EP1064011A1 - Proteines de fixation de metastatine et d'hyaluronate et procedes d'utilisation - Google Patents

Proteines de fixation de metastatine et d'hyaluronate et procedes d'utilisation

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Publication number
EP1064011A1
EP1064011A1 EP99912488A EP99912488A EP1064011A1 EP 1064011 A1 EP1064011 A1 EP 1064011A1 EP 99912488 A EP99912488 A EP 99912488A EP 99912488 A EP99912488 A EP 99912488A EP 1064011 A1 EP1064011 A1 EP 1064011A1
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EP
European Patent Office
Prior art keywords
protein
binding
amino acid
acid sequence
tumor
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP99912488A
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German (de)
English (en)
Inventor
Shawn J. Green
Charles B. Underhill
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Georgetown University
Casi Pharmaceuticals Inc
Original Assignee
Georgetown University
Entremed Inc
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Publication date
Application filed by Georgetown University, Entremed Inc filed Critical Georgetown University
Publication of EP1064011A1 publication Critical patent/EP1064011A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70585—CD44
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This application relates to the fields of immunology, oncology and angiogensis and more particularly to novel anti- tumor compositions comprising hyaluronate (HA) binding proteins and peptides, including HA link modules.
  • HA binding proteins are useful for treating metastatic cancers and angiogenesis-dependent diseases.
  • the HA binding protein is metastatin protein.
  • angiogenesis not only provides the increased nutrients and pathways for the removal of waste needed for the expansion of the tumor, but it also facilitates tumor metastasis by providing a route for tumor cells to leave the primary site and enter the bloodstream (Zetter, 1998).
  • angiogenesis increases the entry of tumor cells into the bloodstream by providing an increased density of immature, highly permeable blood vessels that have thinner basement membranes and fewer intracellular junction complexes than normal mature vessels (Zetter, 1998).
  • angiogenic phenotype is the result of a net balance between both positive and negative regulators of neovascularization (Good et al., 1990; O'Reilly et al., 1994; Parangi et al., 1996; Rastinejad et al., 1989).
  • FGF and BFGF fibroblast growth factors
  • VEGF/VPF vascular endothelial cell growth factor/vascular permeability factor
  • many malignant tumors also generate inhibitors of angiogenesis, including angiostatin and thrombospondin (Chen et al., 1995
  • HA hyaluronate
  • HA is a glycosaminoglycan that is present in connective tissue, synovial fluid, vitreous humor, and cartilage. Studies have shown that HA plays a very intricate role in angiogenesis by exhibiting both pro- and anti-angiogenic properties. For example, as a major component of the extracellular matrix, high molecular weight HA is expected to promote cell invasion by providing the substrate for cell migration and differentiation (Banerjee et al., 1992).
  • vascular growth requires degradation of high molecular weight HA into fragments in order to promote endothelial cell proliferation in vitro and facilitate angiogenesis in vivo (Deed et al.,1997; West et al., 1995).
  • the HA binding protein super- family included CD44, cartilage link protein, hyaluronectin, aggrecan, versican, receptor hyaluronan-mediated motility
  • RHAMM inter-alpha trypsin inhibitor
  • IHABP intracellular hyaluronan binding protein
  • I-CAM 1 I-CAM 1
  • TSG-6 TSG-6
  • HA receptors such as CD44
  • tumor associated endothelial cells and endothelial cells from placental tissue express HA binding proteins.
  • CD44 HA binding proteins
  • increased CD44 expression often correlates with increased metastatic invasiveness of breast, bladder, and melanoma cancer cells.
  • tumor cells utilize the interaction between cell surface CD44 and extracellular matrix HA for intravasation from the primary tumor site and successful extravasation to a distant target organ (Thomas et al., 1992; Goebele et al., 1996).
  • B16BL6 melanoma cells were shown to extravasate to the lungs via binding to pulmonary endothelium-associated HA.
  • HA heumatoid arthritis
  • osteoarthritis OA
  • avascular cartilage differentiates to hypertrophic cartilage that undergoes erosion and vascularization leading to bone formation.
  • the present invention relates to novel protein tumor inhibitors, and methods for their use.
  • the proteins are potent inhibitors of endothelial cell migration and tumor cell metastasis. Systemic therapy with the inhibitors exhibits strong anti -tumor activity.
  • the compositions of the present invention comprise HA binding proteins.
  • HA binding proteins are proteins or peptides that can comprise at least a portion of a proteoglycan tandem repeat domain, and are useful for the inhibition of tumor metastasis and the inhibition of tumor growth in humans and animals.
  • HA binding proteins can also comprise an HA binding link module.
  • the HA binding protein is metastatin protein.
  • Metastatin protein is an approximately 38 kDa fragment of a cartilage link protein, as determined by polyacrylamide gel electrophoresis (PAGE) under non-reducing conditions.
