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WO2000054801A1 - Compositions et procedes d'utilisation de ligands recepteurs du type ldl pour le traitement du cancer et de maladies angiogeniques - Google Patents

Compositions et procedes d'utilisation de ligands recepteurs du type ldl pour le traitement du cancer et de maladies angiogeniques Download PDF

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Publication number
WO2000054801A1
WO2000054801A1 PCT/US2000/007154 US0007154W WO0054801A1 WO 2000054801 A1 WO2000054801 A1 WO 2000054801A1 US 0007154 W US0007154 W US 0007154W WO 0054801 A1 WO0054801 A1 WO 0054801A1
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Prior art keywords
inhibitor
protein
low density
proteinase
cell proliferation
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PCT/US2000/007154
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English (en)
Inventor
Adonia E. Papathanassiu
Shawn J. Green
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Entremed, Inc.
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Publication date
Application filed by Entremed, Inc. filed Critical Entremed, Inc.
Priority to CA002367247A priority Critical patent/CA2367247A1/fr
Priority to EP00925874A priority patent/EP1161258A1/fr
Priority to AU44499/00A priority patent/AU4449900A/en
Priority to JP2000604873A priority patent/JP2002539174A/ja
Publication of WO2000054801A1 publication Critical patent/WO2000054801A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/55—Protease inhibitors
    • A61K38/57—Protease inhibitors from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • 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.
  • 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 va ⁇ ety 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
  • Cancer is characte ⁇ zed 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 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, endomet ⁇ um and placenta.
  • 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”.
  • 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.
  • Endothelial cells and pe ⁇ cytes 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
  • 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.
  • cellular proliferation particularly endothelial cell proliferation, and most particularly angiogenesis
  • 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
  • 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 capilla ⁇ es in angiogenic processes, such as wound healing and reproduction
  • the compositions and methods for inhibiting cellular proliferation should preferably be non-toxic and produce few side effects.
  • compositions and methods are provided that are effective m inhibiting abnormal or undesirable cell proliferation, particularly endothelial cell proliferation and angiogenesis related to neovascula ⁇ zation and tumor growth.
  • the compositions comp ⁇ se 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.
  • LDLR low density lipoprotem receptor
  • Representative gands useful for the present invention comp ⁇ se proteins or peptides and fragments thereof belonging to the following catego ⁇ es: 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 cluste ⁇ n
  • mat ⁇ x proteins i.e. thrombospondin
  • proteins or peptides comp ⁇ sing 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, lactofe ⁇ n, PAI-1, horse leukocyte elastase inhibitor, protein C inhibitor, Cl -inhibitor, alpha2 ⁇ ant ⁇ plasm ⁇ n, alpha 1 -proteinase inhibitor, and alphal-antichymotryps , hepa ⁇ n cofactor II.
  • APP beta-amyloid precursor protein
  • PN2 proteinase nexm II
  • PN1 proteinase nexm I
  • pro-uPA proteinase nexm II
  • PN1 proteinase nexm I
  • pro-uPA proteinase nexm III
  • Prefe ⁇ ed LDLR hgands comp ⁇ se proteins belonging to the protein family of serpins (se ⁇ ne protease inhibitors) those containing
  • the protein, peptide or fragment thereof contains all or an active portion of the above identified proteins
  • active portion means a portion of a protein that inhibits cancer, preferably by inhibiting abnormal or undesirable cell proliferation.
  • homologs, peptides, or protein fragments, or combinations thereof of the above-identified proteins, that inhibit abnormal or undesirable cell proliferation are also included in the present invention.
  • the protein, peptide, or protein fragment is a protein, peptide or protein fragment of an LDLR binding ligand.
  • the methods and compositions desc ⁇ bed herein are useful for inhibiting tumor growth and metastasis by blocking tumor vascula ⁇ zation.
