senne proteases. Their tertiary structure consists of

3 beta sheets (named A, B, and C), several -hehces (designated A through I), and a flexible reactive loop that contains highly vanable residues The reactive site is located in the reactive loop, approximately 30- 40 am o acids from the carboxy-terminal region of the protein, and includes a peptide bond that mimics the normal substrate of the target protease Upon binding to a protease, serpins undergo a profound conformational change in which the active loop becomes inserted into the beta sheet A Serp family hgands compπse tissue factor pathway inhibitor 1 (TFPI-1) and tissue factor pathway inhibitor 2 (TFPI-2), antithrombm III, amyloid protein precursoi (APP), amyloid beta precursor protein, collagen VI, bovine pancreatic trypsm inhibitor (BPTI), α,- protemase inhibitor, 0.,-antichymotrypsin, α ,-antιplasmm, hepann cofactor

II, protein C inhibitor (PCI), proteinase nexm-1 (PN-1), and the plasmmogen activator inhibitors (PAI-1 and PAI-2) One protein representative of protease inhibitors that bind

LDLR is TFPI TFPI is a glycoprotein having a molecular weight of approximately 32 to 45 kilodaltons. TFPI is composed of approximately 276 ammo acids organized in a structure that includes an acidic amino terminus followed by three Kumtz-type protease inhibitor domains, refeπed to as Kunitz- 1, Kunιtz-2, and Kunιtz-3, and a basic carboxyl terminal region.

TFPI, also known to those skilled in the art as hpoprotein- associated coagulation inhibitor, is a protease inhibitor that plays an important role in the regulation of tissue factor-induced blood coagulation.

Generally, TFPI is found in plasma, in platelets, and on endothe um. Typically, at a site of blood vessel injury after the bleeding has stopped, the concentration of TFPI is three times higher than the normal levels found in plasma. Intravascular TFPI exists in several forms, which are known to those skilled in the art. The predominant forms of plasma TFPI have molecular weights of 34 and 41 KDa but other forms with higher molecular weights are also present. TFPI is synthesized in endothelial cells and is exocytosed toward the surface of the cells where it remains bound to hepann sulfate proteoglycans (HSPGs) (see Naπta et al. Journ. Biol. Chemistry 270(42):24800-24804 (1995)).

Other proteins that contain Kunitz type domains and may be used in the compositions of the present invention compπse amyloid precursor protein (APP), collagen VI and bovine pancreatic trypsin inhibitor (BPTI) APP contains Kunitz protease inhibitor domains and exists is at least four different forms that differ m the number of ammo acids ranging from approximately 700-800 residues, (see Niwano et al. J. Lab Chn. Med., 125(2):215-256 (1995)) Collagen VI is found in blood vessel walls, has a tπple-hehcal structure and contains a Kunitz type protease inhibitor domain (see Kehrel, Semin Thromb Hemost. 21(2).123-9 (1995)). Bovine pancreatic trypsin inhibitor (BPTI) is a 58- residue protein with three disulfide bonds that belongs to the Kunitz family of senne proteinase inhibitors (see Moss et al J Gen Physwl 108(6) 473-84 (1996))

In an alternative embodiment, a preferred category of LDLR binding ligands compnses ligand complexes Whereas ligand complexes are internalized by LDL receptor-like proteins, the individual components of the complexes are typically not internalized by themselves. For example, neither plasmmogen activator inhibitor 1 (PAI-1), nor urokmase- type plasmmogen activator (uPA) individually bind to LRP. Upon complexmg with each other however, PAI-1 undergoes a conformational change that allows the uPA/PAI-1 complex to bind to LRP via the PAI-1 component. Such ligand complexes compπse for example protease/serpm complexes such as thrombm/antithrombin or uPA/antithrombm. Additional ligand complexes included, but limited within the scope of the present invention comprise uPA PAI-1, tPA/PAI-1, uPA/PN-1 (proteinase nexin-

1), elastase/ ,proteinase inhibitor, trypsin/α, -proteinase inhibitor, thrombin/heparin cofactor II and thrombin PAI-1, uP A/PCI (protein C inhibitor), uPA/antithrombin III, uPA/Cl -inhibitor, low molecular mass uPA/PAI-1, low molecular mass uP A/PCI, tP A/PCI, thrombin/PCI, thrombin/PN-1. Serine proteinase/serpin complexes are described for example by Kasza et al. Eur. J. Biochem. 248:270-281 (1997).