  • Metastatin protein inhibits endothelial cell migration in vitro and tumor metastasis in vivo.
  • Metastatin also inhibits tumor cell growth in vivo in a chick CAM assay.
  • an exemplary HA binding peptide is an approximately 10 amino acid fragment of metastatin protein of the sequence QYPITKPREP (SEQ ID NO: 2) corresponding to approximately amino acids 216 to 225 of the human cartilage link protein.
  • the kown amino acid sequence of a human cartilage link protein is provided in SEQ ID NO: 1.
  • HA binding proteins and fragments thereof can be animal or human in origin.
  • the proteins, and fragments thereof, of the present invention can also be produced synthetically by chemical reaction or by recombinant techniques in conjunction with expression systems.
  • the anti-cancer proteins can also be produced by enzymatically cleaving different molecules, including metastatin precursors, containing sequence homology or identity with segments of metastatin to generate peptides having anti- metastatic activity.
  • the present invention also includes HA binding proteins, including HA link module peptides, that can be labeled isotopically or with other molecules or proteins for use in the detection and visualization of HA binding link module sites with state of the art techniques, including, but not limited to, positron emission tomography, autoradiography, flow cytometry, radioreceptor binding assays, and immunohistochemistry.
  • HA binding proteins also act as agonists and antagonists at the HA binding link module receptor, thereby enhancing or blocking the biological activity of HA binding proteins.
  • the present invention encompasses nucleic acid sequences comprising corresponding nucleotide codons that code for the above disclosed HA binding proteins.
  • the nucleic acid molecules encoding the HA binding proteins are useful for the recombinant production of the HA binding proteins and are also useful as molecular probes or primers for the detection of ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) involved in transcription and translation of HA binding proteins. These molecular probes or primers provide means to detect and measure biosynthesis of HA binding proteins in tissues and cells.
  • RNA ribonucleic acid
  • DNA deoxyribonucleic acid
  • compositions provided herein additionally include antibodies that are specific for the hyaluronic acid binding link module compositions described herein and active portions thereof, or that inhibit the binding of antibodies specific for hyaluronic acid binding link module peptides.
  • These antibodies can be polyclonal antibodies or monoclonal antibodies.
  • the antibodies are useful as in vitro research tools for studying cell migration and tumor metastasis and also for isolating large quantities of hyaluronic acid binding link module polypeptides.
  • Antibodies specific for HA binding proteins may be used in diagnostic kits for the detection of the presence and quantity of HA binding proteins, and may also be administered to a human or animal to block the binding of endogenous HA binding proteins, thereby increasing endothelial cell migration and tumor growth.
  • the present invention also includes methods of treating or preventing metastatic diseases and angiogenesis-related diseases, particularly metastatic or angiogenesis-dependent cancer, in patients, and for curing metastatic or angiogenesis-dependent cancer in patients.
  • Prevention or treatment may be accomplished by administering substantially purified HA binding proteins, or active fragments thereof, or HA binding protein agonists or antagonists, and/or HA binding protein antisera or antisera directed against HA binding protein antisera to a patient.
  • Additional treatment methods include administration of HA binding proteins, HA binding link module fragments, HA binding link module antisera, or HA binding link module receptor agonists and antagonists linked to cytotoxic agents.
  • the present invention also relates to methods of using the HA proteins and peptide fragments, corresponding nucleic acid sequences, and antibodies that bind specifically to the inhibitor and its peptides, to diagnose metastatic and angiogenesis-related diseases and disorders. These methods include the detection of HA binding proteins in body fluids and tissues for the purpose of diagnosis or prognosis of metastatic and angiogenesis-related diseases such as cancer. The present invention also includes the detection of HA binding link module binding sites and receptors in cells and tissues.
  • the present invention also includes diagnostic methods and kits for detection and measurement of HA binding proteins in biological fluids and tissues, and for localization of HA binding proteins in tissues.
  • the diagnostic method and kit can be in any configuration well known to those of ordinary skill in the art.
  • Antibodies specific for HA binding proteins can be used in diagnostic kits to detect the presence and quantity of HA binding proteins which is diagnostic or prognostic for the occurrence or recurrence of metastatic cancer or other diseases or conditions mediated by endothelial cell migration.
  • the present invention also includes diagnostic methods and kits for detecting the presence and quantity of antibodies that bind HA binding proteins in body fluids.
  • the diagnostic method and kit can be in any configuration well known to those of ordinary skill in the art.
  • composition comprising an isolated HA binding protein, which inhibits tumor growth and metastasis. It is another object of the present invention to provide a composition comprising an approximately 38 kDa fragment of the cartilage link protein referred to as metastatin protein that inhibits tumor growth and metastasis.