  • the methods provided herein for treating diseases and processes mediated by undesired and uncontrolled cell proliferation, such as cancer involve administe ⁇ ng to a human or animal the composition desc ⁇ bed 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 prevascula ⁇ zed metastasized tumors is useful for preventing the growth or expansion of such tumors
  • Yet another object of the present invention is to provide methods and compositions comp ⁇ sing 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 comp ⁇ sing low density lipoprotem receptor hgands
  • Yet another object of the present invention is to provide antiangiogemc compositions comp ⁇ smg low density lipoprotem receptor hgands, wherein the hgands comp ⁇ se protemases, proteinase inhibitors, hpoprotems and matnx proteins optionally combined with pharmaceutically acceptable earners that may be administered intramuscularly, intravenously, transdermally, orally, or subcutaneously
  • 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 neovascula ⁇ zation, arterio venous malformations, corneal diseases, rubeosis, neovascular glaucom
  • 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 va ⁇ ous 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 cluste ⁇ n on bFGF-induced endothelial cell proliferation
  • compositions and methods for the treatment of diseases and processes that are mediated by, or associated with, abnormal or undesirable cellular proliferation comprise isolated naturally occur ⁇ ng 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)
  • LDLR low density lipoprotem receptors
  • the compositions may optionally comp ⁇ se 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
  • the LDLR ligand compositions of the present invention comp ⁇ se proteins belonging to the catego ⁇ es 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)
  • uPA/PAI urokmase-type plasmmogen activator/plasminogen activator inhibitor 1
  • thrombm/antithrombin thrombm/antithrombin
  • hpoprotems i.e. apohpoprotem B
  • mat ⁇ x proteins i.e. thrombospondin
  • 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, lactofe ⁇ n, PAI-1, horse leukocyte elastase inhibitor, protein C inhibitor, Cl -inhibitor, alpha2- antiplasmin, alpha 1 -proteinase inhibitor, alphal-antichymotrypsm, hepa ⁇ n cofactor II, and antithrombm III.
  • APP beta-amyloid precursor protein
  • PN2 proteinase nexm II
  • PN1 proteinase nexm I
  • pro-uPA pro-uPA
  • lipoprotem lipase lactofe ⁇ n
  • PAI-1 horse le
  • compositions of the present invention may be optionally combined with a pharmaceutical earner.
  • a pharmaceutical earner may be optionally combined with a pharmaceutical earner.
  • “earner” as used herein comp ⁇ ses 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
  • KLH keyhole limpet hemocyamn
  • BSA bovme serum albumin
  • the low density lipoprotem receptor ligand compositions of the present invention can further comp ⁇ se 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 ⁇ -(l,4) linked acetylated mannan ("Acemannan”), TlTERMAX® (polyoxyethylene- polyoxypropylene cop
  • 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.
  • compositions desc ⁇ bed 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.
  • 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 (.
  • the carboxy terminal has a free carboxyl group
  • the term "ammo terminus” 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
  • carboxy terminus 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
  • 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
  • that amino acid is positioned closer to the carboxy terminal of the peptide than the preceding amino acid
  • residue is used herein to refer to an ammo acid
  • ammo acid may be a naturally occur ⁇ ng 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).
  • an amide bond mimetic includes peptide backbone modifications well known to those skilled in the art.
  • 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 pu ⁇ ty 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 pu ⁇ fication utilized.
  • 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 desc ⁇ bed herein
  • Techniques for solid phase synthesis are known to those skilled m the art
  • 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
  • recombinant peptides can be pu ⁇ fied 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 prefe ⁇ ed, and 80 to 95% or greater homogeneity are most preferred for use as therapeutic agents
  • 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
  • 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
  • suitable biologically active compounds include enzymes, antibodies, antigens and proteins
  • body fluid includes, but is not limited to, saliva, gmgival secretions, cerebrospinal fluid, gastrointestinal fluid, mucous, urogemtal secretions, synovial fluid, blood, serum, plasma, u ⁇ ne, 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 lipoprotem receptor includes a family of proteins comp ⁇ sing low density lipoprotem (LDL), low density lipoprotem receptor-related protein (LRP), very low density lipoprotem receptor (VLDLR), ⁇ 2 macroglobuhn receptor/low - density- poprotein-receptor-related protein ( 2 MR LRP), gp330/megahn apohpoprotem E receptor 2 (apoER2) and receptor LR8B.
  • LDL-receptor family of proteins is characte ⁇ zed by transmembrane proteins comp ⁇ sing approximatey four members: LDL, LRP, VLDLR, and gp330/megalm (see St ⁇ ckland et al. FASEB 9:890-898
  • LDL-receptor family exhibit similar structural patterns and perform related functions such as the mediation of endocytosis and cellular degradation of a diverse va ⁇ ety 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 clath ⁇ n-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.
  • TFPI was reduced m the presence of LDL (an LDLR ligand).