The LDLR ligands of the present invention may be isolated from body fluids including, but not limited to, serum, urine, and ascites, or may be synthesized by chemical or biological methods, such as cell culture, recombinant gene expression, and peptide synthesis. Recombinant techniques include gene amplification from DNA sources using the polymerase chain reaction (PCR), and gene amplification from RNA sources using reverse transcriptase/PCR. LDLR ligands are extracted from body fluids by known protein extraction methods, particularly the method described by Novotny, W.F., et al, J. Biol. Chem. 264:18832-18837 (1989).

Peptides or Protein Fragments

Peptides or protein fragments comprising LDLR binding ligands can be produced from the proteins described above and tested for antiprohferative or antiangiogenic activity using techniques and methods known to those skilled in the art. For example, full length recombinant TFPI (rTFPI) can be produced using the Baculovirus gene expression system. Full length proteins can be cleaved into individual domains or digested using various methods such as, for example, the method described by Enjyoji et al. (Biochemistry 34:5725-5735 (1995)). In accordance with the method of Enjyoji et al., rTFPI is treated with a digestion enzyme, human neutrophil elastase, and the digest purified using a heparin column.

Human neutrophil elastase cleaves TFPI at Leu^9 into two fragments: one containing Kunitz- 1 and the other containing Kunitz-2 and Kunitz-3. To produce additional fragments, the fragment containing Kunitz-2 and Kunitz-3 (Kunitz-2/Kunitz-3) is preferably treated with a digestion compound, hydroxylamine, according to the method of Balian et al.

(Biochemistry 11:3798-3806 (1972)), and the digest purified using a heparin column. Hydroxylamine cleaves the fragment containing Kunitz-2 and Kunitz-3 into two fragments one containing Kunitz-3 and the other containing the Kunitz-2 domain

Alternatively, fragments are prepared by digesting the entire protein, or large fragments thereof exhibiting anti-prohferative activity, to remove one amino acid at a time Each progiessively shorter fragment is then tested for anti-prohferative activity Similarly, fragments of vaπous lengths may be synthesized and tested for anti-prohferative activity By increasing or decreasing the length of a fragment, one skilled m the art may determine the exact number, identity, and sequence of amino acids within the protein that are required for anti-prohferative activity using routine digestion, synthesis, and screening procedures known to those skilled in the art

Anti-prohferative activity is evaluated in situ by testing the ability of the fragments to inhibit the proliferation of new blood vessel cells, referred to herein as the inhibition of angiogenesis A suitable assay is the chick embryo choπoallantoic membrane (CAM) assay descnbed by Crum et al , Science 230 1375 (1985) and descπbed m U S Patent No 5,001,116 The CAM assay is bπefly descnbed as follows Fertilized chick embryos are removed from their shell on day 3 or 4, and a methylcellulose disc containing the fragment of interest is implanted on the chonoallantoic membrane The embryos are examined 48 hours later and, if a clear avascular zone appears around the methylcellulose disc, the diameter of that zone is measured The larger the diameter of the zone, the greater the anti -angiogenic activity Another suitable assay is the HUVEC assay as descnbed in Example 2

The active fragment is preferably a fragment containing that portion of the ligand that is necessary foi binding LDLR In particulai, ligands belonging to the family of protease inhibitors, having either Kunitz or non-Ku tz domains, are prefeπed Serpm ligands, include but are not limited to, tissue factor pathway inhibitor 1 (TFPI-1), tissue factor pathway inhibitor 2 (TFPI-2), antithrombm III, amyloid protein precursor (APP), amyloid beta precursor protein, collagen VI, bovine pancreatic trypsin inhibitor (BPTI), 0.,-proteinase inhibitor, alphal-antichymotrypsin, alpha2- antiplasmm, hepann cofactor II, protein C inhibitor (PCI), proteinase nexin- 1 (PN-1), and the plasmmogen activator inhibitors (PAI-1 and PAI-

2) As discussed above, one of skill in the art will recognize that, individual substitutions, deletions or additions which alter, add or delete a single ammo acid or a small percentage of ammo acids (typically less than 5%, more typically less than 1%) in an encoded sequence are conservatively modified vanations where the alterations result in the substitution of an ammo acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar am o acids are well known in the art. Accordingly, also included in the present invention are peptides having conservatively modified vanations m compaπson to the claimed peptides, wherein the chemical reactivity of the peptide is not significantly different from that of the claimed peptide.