  • composition comprising an active fragment of metastatin protein. It is a further object of the present invention to provide a composition comprising at least a portion of an HA binding link module.
  • nucleic acids that correspond to the HA binding link proteins and peptides herein. It is another object of the present invention is to provide a composition consisting of antibodies to HA binding link proteins and peptides that are selective for specific regions thereof.
  • FIG. 1 Inhibition of Growth Factor Induced Migration of Human Umbilical Vein Endothelial Cells (HUVECs).
  • Metastatin protein was applied to HUVECs concurrently stimulated with 5 ng/ml bFGF. Application of the 38 kDa fraction inhibited HUVEC migration in a dose dependent manner. Each bar represents the mean + SEM.
  • FIG. 2 A-B Inhibition of Pulmonary Metastases in the B16BL6 Melanoma Experimental Metastatic Tumor Model. After injection of B16BL6 melanoma cells (5 x 10 4 ) into the tail vein of C57BL/6J mice, the mice were treated with increasing doses of metastatin. Control mice were treated with PBS, BSA or metastatin pre-incubated with an 100-fold excess of hyaluronate, and heat denatured metastatin. Administration of metastatin reduced the number of pulmonary metastases in a dose-dependent manner, seen graphically in Fig. 2A, and photographically in Fig. 2B.
  • Figure 3 A-B Inhibition of pulmonary metastases in the Lewis lung carcinoma experimental metastatic tumor model.
  • mice C57BL/6J mice, the mice were treated with increasing doses of metastatin. Control mice were treated with PBS. Administration of metastatin non-reducing the number of pulmonary metastases in a dose-dependent manner.
  • Figure 4 Inhibition of tumor growth in the chick CAM assay.
  • Metastatin reduced tumor weight by roughly 70% as compared to the PBS control in the modified CAM assay.
  • HA binding proteins hyaluronate binding proteins
  • substantially sequence homology means at least approximately 70% homology between a known amino acid residue sequence in a HA binding protein or peptide, preferably at least approximately 80% homology, more preferably at least approximately 90% homology. Homology can be determined by sequence identity, or by using a well-known computer program, such as DNA Star or GeneJockey, on default setting parameters.
  • the phrases “isolated”, “biologically pure” or “substantially pure” refer to material which is substantially or essentially free from components which normally accompany it as found in its native state.
  • the isolated, HA binding proteins of the invention 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.
  • the compositions provided herein comprise HA binding proteins. These compositions are proteins that can comprise at least a portion of a proteoglycan tandem repeat domain and are useful for the treatment of cancer as well as non-cancerous angiogenesis dependent diseases in humans and animals.
  • portions of the proteoglycan tandem repeat domain mediate the binding of the HA binding protein or peptide to HA and provide the anti-tumor activity of these molecules.
  • the present invention also includes compositions wherein the HA binding protein is metastatin protein.
  • “Metastatin protein” is an approximately 38 kDa fragment of a cartilage link protein, as determined by SDS-PAGE under non-reducing conditions, that has anti-tumor activity.
  • the amino terminal portion of a preferred metastatin protein is TVYLHAAQTG (SEQ ID NO:3).
  • the complete amino acid sequence of a human cartilage link protein is provided in SEQ ID NO: l.
  • Metastatin protein inhibits endothelial cell migration in vitro and tumor metastasis in vivo. Metastatin also inhibits tumor cell growth in vivo in a chick CAM assay.
  • an exemplary HA binding peptide is an approximately 10 amino acid fragment of metastatin protein of the sequence QYPITKPREP (SEQ ID NO: 2) corresponding to approximately amino acids 216 to 225 of the human cartilage link protein.
  • HA binding protein or peptide includes shortened proteins or peptides wherein one or more amino acid is removed from either or both ends of a HA binding protein, or from an internal region of the protein, yet the resulting molecule retains its anti-tumor activity.
  • the term "HA binding protein or peptide” also includes lengthened proteins or peptides wherein one or more amino acids is added to either or both ends of an HA binding protein or peptide, or to an internal location, yet the resulting molecule retains anti-tumor activity.
  • One example of such a modification is the addition of tyrosine to the first position. Tyrosine labeled molecules may be further labeled with iodine for use in assays. Labeling with other radioisotopes or chemicals such as ricin may also be useful in providing a molecular tool for destroying the target cell containing metastatin receptors. 12
  • silent substitutions of amino acids are well known in the art and are intended to fall within the scope of the appended claims. Silent substitutions occur when the replacement of an amino acid with a structurally or chemically similar amino acid does not significantly alter the structure, conformation or activity of the protein.