  • LDL an LDLR ligand
  • LDLR ligand-like low-density lipoprotein
  • Ligands that are known to be endocytosed via the LDL receptor family of proteins comp ⁇ se members of the following catego ⁇ es: protemases (e.g., tPA), proteinase inhibitors (e.g., serpins, TFPI), protemase/mhibitor complexes (uPA/PAI, thrombm/antithrombin), hpoprotems (e.g.
  • LDLR ligands compnse oc 2 -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, ⁇ 2 -ant ⁇ plasm ⁇ n, alphal- proteinase inhibitor, alphal-antichymotrypsm, hepa ⁇ n cofactor II, tissue- type plasmmogen activator, antithrombm III, tissue factor pathway inhibitor, apohpoprotem B, apohpoprotem J, cluste ⁇ n, thrombospondin, and active fragments thereof.
  • LDLR ligands compnse oc 2 -macroglobulm, beta-amyloid precursor protein, proteinase nexin II, proteinase nexin I, pro-u
  • Serpins are a family of structurally similar proteins known to irreversibly inhibit a wide range of senne proteases. Their tertiary structure consists of
  • beta sheets named A, B, and C
  • several -hehces designated A through I
  • 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
  • serpins Upon binding to a protease, serpins undergo a profound conformational change in which the active loop becomes inserted into the beta sheet
  • PCI protein C inhibitor
  • PN-1 proteinase nexm-1
  • PAI-1 and PAI-2 proteinase nexm-1
  • 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, refe ⁇ ed to as Kunitz- 1, Kun ⁇ tz-2, and Kun ⁇ tz-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.
  • 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 Na ⁇ ta et al. Journ. Biol. Chemistry 270(42):24800-24804 (1995)).
  • HSPGs hepann sulfate proteoglycans
  • APP amyloid precursor protein
  • collagen VI and bovine pancreatic trypsin inhibitor (BPTI)
  • BPTI bovine pancreatic trypsin inhibitor
  • 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 t ⁇ ple-hehcal structure and contains a Kunitz type protease inhibitor domain (see Kehrel, Semin Thromb Hemost. 21(2).123-9 (1995)).
  • Bovine pancreatic trypsin inhibitor 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))
  • 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.
  • PAI-1 plasmmogen activator inhibitor 1
  • uPA urokmase- type plasmmogen activator
  • Such ligand complexes comp ⁇ se 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-
  • 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 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.
  • full length recombinant TFPI rTFPI
  • 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)).
  • Enjyoji et al. Biochemistry 34:5725-5735 (1995)
  • 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.
  • 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.
  • 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 va ⁇ ous 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
  • a suitable assay is the chick embryo cho ⁇ oallantoic membrane (CAM) assay descnbed by Crum et al , Science 230 1375 (1985) and desc ⁇ bed m U S Patent No 5,001,116
  • the CAM assay is b ⁇ efly 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
  • ligands belonging to the family of protease inhibitors having either Kunitz or non-Ku tz domains, are prefe ⁇ ed 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-
  • the naturally occur ⁇ ng 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
  • a physiologically acceptable formulation such as m a pharmaceutically acceptable earner
  • the protein, peptide or protein fragment is combined with a pharmaceutically acceptable excipient to form a therapeutic composition
  • 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.
  • compositions may be in the form of a solid, liquid or aerosol
  • 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
  • liquid compositions include formulations adapted for injection subcutaneously, intravenously, mtra-artally, and formulations for topical and intraocular administration.
  • aerosol formulations include inhaler formulations for administration to the lungs
  • composition may be administered by standard routes of administration
  • the composition may be administered by topical, oral, rectal, nasal or parenteral (for example, intravenous, subcutaneous, or mtermuscular) routes
  • the composition may be incorporated into sustained release mat ⁇ ces 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 pe ⁇ od of time
  • a sustained release mat ⁇ x is a mat ⁇ x 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 mat ⁇ x 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
  • 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
  • antiprohferative compositions of the present invention may be administered parenterally or orally in a range of approximately 1 0 ⁇ g 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 expe ⁇ mentation and based upon the training and expenence of one skilled in the art
  • compositions may be administered in combination with other compositions and procedures for the treatment of diseases
  • 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
  • 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
  • the antibodies are specific for ligands comp ⁇ sing protemases, proteinase inhibitors, serpins, prote ase/inhibitor complexes, thrombin/antithrombm, hpoprotems, and mat ⁇ x 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 hyb ⁇ domas generated by fusing sensitized spleen cells with a myeloma cell line, such as munne SP2/O myeloma cells
  • Hybndomas are chemically selected by plating the cells in a selection medium containing hypoxanthine, aminopte ⁇ n and thymidme (HAT)
  • Hybndomas are subsequently screened for the ability to produce monoclonal antibodies against LDLR ligands
  • Hyb ⁇ domas producing antibodies that bind to the LDLR ligands are cloned, expanded and stored frozen for future production
  • the preferred hyb ⁇ doma 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 desc ⁇ bed 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 desc ⁇ bed 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 desc ⁇ bed 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.