Formulations

The naturally occurπng or synthetic protein, peptide, or protein fragment, containing all or an active portion of an LDLR ligand can be prepared in a physiologically acceptable formulation, such as m a pharmaceutically acceptable earner, using known techniques For example, the protein, peptide or protein fragment is combined with a pharmaceutically acceptable excipient to form a therapeutic composition Alternatively, the gene for the protein, peptide, or protein fragment, containing all or an active portion of an LDLR ligand, is delivered in a vector for continuous administration using gene therapy techniques The vector may be administered in a vehicle having specificity for a target site, such as a tumor. The composition may be in the form of a solid, liquid or aerosol Examples of solid compositions include pills, creams, and implantable dosage units Pills may be administered orally Therapeutic creams may be administered topically Implantable dosage units may be administered locally, for example, at a tumor site, or may be implanted for systematic release of the therapeutic composition, for example, subcutaneously Examples of liquid compositions include formulations adapted for injection subcutaneously, intravenously, mtra-artenally, and formulations for topical and intraocular administration. Examples of aerosol formulations include inhaler formulations for administration to the lungs

The composition may be administered by standard routes of administration In general, the composition may be administered by topical, oral, rectal, nasal or parenteral (for example, intravenous, subcutaneous, or mtermuscular) routes In addition, the composition may be incorporated into sustained release matπces such as biodegradable polymers, the polymers being implanted in the vicinity of where delivery is desired, for example, at the site of a tumor The method includes administration of a single dose, administration of repeated doses at predetermined time intervals, and sustained administration for a predetermined peπod of time

A sustained release matπx, as used herein, is a matπx made of matenals, usually polymers which are degradable by enzymatic or acid/base hydrolysis or by dissolution Once inserted into the body, the matnx is acted upon by enzymes and body fluids The sustained release matπx desirably is chosen by biocompatible matenals such as hposomes, polylactides (polylactide acid), polyglyco de (polymer of glycohc acid), polylactide co-glycohde (copolymers of lactic acid and glycohc acid), polyanhydndes, poly(ortho)esters, polypeptides, hyaluronic acid, collagen, chondroitin sulfate, carboxyhc acids, fatty acids, phospholipids, polysacchandes, nucleic acids, polyammo acids, ammo acids such phenylalanme, tyrosme, lsoleucme, polynucleotides, polyvmyl propylene, polyvmylpyrrohdone and sihcone A preferred biodegradable matnx is a matnx of one of either polylactide, polyglycohde, or polylactide co- glycohde (co-polymers of lactic acid and glycohc acid)

The dosage of the composition will depend on the condition being treated, the particular composition used, and other clinical factors such as weight and condition of the patient, and the route of administration Further, the term "effective amount" refers to the amount of the composition which, when administered to a human or animal, inhibits undesirable cell proliferation, particularly endothelial cell proliferation, causing a reduction in cancer or inhibition in the spread and proliferation of cancer The effective amount is readily determined by one of skill in the art following routine procedures

For example, antiprohferative compositions of the present invention may be administered parenterally or orally in a range of approximately 1 0 μg to 1 0 mg per patient, though this range is not intended to be limiting The actual amount of antiprohferative composition required to elicit an appropnate response will vary for each individual patient depending on the potency of the composition administered, the condition being treated and on the response of the individual Consequently, the specific amount administered to an individual will be determined by routine expeπmentation and based upon the training and expenence of one skilled in the art

The composition may be administered in combination with other compositions and procedures for the treatment of diseases For example, unwanted cell proliferation may be treated conventionally with surgery, radiation or chemotherapy in combination with the administration of the composition, and additional doses of the composition may be subsequently administered to the patient to stabilize and inhibit the growth of any residual unwanted cell proliferation