  • modifications of the protein, its subunits and peptide fragments include substitutions of naturally occurring amino acids at specific sites with other molecules, including but not limited to naturally and non-naturally occurring amino acids. Such substitutions may modify the bioactivity of HA binding proteins, such as by increasing or decreasing HA binding and anti-tumor activity, and produce biological or pharmacological agonists or antagonists.
  • the HA binding proteins may be isolated from body fluids and tissues including, but not limited to, cartilage, connective tissue, synovial fluid, and vitreous humor, blood, serum or plasma. HA binding proteins may also be produced from recombinant sources, genetically altered cells implanted into animals, tumors, cell cultures and other sources. The HA binding proteins may be produced by polypeptide synthesis, or derived by in vitro enzymatic catalysis of larger, encompassing polypeptides, to yield active HA binding proteins. Recombinant techniques include gene amplification from DNA sources using amplification techniques such as the polymerase chain reaction (PCR), and gene amplification from RNA sources using amplification techniques such as reverse transcriptase/PCR.
  • PCR polymerase chain reaction
  • the HA binding proteins described herein have a variety of uses.
  • the HA binding proteins may be employed to treat metastatic and angiogenesis-dependent cancers and other angiogenesis-related diseases.
  • metastatic disease is metastatic cancer.
  • Angiogenesis-related diseases include, but are not limited to, angiogenesis-dependent cancer, including, for example, solid tumors, blood born tumors such as leukemias, and tumor metastases; benign tumors, for example hemangiomas, 13
  • acoustic neuromas acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas
  • rheumatoid arthritis psoriasis
  • ocular angiogenic diseases for example, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis; Osier-
  • the HA binding proteins of the present invention are useful in the treatment of disease of excessive or abnormal stimulation of endothelial cells. These diseases include, but are not limited to, intestinal adhesions, atherosclerosis, scleroderma, and hypertrophic scars, i.e., keloids. They are also useful in the treatment of diseases that have angiogenesis as a pathologic consequence such as cat scratch disease (Rochele minalia quintosa) and ulcers (Helobacter pylori).
  • the HA binding proteins may also be used to develop affinity columns for isolating antibodies directed toward the HA binding proteins. Those antibodies may be isolated and purified, followed by amino acid sequencing. Also, polypeptides that bind to the HA binding protein or peptide antibodies with high specificity and avidity may be labeled with a label or reporter group and employed for visualization and quantitation in the assays described herein using detection techniques such as autoradiographic and membrane binding techniques.
  • the reporter group or label is commonly a fluorescent or radioactive group or an enzyme.
  • HA binding proteins and peptides can also be employed to develop affinity columns for isolation of HA binding link module receptors. Isolation and purification of such receptors may be followed by amino acid sequencing. Using this information, the gene or genes coding for the receptors can be identified and isolated. Next, cloned nucleic acid sequences may be developed for insertion into vectors capable of expressing the receptors. 14
  • the HA binding protein and peptide compositions of the present invention can be made by automated protein synthesis methodologies well known to one skilled in the art. Alternatively, the HA binding protein and peptide of the present invention may be isolated from larger known proteins, such as cartilage link protein.
  • Metastatin protein, and active fragments thereof are useful for treating metastatic cancers and tumors as well as angiogenesis- related cancers and diseases.
  • the metastatin compositions, and active fragments thereof are also useful for curing metastatic and angiogenesis-dependent cancers and tumors.
  • the unexpected and surprising ability of these novel compounds to treat and cure cancers and tumors answers a long felt unfulfilled need in the medical arts, and provides an important benefit to mankind.
  • Applicants' invention also encompasses nucleic acid sequences that correspond to and code for the HA binding proteins, including metastatin protein, of the present invention.
  • the biologically active protein molecules and nucleic acid sequences corresponding to the proteins are useful for modulating endothelial processes in vivo, and for diagnosing and treating endothelial cell- related diseases, for example by gene therapy.
  • Nucleic acid sequences that correspond to, and code for, the HA binding proteins and metastatin can be prepared based upon the knowledge of the amino acid sequence, and the art recognized correspondence between codons (sequences of three nucleic acid bases), and amino acids. Because of the degeneracy of the genetic code, wherein the third base in a codon may vary, yet still code for the same amino acid, many different possible coding nucleic acid sequences are derivable for any particular protein or peptide fragment.
  • the nucleic acid sequence may be derived from a gene bank using oligonucleotides probes designed based on the N-terminal amino acid sequence and well known techniques for cloning genetic material. Nucleic acid sequences are synthesized using automated systems well known in the art. Either the entire sequence may be synthesized or a series of smaller oligonucleotides are made and subsequently ligated together to yield the full length sequence.
  • the genes for HA binding proteins may also be isolated from cells or tissue (such as cartilage) that express high levels of HA binding proteins by (1) isolating messenger RNA from the tissue, (2) using reverse transcriptase to generate the corresponding DNA sequence and then (3) using PCR with the appropriate primers to amplify the DNA sequence coding for the active hyaluronic acid binding link module amino acid sequence.