  • 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.
  • the antibodies may be conjugated to biot and the antibody-biot conjugate detected using labeled avidm or streptavidm
  • the antibodies may be conjugated to a hapten and the antibody- hapten conjugate detected using labeled anti-hapten antibody
  • Sensitive immunoassays employing one or more of the antibodies desc ⁇ bed 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.
  • the sample may include, but is not limited to, blood, saliva, semen, tears, and u ⁇ ne.
  • 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, ⁇ -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.
  • 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.
  • abnormal or undesirable endothelial cell proliferation including, but not limited to, hemangioma, solid tumors, leukemia, metastasis, telangiectasia psoriasis scleroderma
  • the method and composition are particularly useful for treating angiogenesis-related disorders and diseases by inhibiting angiogenesis.
  • 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.
  • 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 spi ⁇ t of the present invention and/or the scope of the appended claims.
  • HUVECs Human umbilical vascular endothelial cells
  • EGM Human umbilical vascular endothelial cells
  • EBM Human umbilical vascular endothelial cells
  • EGM Human umbilical vascular endothelial cells
  • EBM Human umbilical vascular endothelial cells
  • EGM Human umbilical vascular endothelial cells
  • EBM Human umbilical vascular endothelial cells
  • TFPI tissue factor pathway inhibitor
  • LDL low density hpoprotems
  • 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
  • the proliferation assay involved the routine cultu ⁇ ng 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 ⁇ L 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
  • RAP receptor associated protein
  • TFPI at 275nM concentration inhibited bFGF-induced proliferation of HUVECs by 100%
  • RAP Ame ⁇ can Red Cross (Rockville, MD)
  • the same concentration of TFPI inhibited bFGF-dnven replication of HUVECs only by approximately 50%.
  • 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.
  • 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
  • Figure 2 The results of this expenment are provided graphically in Figure 2.

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Abstract

L'invention concerne des compositions et des procédés efficaces pour l'inhibition de la prolifération de cellules anormales ou indésirables, en particulier de cellules endothéliales et d'angiogénèse en rapport avec une néoformation de vaisseaux sanguins et un développement de tumeurs. Les compositions comprennent des protéines, peptides, ou fragments de protéines synthétiques ou se présentant à l'état naturel, capables de lier des récepteurs de lipoprotéine faible densité (ou du type lipoprotéine faible densité). Les compositions peuvent être administrées par l'intermédiaire d'un support pharmaceutiquement acceptable. Les procédés concernent l'administration des compositions précitées à un être humain ou à un animal, en un dosage suffisant pour inhiber la prolifération des cellules, en particulier, des cellules endothéliales. Ces procédés sont utilisés pour le traitement de maladies et de processus, tels que le cancer, à médiation par prolifération cellulaire non désirée et non contrôlée, en particulier par inhibition d'angiogénèse. L'administration des compositions de l'invention à un être humain ou à un animal présentant des tumeurs métastasées prévascularisées est utilisée pour prévenir la croissance ou l'expansion de telles tumeurs.