LDL Receptor Ligand Antibodies

The present invention further compnses LDLR ligand antibodies that may be used for diagnostic as well as therapeutic purposes The antibodies provided herein are monoclonal or polyclonal antibodies having binding specificity for LDLR ligands The preferred antibodies are monoclonal antibodies, due to their higher specificity for the ligands The antibodies exhibit minimal or no crossreactivity with other proteins or peptides Preferably, the antibodies are specific for ligands compπsing protemases, proteinase inhibitors, serpins, prote ase/inhibitor complexes, thrombin/antithrombm, hpoprotems, and matπx proteins

Monoclonal antibodies are prepared by immunizing an animal, such as a mouse or rabbit, with a whole or immunogenic portion of an LDLR ligand, such as antithrombm III Spleen cells are harvested from the immunized animals and hybπdomas generated by fusing sensitized spleen cells with a myeloma cell line, such as munne SP2/O myeloma cells

(ATCC, Manassas, VA) The cells are induced to fuse by the addition of polyethylene glycol Hybndomas are chemically selected by plating the cells in a selection medium containing hypoxanthine, aminopteπn and thymidme (HAT) Hybndomas are subsequently screened for the ability to produce monoclonal antibodies against LDLR ligands Hybπdomas producing antibodies that bind to the LDLR ligands are cloned, expanded and stored frozen for future production The preferred hybπdoma produces a monoclonal antibody having the IgG isotype, more preferably the IgGl isotype

The polyclonal antibodies are prepared by immunizing animals, such as mice or rabbits with LDLR ligands as descπbed above Blood sera is subsequently collected from the animals, and antibodies in the sera screened for binding reactivity against the LDLR ligands, preferably the antigens that are reactive with the monoclonal antibody descπbed above.

Either the monoclonal antibodies or the polyclonal antibodies, or both may be labeled directly with a detectable label for identification and quantitation of LDLR ligands m a biological or environmental sample as descπbed below. Labels for use in immunoassays are generally known to those skilled in the art and include enzymes, radioisotopes, and fluorescent, luminescent and chromogemc substances including colored particles, such as colloidal gold and latex beads. The antibodies may also be bound to a solid phase to facilitate separation of antibody-antigen complexes from non-reacted components in an immunoassay. Exemplary solid phase substances include, but are not limited to, microtiter plates, test tubes, magnetic, plastic or glass beads and slides. Methods for coupling antibodies to solid phases are well known to those skilled m the art.

Alternatively, the antibodies may be labeled indirectly by reaction with labeled substances that have an affinity for immunoglobulin, such as protein A or G or second antibodies. The antibodies may be conjugated with a second substance and detected with a labeled third substance having an affinity for the second substance conjugated to the antibody. For example, the antibodies may be conjugated to biot and the antibody-biot conjugate detected using labeled avidm or streptavidm Similarly, the antibodies may be conjugated to a hapten and the antibody- hapten conjugate detected using labeled anti-hapten antibody These and other methods of labeling antibodies and assay conjugates are well known to those skilled in the art

Sensitive immunoassays employing one or more of the antibodies descπbed above are provided by the present invention The immunoassays are useful for detecting the presence or amount of LDLR ligands in a vanety of samples, particularly biological samples, such as human or animal biological fluids or The samples may be obtained from any source in which the LDLR ligands may exist. For example, the sample may include, but is not limited to, blood, saliva, semen, tears, and uπne. The antibody-antigen complexes formed the immunoassays of the present invention are detected using immunoassay methods known to those skilled in the art, including sandwich immunoassays and competitive immunoassays. The antibody-antigen complexes are exposed to antibodies similar to those used to capture the antigen, but which have been labeled with a detectable label. Suitable labels include: chemiluminescent labels, such as horseradish peroxidase; electrochemiluminescent labels, such as ruthenium and aequorin; bioluminescent labels, such as luciferase; fluorescent labels such as FITC; and enzymatic labels such as alkaline phosphatase, β-galactosidase, and horseradish peroxidase.