  • the nucleic acid molecules are useful for production of recombinant polypeptides. Because recombinant methods of polypeptide production produce large quantities of polypeptide that require less purification, recombinant polypeptides are often less expensively produced than polypeptides produced using traditional isolation or purification techniques.
  • nucleic acid sequences encoding the hyaluronic acid binding modules can be inserted into a vector, such as a plasmid, and recombinantly expressed in a living organism to produce recombinant peptides in accordance with methods well known to those skilled in the art.
  • One example of a method of producing HA binding proteins using recombinant DNA techniques entails the steps of (1) identifying and purifying a hyaluronic acid binding link module as discussed below, (2) determining the N-terminal amino acid sequence of the purified inhibitor, (3) synthetically generating a DNA oligonucleotide probe that corresponds to the N-terminal amino acid sequence, (4) generating a DNA gene bank from human or other mammalian DNA, (5) probing the gene bank with the DNA oligonucleotide probe, (6) selecting clones that hybridize to the oligonucleotide, (7) isolating the inhibitor gene from the clone, (8) inserting the gene into an appropriate vector such as an expression vector, (9) inserting the gene-containing vector into a microorganism or other expression system capable of expressing the inhibitor gene, and (10) isolating the recombinantly produced inhibitor.
  • the above techniques are more fully described in laboratory manuals such as "Molecular Cloning: A
  • HA binding proteins or biologically active fragments thereof, is by peptide synthesis.
  • the fragment can be synthesized by techniques well known in the art, as exemplified by "Solid Phase Peptide Synthesis: A Practical Approach” E. Atherton and R.C. Sheppard, IRL Press, Oxford England.
  • multiple fragments can be synthesized which are subsequently linked together to form larger fragments.
  • These synthetic peptide fragments can also be made with amino acid substitutions at specific locations in order to test for agonistic and antagonistic activity in vitro and in vivo.
  • the isolated, recombinant or synthetic HA binding proteins described herein are useful for generating antibodies specific for the HA binding proteins. These antibodies that specifically bind to the HA binding proteins can be used in diagnostic methods and kits that are well known to those of ordinary skill in the art to detect or quantify the HA binding proteins in a body fluid or tissue. Results from these tests can be used to diagnose or predict the occurrence or recurrence of a cancer and other angiogenesis mediated diseases.
  • passive antibody therapy using antibodies that specifically bind hyaluronic binding link modules can be employed to modulate metastatic processes such as metastatic cancer as well as angiogenesis-dependent processes such as reproduction, development, wound healing, tissue repair, and angiogenesis- dependent diseases.
  • antisera directed to the Fab regions of hyaluronic acid binding link module antibodies can be administered to block the ability of endogenous hyaluronic acid binding link module antisera to bind HA binding proteins.
  • Antibodies specific for HA binding proteins may be either polyclonal or monoclonal, and are made according to techniques and protocols well known in the art.
  • the preferred method of making monoclonal antibodies is a modified version of the method 17
  • the monoclonal antibodies are utilized in well known immunoassay formats, such as competitive and non-competitive immunoassays, including ELISA, sandwich immunoassays and radioimmunoassays (RIAs), to determine the presence or absence of the HA binding proteins of the present invention in body fluids and tissues.
  • body fluids include, but are not limited to, blood, serum, synovial fluid, peritoneal fluid, pleural fluid, cerebrospinal fluid, uterine fluid, saliva, mucus and vitreous humor.
  • body tissues include, but are not limited to, connective tissue and cartilage.
  • Polyclonal antisera are also raised using established techniques known to those skilled in the art.
  • polyclonal antisera may be raised in mice, rabbits, rats, goats, sheep, guinea pigs, chickens, and other animals, most preferably mice and rabbits by administering the antigen to the animals.
  • Isolated, recombinant or synthetic HA binding proteins conjugated to a carrier molecule such as bovine serum albumin may be combined with an adjuvant mixture, emulsified and injected subcutaneously at multiple sites on the back, neck, flanks, and sometimes in the footpads of the animals.
  • Booster injections are made at regular intervals, such as every two to four weeks.
  • Blood samples are obtained by venipuncture, for example using the marginal ear veins after dilation, approximately seven to ten days after each injection.
  • the blood samples are allowed to clot and are centrifuged, and the serum removed, aliquoted, and stored under refrigeration for immediate use or frozen for subsequent analysis.
  • All serum samples from generation of polyclonal antisera or media samples from production of monoclonal antisera are analyzed for determination of titer. Titer is established through several means, for example, using dot blots and density analysis, and also with precipitation of radiolabeled peptide-antibody complexes using protein A, secondary antisera, cold ethanol or charcoal-dextran followed by activity measurement with a gamma counter. The highest titer antisera are also purified on affinity columns which are commercially available.