PCT/US2000/007154 1999-03-17 2000-03-17 Compositions et procedes d'utilisation de ligands recepteurs du type ldl pour le traitement du cancer et de maladies angiogeniques WO2000054801A1 (fr)

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CA002367247A CA2367247A1 (fr) 1999-03-17 2000-03-17 Compositions et procedes d'utilisation de ligands recepteurs du type ldl pour le traitement du cancer et de maladies angiogeniques
EP00925874A EP1161258A1 (fr) 1999-03-17 2000-03-17 Compositions et procedes d'utilisation de ligands recepteurs du type ldl pour le traitement du cancer et de maladies angiogeniques
AU44499/00A AU4449900A (en) 1999-03-17 2000-03-17 Compositions and methods of use of ldl-like receptor ligands for the treatment of cancer and angiogenic-based disease
JP2000604873A JP2002539174A (ja) 1999-03-17 2000-03-17 癌および血管新生関連の疾病の治療のためのｌｄｌ様受容体リガンドを使用する組成物および方法

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EP1075840A2 (fr) * 1999-08-13 2001-02-14 Aventis Behring GmbH Utilisations thérapeutiques et prophylactiques de l'antithrombine III
WO2001027079A2 (fr) * 1999-10-14 2001-04-19 Entremed, Inc. Compositions et methodes d'utilisation de ligands liant des elements de la voie de coagulation sanguine pour le traitement du cancer et de maladies induites par le processus d'angiogenese
EP1224941A2 (fr) * 2001-01-17 2002-07-24 Aventis Behring GmbH Utilisation de l'antithrombine iii pour des maladies provoquees par l'angiogenese
WO2003015615A2 (fr) 2001-08-15 2003-02-27 Brown University Research Foundation Traitement de dystrophies musculaires et de troubles connexes
WO2003039575A2 (fr) * 2001-11-09 2003-05-15 Neuronova Ab Role fonctionnel et utilisation therapeutique potentielle de proteines s, gas6 et reelin en relation avec des cellules souches neuronales adultes
WO2004050125A1 (fr) * 2002-12-03 2004-06-17 Koken Co.,Ltd. Inhibiteur de proliferation et/ou d'infiltration de cellules tumorales
US6984389B2 (en) 2002-04-25 2006-01-10 University Of Connecticut Health Center Using heat shock proteins to improve the therapeutic benefit of a non-vaccine treatment modality
US7179462B2 (en) 2000-06-02 2007-02-20 University Of Connecticut Health Center α (2) macroglobulin receptor as a heat shock protein receptor and uses thereof
US7186515B1 (en) 2000-06-02 2007-03-06 University Of Connecticut Health Center Alpha(2) macroglobulin receptor as a heat shock protein receptor and uses thereof
WO2007097961A1 (fr) * 2006-02-16 2007-08-30 Massachusetts Eye & Ear Infirmary Utilisation d'inhibiteurs de l'azurocidine dans la prévention et le traitement d'une fuite vasculaire oculaire
US7329642B2 (en) 2002-05-17 2008-02-12 Board Of Regents, University Of Texas System β-2-glycoprotein is an inhibitor of angiogenesis
US7449557B2 (en) 2000-06-02 2008-11-11 University Of Connecticut Health Center Complexes of alpha (2) macroglobulin and antigenic molecules for immunotherapy
US7666581B2 (en) 2001-08-20 2010-02-23 University Of Connecticut Health Center Methods for preparing compositions comprising heat shock proteins useful for the treatment of cancer and infectious disease
US8029808B2 (en) 2002-04-25 2011-10-04 University Of Connecticut Using heat shock proteins to improve the therapeutic benefit of a non-vaccine treatment modality
US8658596B2 (en) 1999-11-18 2014-02-25 Brown University Research Foundation Biglycan and related therapeutics and methods of use
US8691766B2 (en) 1999-11-18 2014-04-08 Brown University Research Foundation Biglycan and related therapeutics and methods of use
US9958458B2 (en) 2010-12-27 2018-05-01 Brown University Therapeutic and diagnostic methods involving biglycan and utrophin
US9969785B2 (en) 2010-05-17 2018-05-15 Brown University Biglycan mutants and related therapeutics and methods of use

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Cited By (29)

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Publication number Priority date Publication date Assignee Title
EP1075840A3 (fr) * 1999-08-13 2001-05-16 Aventis Behring GmbH Utilisations thérapeutiques et prophylactiques de l'antithrombine III
EP1075840A2 (fr) * 1999-08-13 2001-02-14 Aventis Behring GmbH Utilisations thérapeutiques et prophylactiques de l'antithrombine III