The labeled complex is then detected using a detection technique or instrument specific for detection of the label employed. Soluble antigen or antigens may also be incubated with magnetic beads coated with non-specific antibodies in an identical assay format to determine the background values of samples 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 mediated by abnormal or undesirable cellular proliferation, particularly abnormal or undesirable endothelial cell proliferation, including, but not limited to, hemangioma, solid tumors, leukemia, metastasis, telangiectasia psoriasis scleroderma, pyogenic granuloma, myocardial angiogenesis, plaque neovascularization, coronary collaterals, ischemic limb angiogenesis, corneal diseases, rubeosis, neovascular glaucoma, diabetic retinopathy, retrolental fibroplasia, arthritis, diabetic neovascularization, macular degeneration, wound healing, peptic ulcer, fractures, keloids, vasculogenesis, hematopoiesis, ovulation, menstruation, and placentation.

The method and composition are particularly useful for treating angiogenesis-related disorders and diseases by inhibiting angiogenesis.

The methods and compositions described herein are particularly useful for treating cancer, arthritis, macular degeneration, and diabetic retinopathy. Administration of the compositions to a human or animal having prevascularized metastasized tumors is useful for preventing the growth or expansion of such tumors.

The compositions and methods are further illustrated by the following non-limiting examples, which are not to be construed in any way as imposing limitations upon the scope thereof. On the contrary, it is to be clearly understood that resort may be had to various other embodiments, modifications, and equivalents thereof which, after reading the description herein, may suggest themselves to those skilled in the art without departing from the spiπt of the present invention and/or the scope of the appended claims.

EXAMPLE 1 Human Umbilical Vascular Endothelial Cell Assay

Human umbilical vascular endothelial cells (HUVECs) and their media (EGM and EBM) were purchased from Clonetics (San Diego, CA). HUVECs were routinely cultured to confluency in EGM. The cells were trypsmized and plated in a 96-well plate at 5,000 cells per lOOμl EBM supplemented with 2% serum and antibiotics. The cells were allowed to adhere to the plate for at least 2 hrs Then, bFGF at 10 ng/ml and vanous concentrations of an antiangiogemc agent were added to the wells. The cells were cultured for 48 hrs at 37°C m a 5% CO, atmosphere. Cell proliferation was determined using a undme incorporation method (Boennger Mannheim Corporation, Indianapolis, IN).

EXAMPLE 2

Endothelial Cell Proliferation Assay in the

Presence of TFPI and LDL Proliferation assays familiar to those skilled m the art using human umbilical vein endothelial cells HUVECs were conducted using tissue factor pathway inhibitor (TFPI) and low density hpoprotems (LDL) (LDL is a LDLR ligand known to induce growth of endothelial cells)

Materials and Methods

The matenals for this expenment included HUVECs and media for their proliferation, Endothelial Cell Basal Medium (EBM) and Endothelial Cell Growth Medium (EGM), (Clonetics, San Diego, CA) Also used was full length TFPI (Amencan Diagnostica Inc , Greenwich, CT) In addition, a cell proliferation ELISA BrdU (Boehnnger Mannheim

Corporation, Indianapolis, IN), bFGF (R&D, Minneapolis, MN) and LDL (Perlmmune, Inc. Rockville MD).

The proliferation assay involved the routine cultuπng of HUVECs to confluency in EGM media. The cells were trypsmized and plated in a 96-well plate at 5000 cells per well per 100 μL EBM media.

The cells were allowed to adhere to the plate for at least two hours. Next, bFGF at 10 ng/ml and full length TFPI at vanous concentrations was added to the wells The cells were cultured for 48 hours after which cell proliferation was determined using a standard undme incorporation method

Results

In the absence of LDL, 150 nM TFPI inhibited bFGF- mduced proliferation of HUVECs by 50% Under the same expeπmental conditions but in the presence of 100 μg/ml LDL, the same concentration of TFPI inhibited proliferation of HUVECs by only 30% This partial reduction in activity of TFPI demonstrates that a LDLR-like protein mediates the antiprohferative effect of TFPI Though not wishing to be bound by the following theory, engagement of the receptor by LDL results in partial internalization of the receptor which prevents its hgation with TFPI and the induction of an antimitotic signal

EXAMPLE 3 Endothelial Cell Proliferation Assay in the Presence of TFPI and VLDLR Antibodies Previous expenments conducted by the inventors demonstrated that 550 nM TFPI inhibited bFGF-mduced proliferation of HUVECs by 90%