  • Bispecific antibodies have two antigen binding domains wherein each domain is directed against a different epitope.
  • Phage display technology may be used to select antibody genes having binding activities for hyaluronic acid link modules from PCR- amplified v genes of lymphocytes from naive libraries or humans screened for having antibodies to the hyaluronic acid link binding modules.
  • the HA binding proteins and antibodies described above are useful for purposes such as in vivo and in vitro diagnostics and laboratory research using the methods and assays described below.
  • Various types of labels and methods of conjugating the labels to the HA binding proteins and antibodies are well known to those skilled in the art. Several specific labels are set forth below.
  • the HA binding proteins and antibodies are conjugated to a radiolabel such as, but not restricted to, 32p ? 3H, i C, 35s, 1251, or 131l. Detection of a label can be by methods such as scintillation counting, gamma ray spectrometry or autoradiography.
  • Bioluminescent labels such as derivatives of firefly luciferin, are also useful.
  • the bioluminescent substance is covalently bound to the hyaluronic acid binding link module or antibody by conventional methods, and the labeled hyaluronic acid binding link module or antibody is detected when an enzyme, such as luciferase, catalyzes a reaction with ATP causing the bioluminescent molecule to emit photons of light.
  • Fluorogens may also be used as labels.
  • fluorogens include fluorescein and derivatives, phycoerythrin, allo-phycocyanin, phycocyanin, rhodamine, and Texas Red.
  • the fluorogens are generally detected by a fluorescence detector.
  • the HA binding proteins and antibodies can alternatively be labeled with a chromogen to provide an enzyme or affinity label.
  • the hyaluronic acid binding link module or antibody can be biotinylated so that it can be utilized in a biotin-avidin reaction, which may also be coupled to a label such as an enzyme or fluorogen.
  • the HA binding protein and peptide or antibody can be labeled with peroxidase, alkaline phosphatase or other enzymes giving a chromogenic or fluorogenic reaction upon addition of substrate.
  • peroxidase alkaline phosphatase or other enzymes giving a chromogenic or fluorogenic reaction upon addition of substrate.
  • Additives such as 5-amino-2,3-dihydro- 1 ,4-phthalazinedione (also known as LuminolTM) (Sigma Chemical
  • p-hydroxybiphenyl also known as p-phenylphenol
  • rate enhancers such as p-hydroxybiphenyl (also known as p-phenylphenol)
  • p-hydroxybiphenyl also known as p-phenylphenol
  • luminogeneic or fluorogenic dioxetane derivatives of enzyme substrates can also be used.
  • labels can be detected using enzyme-linked immunoassays (ELISA) or by detecting a color change with the aid of a spectrophotometer.
  • HA binding proteins or antibodies may be labeled with colloidal gold for use in immunoelectron microscopy in accordance with methods well known to those skilled in the art.
  • Angiogenesis-related diseases include, but are not limited to, angiogenesis-dependent cancer, including, for example, solid tumors, blood born tumors such as leukemias, and tumor metastases; benign tumors, for example hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas; rheumatoid arthritis; psoriasis; ocular angiogenic diseases, for example, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis; Osier- Webber Syndrome; myocardial angiogenesis; plaque neovascularization; telangiectasia; hemophiliac joints; 20
  • HA binding proteins of the present invention are useful in the treatment of disease of excessive or abnormal stimulation of endothelial cells. These diseases include, but are not limited to, intestinal adhesions, atherosclerosis, scleroderma, and hypertrophic scars, i.e., keloids.
  • a particularly important aspect of the present invention is the discovery of a novel and effective method for inhibiting cancer metastasis and growth in patients. It was unexpectedly found that metastatin protein inhibits the growth of B 16 tumors in the chick CAM assay in vivo and the administration of metastatin causes inhibition of pulmonary metastases in vivo. Additionally, the metastatin peptide having the amino acid sequence of SEQ ID NO: 1
  • the present invention also includes formulations effective for treating or curing metastatic cancers and tumors.
  • the HA binding proteins can also be used as a birth control agent by reducing or preventing uterine vascularization required for embryo implantation.
  • the present invention provides an effective birth control method when an amount of the HA binding protein or peptide sufficient to prevent embryo implantation is administered to a female.
  • an amount of the link module sufficient to block embryo implantation is administered before or after intercourse and fertilization have occurred, thus providing an effective method of birth control, possible a "morning after" method. While not wanting to be bound by this statement, it is believed that inhibition of vascularization of the uterine endometrium interferes with implantation of the blastocyst.