WO2001027079A2 (fr) * 1999-10-14 2001-04-19 Entremed, Inc. Compositions et methodes d'utilisation de ligands liant des elements de la voie de coagulation sanguine pour le traitement du cancer et de maladies induites par le processus d'angiogenese
WO2001027079A3 (fr) * 1999-10-14 2013-04-11 Entremed, Inc. Compositions et methodes d'utilisation de ligands liant des elements de la voie de coagulation sanguine pour le traitement du cancer et de maladies induites par le processus d'angiogenese
US8691766B2 (en) 1999-11-18 2014-04-08 Brown University Research Foundation Biglycan and related therapeutics and methods of use
US8658596B2 (en) 1999-11-18 2014-02-25 Brown University Research Foundation Biglycan and related therapeutics and methods of use
US7449557B2 (en) 2000-06-02 2008-11-11 University Of Connecticut Health Center Complexes of alpha (2) macroglobulin and antigenic molecules for immunotherapy
US7186515B1 (en) 2000-06-02 2007-03-06 University Of Connecticut Health Center Alpha(2) macroglobulin receptor as a heat shock protein receptor and uses thereof
US7179462B2 (en) 2000-06-02 2007-02-20 University Of Connecticut Health Center α (2) macroglobulin receptor as a heat shock protein receptor and uses thereof
EP1224941A3 (fr) * 2001-01-17 2003-02-26 Aventis Behring GmbH Utilisation de l'antithrombine iii pour des maladies provoquees par l'angiogenese
EP1224941A2 (fr) * 2001-01-17 2002-07-24 Aventis Behring GmbH Utilisation de l'antithrombine iii pour des maladies provoquees par l'angiogenese
US8822418B2 (en) 2001-08-15 2014-09-02 Brown University Treatment of muscular dystrophies and related disorders
EP1423133A2 (fr) * 2001-08-15 2004-06-02 Brown University Research Foundation Traitement de dystrophies musculaires et de troubles connexes
WO2003015615A2 (fr) 2001-08-15 2003-02-27 Brown University Research Foundation Traitement de dystrophies musculaires et de troubles connexes
EP1423133B1 (fr) * 2001-08-15 2009-01-14 Brown University Research Foundation Traitement de dystrophies musculaires et de troubles connexes
US7666581B2 (en) 2001-08-20 2010-02-23 University Of Connecticut Health Center Methods for preparing compositions comprising heat shock proteins useful for the treatment of cancer and infectious disease
WO2003039575A3 (fr) * 2001-11-09 2003-10-16 Neuronova Ab Role fonctionnel et utilisation therapeutique potentielle de proteines s, gas6 et reelin en relation avec des cellules souches neuronales adultes
WO2003039575A2 (fr) * 2001-11-09 2003-05-15 Neuronova Ab Role fonctionnel et utilisation therapeutique potentielle de proteines s, gas6 et reelin en relation avec des cellules souches neuronales adultes
US8591890B2 (en) 2002-04-25 2013-11-26 University Of Connecticut Health Center Using heat shock proteins to improve the therapeutic benefit of a non-vaccine treatment modality
US8029808B2 (en) 2002-04-25 2011-10-04 University Of Connecticut Using heat shock proteins to improve the therapeutic benefit of a non-vaccine treatment modality
US6984389B2 (en) 2002-04-25 2006-01-10 University Of Connecticut Health Center Using heat shock proteins to improve the therapeutic benefit of a non-vaccine treatment modality
US9248172B2 (en) 2002-04-25 2016-02-02 University Of Connecticut Health Center Using heat shock proteins to improve the therapeutic benefit of a non-vaccine treatment modality
US9352019B2 (en) 2002-04-25 2016-05-31 University Of Connecticut Health Center Using heat shock proteins to improve the therapeutic benefit of a non-vaccine treatment modality
US7642234B2 (en) 2002-05-17 2010-01-05 Board Of Regents The University Of Texas Systems Beta-2-glycoprotein 1 is an inhibitor of angiogenesis
US7329642B2 (en) 2002-05-17 2008-02-12 Board Of Regents, University Of Texas System β-2-glycoprotein is an inhibitor of angiogenesis
WO2004050125A1 (fr) * 2002-12-03 2004-06-17 Koken Co.,Ltd. Inhibiteur de proliferation et/ou d'infiltration de cellules tumorales
WO2007097961A1 (fr) * 2006-02-16 2007-08-30 Massachusetts Eye & Ear Infirmary Utilisation d'inhibiteurs de l'azurocidine dans la prévention et le traitement d'une fuite vasculaire oculaire
US9969785B2 (en) 2010-05-17 2018-05-15 Brown University Biglycan mutants and related therapeutics and methods of use
US9958458B2 (en) 2010-12-27 2018-05-01 Brown University Therapeutic and diagnostic methods involving biglycan and utrophin

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