When HUVECs were pre-incubated in the presence of lμM VLDLR antibodies (Amencan Red Cross (Rockville, MD)) for an hour at 37°C, the same concentration of TFPI inhibited bFGF-induced proliferation of HUVECs by 50% This partial reduction m the TFPI activity demonstrates that, at least partially, VLDLR mediates the antiprohferative property of

TFPI

EXAMPLE 4 Proliferation Assa\ in the Presence of Receptoi Associated Protein

Proliferation assays as descnbed in Examples 1 and 2 were conducted to assess the effect of receptor associated protein (RAP) on proliferation of endothelial cells RAP is a 39 Kd protein that binds with high affinity to the members of the LDL receptor family of proteins and antagonizes their ligand binding properties

TFPI at 275nM concentration inhibited bFGF-induced proliferation of HUVECs by 100% Under the same conditions and in the presence of 100 nM RAP (Ameπcan Red Cross (Rockville, MD)), the same concentration of TFPI inhibited bFGF-dnven replication of HUVECs only by approximately 50%. Again, partial neutralization of TFPI's activity by RAP, demonstrates that a LDLR-hke protein is involved in the transduction of TFPI's antimitotic signal. The results of this expenment are provided graphically in Figure 1.

EXAMPLE 5 Endothelial Cell Migration Assay in the Presence of TFPI and RAP

Proliferation assays as descnbed in Examples 1 and 2 were conducted to assess the effect of RAP on proliferation of endothelial cells.

RAP is a 39 Kd protein that binds with high affinity to the members of the

LDL receptor family of proteins and antagonizes their ligand binding properties.

TFPI inhibits bFGF-mduced migration of HUVECs in a dose- dependent manner Presence of RAP in the culture media at 500 nM concentration neutralized TFPI's antimigratory activity The results of this expenment are provided graphically in Figure 2.

EXAMPLE 6 Endothelial Cell Proliferation Assay in the Presence of Clustenn Clustenn or apohpoprotem J is a 80 Kd multifunctional protein involved m the terminal complement pathway Clustenn is synthesized by endothelial cells and internalized by gp330/megahn receptor and LRP (Zlokovic et al Proc Natl. Acad Sci. USA 1996 93(9), 4229-4234, and Morales et al 1996 Biol Reprod 55(3), 676-683)

Proliferation assays conducted as descnbed above in Examples 1 and 2, demonstrate that clustenn inhibits bFGF-mduced endothelial cell proliferation in a dose-dependent manner. Results of this expenment are provided graphically in Figure 3.

It should be understood, of course, that the foregoing relates only to preferred embodiments of the present invention and that numerous modifications or alterations may be made therein without departing from the spiπt and the scope of the invention as set forth in the appended claims.

Claims

CLAIMSWe claim:

1 . A method of treating a human or animal having undesirable cell proliferation compnsmg, admimstenng to the human or animal an effective amount of a composition compnsmg an isolated protein or peptide, or antiprohferative fragment thereof, wherein the protein or peptide is capable of binding to a low density lipoprotem receptor, and wherein the effective amount is sufficient to inhibit the undesirable cell proliferation.

2. The method of Claim 1, wherein the low density lipoprotem receptor compnses low density lipoprotem, low density lipoprotem receptor-related protein, very low density lipoprotem receptor, gp330/megahn and active fragments thereof.

3 The method of Claim 1, wherein isolated protein or peptide compnses a protein selected from the group consisting of protemases, proteinase inhibitors, serpins, proteinase/mhibitor complexes, thrombm/antithrombin, hpoprotems, matnx proteins and active fragments thereof

4 The method of Claim 1 , wherein isolated protein or peptide is selected from the group consisting of alpha2-macroglobulm, beta-amyloid precursor protein, proteinase nexm I, proteinase nexm II, pro-uPA, lipoprotem lipase, lactofeπn, PAI-1, horse leukocyte elastase inhibitor, protein C inhibitor, Cl-inhibitor, alpha2-antιplasmm, alphal- proteinase inhibitor, alphal-antichymotrypsin, hepann cofactor II, tissue- type plasmmogen activator, antithrombm III, tissue factor pathway inhibitor, apohpoprotem B, apohpoprotem J, clustenn, thrombospondin, tissue factor pathway inhibitor 1, tissue factor pathway inhibitor 2, amyloid protein precursor, amyloid beta precursor protein, collagen VI, bovine pancreatic trypsin inhibitor, and active fragments thereof

5. The method of Claim 3, wherein the protemase/inhibitor complexes compnse urokinase-type plasmmogen activator/plasmmogen activator inhibitor 1 complex, or thrombin antithrombm complex.