  • Administration methods may include, but are not limited to, pills, injections (intravenous, subcutaneous, 21
  • HA binding proteins will interfere with normal enhanced vascularization of the placenta, and also with the development of vessels within a successfully implanted blastocyst and developing embryo and fetus.
  • blockade of HA binding protein and peptide receptors with HA binding protein and peptide analogs which act as receptor antagonists may promote endothelialization and vascularization.
  • Such effects may be desirable in situations of inadequate vascularization of the uterine endometrium and associated infertility, wound repair, healing of cuts and incisions, treatment of vascular problems in diabetics, especially retinal and peripheral vessels, promotion of vascularization in transplanted tissue including muscle and skin, promotion of vascularization of cardiac muscle especially following transplantation of a heart or heart tissue and after bypass surgery, promotion of vascularization of solid and relatively avascular tumors for enhanced cytotoxin delivery, and enhancement of blood flow to the nervous system, including but not limited to the cerebral cortex and spinal cord.
  • HA binding proteins may be used in combination with other compositions and procedures for the treatment of the above described diseases and conditions.
  • a tumor may be treated conventionally with surgery, radiation or chemotherapy combined with HA binding proteins and subsequently HA binding proteins may be administered to the patient to extend the dormancy of micrometastases and to stabilize any residual primary tumor.
  • proteins and protein fragments with the HA binding activity described above can be provided as isolated and substantially purified proteins and protein fragments in pharmaceutically acceptable formulations using formulation methods known to those of ordinary skill in the art. These formulations can be administered by standard routes. In general, the combinations may be administered by the topical, transdermal, 22
  • the hyaluronic acid binding link module may be inco ⁇ orated into biodegradable polymers allowing for sustained release of the compound, the polymers being implanted in the vicinity of where drug delivery is desired, for example, at the site of a tumor or implanted so that the hyaluronic acid binding link module is slowly released systemically.
  • Osmotic minipumps may also be used to provide controlled delivery of high concentrations of HA binding proteins through cannulae to the site of interest, such as directly into a metastatic growth or into the vascular supply to that tumor.
  • the biodegradable polymers and their use are described, for example, in detail in Brem et al. (1991), which is hereby inco ⁇ orated by reference in its entirety.
  • a HA binding protein and peptide of the present invention will depend on the disease state or condition being treated and other clinical factors such as weight and condition of the human or animal and the route of administration of the compound.
  • For treating humans or animals between approximately 0.5 mg/kilogram to 500 mg/kilogram of the HA binding protein and peptide can be administered.
  • a more preferable range is 1 mg/kilogram to 100 mg/kilogram with the most preferable range being from 2 mg/kilogram to 50 mg/kilogram.
  • it can be administered between several times per day to once a week. It is to be understood that the present invention has application for both human and veterinary use.
  • the methods of the present invention contemplate single as well as multiple administrations, given either simultaneously or over an extended period of time.
  • the HA binding protein and peptide formulations include those suitable for oral, rectal, ophthalmic (including intravitreal or intracameral), nasal, topical (including buccal and sublingual), intrauterine, vaginal or parenteral (including subcutaneous, 23
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
  • Preferred unit dosage formulations are those containing a daily dose or unit, daily sub-dose, as herein above recited, or an appropriate fraction thereof, of the administered ingredient. It should be understood that in addition to the ingredients, particularly mentioned above, the formulations of the present invention may include other agents conventional in the art having regard to the type of formulation in question.
  • the present invention also relates to methods of using HA binding proteins, and active fragments thereof, nucleic acid sequences corresponding to HA binding proteins and active peptide fragments thereof, and 24
  • HA binding proteins antibodies that bind specifically to HA binding proteins and their peptides, to diagnose endothelial cell-related diseases and disorders. Diagnosis is accomplished by detection of HA binding proteins, antibodies thereto, or nucleic acids in body fluids or tissues. Detection may be accompanied by comparison of the detected levels of HA binding proteins, or antibodies thereto, to normal levels of HA binding proteins, or antibodies thereto.
  • Kits for measurement of HA binding proteins are also contemplated as part of the present invention.
  • Antisera that possess the highest titer and specificity and can detect HA binding protein and peptide in extracts of plasma, urine, tissues, and in cell culture media are further examined to establish easy to use kits for rapid, reliable, sensitive, and specific measurement and localization of HA binding proteins.
  • assay kits include but are not limited to the following techniques; competitive and non- competitive assays, radioimmunoassay, bioluminescence and chemiluminescence assays, fluorometric assays, sandwich assays, immunoradiometric assays, dot blots, enzyme linked assays including ELISA, microtiter plates, antibody coated strips or dipsticks for rapid monitoring of urine or blood, and immunocytochemistry.