6. The method of Claim 1, wherein the wherein the low density lipoprotem receptor compnses low density lipoprotem receptor-related protein and wherein the isolated protein or peptide, or antiprohferative fragment thereof compnses alpha2-macroglobuhn.

7. The method of Claim 1, wherein the wherein the low density lipoprotem receptor compnses low density lipoprotem receptor-related protein and wherein the isolated protein or peptide, or antiprohferative fragment thereof compnses antithrombm III.

8 The method of Claim 1, wherein the composition further compnses a pharmaceutically acceptable excipient, earner or sustained-release matnx.

9 The method of Claim 1, wherein the undesirable cell proliferation is undesirable endothelial cell proliferation.

10 The method of Claim 9 wherein the inhibition of endothelial cell proliferation inhibits neovasculanzation

1 1 The method of Claim 1, wherein the undesirable cell proliferation is an angiogenesis-related disease

12 The method of Claim 4, wherein the angiogemc- related disease is a disease selected from the group consisting of cancer, arthritis, macular degeneration, and diabetic retinopathy

13. A method of treating undesired angiogenesis in a human or animal compπsing the steps of admmisteπng to the human or animal with the undesired angiogenesis a composition compπsing an effective amount of an angiogenesis-mhibiting compound compπsing protemases, proteinase inhibitors, serpins, protemase/inhibitor complexes, thrombin/antithrombm, hpoprotems, and matnx proteins.

14. The method of Claim 13, wherein the angiogenesis- inhibiting compound is selected from the group consisting of alpha2- macroglobulin, beta-amyloid precursor protein, proteinase nexin I, proteinase nexin II, pro-uPA, lipoprotein lipase, lactoferin, PAI-1, horse leukocyte elastase inhibitor, protein C inhibitor, Cl -inhibitor, alpha2- antiplasmin, alpha 1 -proteinase inhibitor, alphal-antichymotrypsin, heparin cofactor II, tissue-type plasmmogen activator, antithrombin III, tissue factor pathway inhibitor, apolipoprotein B, apolipoprotein J, clusterin, thrombospondin, tissue factor pathway inhibitor 1, tissue factor pathway inhibitor 2, amyloid protein precursor, amyloid beta precursor protein, collagen VI, and bovine pancreatic trypsin inhibitor and active fragments thereof.

15. The method of Claim 14, further comprising a pharmaceutically acceptable excipient, carrier or sustained-release matrix.

16. The method of Claim 14, wherein the undesired angiogenesis is related to an angiogenic-related disease comprising cancer, arthritis, macular degeneration, and diabetic retinopathy.

17. A composition for treating undesired angiogenesis in a human or animal comprising an effective amount of an angiogenesis- inhibiting compound wherein the compound comprises ligands that bind low density lipoprotein receptor.

18. The composition 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.

19. The composition of Claim 17, wherein the ligands comprise proteinases, proteinase inhibitors, serpins, proteinase/inhibitor complexes, thrombin/antithrombin, lipoproteins, matrix proteins and active fragments thereof.

20. The composition of Claim 17, wherein the ligand is selected from the group consisting of alpha2-macroglobulin, beta-amyloid precursor protein, proteinase nexin I, proteinase nexin II, pro-uPA, lipoprotein lipase, lactoferin, PAI-1, horse leukocyte elastase inhibitor, protein C inhibitor, Cl-inhibitor, alpha2-antiplasmin, alpha 1 -proteinase inhibitor, alpha 1-antichymotrypsin, heparin cofactor II, tissue-type plasmmogen activator, antithrombin III, tissue factor pathway inhibitor, apolipoprotein B, apolipoprotein J, clusterin, thrombospondin, tissue factor pathway inhibitor 1, tissue factor pathway inhibitor 2, amyloid protein precursor, amyloid beta precursor protein, collagen VI, bovine pancreatic trypsin inhibitor and active fragments thereof.