  • competitive and non- competitive assays include radioimmunoassay, bioluminescence and chemiluminescence assays, fluorometric assays, sandwich assays, immunoradiometric assays, dot blots, enzyme linked assays including ELISA, microtiter plates, antibody coated strips or dipsticks for rapid monitoring of urine or blood, and immunocytochemistry.
  • competitive and non- competitive assays include but are not limited to the following techniques; competitive and non- competitive assays, radioimmunoassay, bioluminescence and chem
  • the assay kit provides instructions, antiserum, HA binding protein or peptide, and possibly radiolabeled HA binding proteins or peptides and/or reagents for precipitation of bound antibody complexes.
  • the kit is useful for the measurement of HA binding proteins or peptides in biological fluids and tissue extracts of animals and humans with and without tumors or angiogenesis- dependent diseases.
  • kits for the localization of HA binding proteins or peptides in tissues and cells are also included in the present invention.
  • This HA binding protein and peptide immunohistochemistry kit 25 are also included in the present invention.
  • HA binding protein or peptide antiserum provides instructions, HA binding protein or peptide antiserum, and possibly blocking serum and secondary antiserum linked to a fluorescent molecule such as fluorescein isothiocyanate, or to some other reagent used to visualize the primary antiserum.
  • Immunohistochemistry techniques are well known to those skilled in the art.
  • This HA binding protein and peptide immunohistochemistry kit permits localization of HA binding proteins in tissue sections and cultured cells using both light and electron microscopy. It is used for both research and clinical pu ⁇ oses. For example, tumors are biopsied or collected and tissue sections cut with a microtome to examine sites of hyaluronic acid binding link module production. Such information is useful for diagnostic and possibly therapeutic pu ⁇ oses in the detection and treatment of cancer.
  • the invention further encompasses a method for identifying and quantitating HA binding protein and peptide-specific receptors. Labeling of the HA binding proteins with short lived isotopes enables visualization of receptor binding sites in vivo using positron emission tomography or other modern radiographic techniques. These methods are important for the study of angiogenesis and metastasis in cancer and other angiogenesis- related diseases.
  • Bovine nasal cartilage shredded with a superform blade was disassociated with 4M guanidinium HCI in sodium acetate, dialyzed against water, and lyophilized. Trypsin digestion and purification by HA affinity chromatography yielded cartilage fragments with different molecular weights. These fragments were tested for their ability to inhibit growth factor-induced migration of HUVECs in a migration assay.
  • endothelial cells were grown to confluency on gelatin-coated plates; after wounding with a razor blade, migration was induced by addition of 5 ng/ml bFGF.
  • a cartilage fraction enriched in a 38 kDa protein was shown to suppress HUVEC migration in a dose dependent manner (Fig. 1A). The fraction had no effect on bFGF -driven proliferation of HUVEC cells. Therefore, a toxic effect was excluded as the basis of the anti-migratory activity.
  • HA-affinity chromatography was employed to purify a cartilage-derived preparation with the inhibitory activity.
  • the trypsonized material was loaded onto an HA affinity chromatography column, then washed with increasing concentrations of NaCl up to 3 M. Then, the protein was eluted with 4 M guanadine. Elution of the preparation from the HA-Sepharose column yielded several fractions with different HA-binding abilities. Further investigation indicated that a 38 kDa protein was responsible for the inhibitory effect observed in the migration assay described as follows.
  • HUVECs Confluent monolayers of HUVECs were wounded with a razor blade and cellular debris was removed by washing with PBS. Subsequently, the cells were incubated overnight in EBM containing either bFGF (5 ng/ml) alone or bFGF (5 ng/ml) and various concentrations of the 38 kDa fraction. The cells were then fixed in 25% acetic acid/75% methanol and stained with hematoxylin (Sigma, St. Louis, MO). The number of cells migrating from the wound origin was counted with a light microscope at 100 times magnification using a grid. Each experiment was performed at least twice and each condition was tested in duplicate. The resulting values shown in the figures represent the mean of ten fields.
  • metastatin amino acid sequence which was subsequently shown to belong to a fragment of an HA binding protein bovine cartilage link protein (LP) complex and was termed metastatin.
  • the amino terminal sequence was TVYLHAAQTG (SEQ ID NO:3).
  • purified metastatin protein was immunoreactive toward an antibody raised against LP (Fig. IB).
  • Metastatin protein inhibited bFGF-induced migration of HUVECs with an IC 50 value of 25 nM.
  • the inhibitory activity of metastatin was attributed to its strong HA-binding affinity, since precipitation with excess HA neutralized the anti- migratory action of metastatin (data not shown).
  • B 16BL6 melanoma cells (5x 10 4 ) were injected into the tail vein of C57BL/6J mice. Subsequently, the animals were treated intraperitoneally with increasing doses of metastatin protein. The treatment was initiated three days after the injection of tumor cells. Fourteen days after tumor cell inoculation, the animals were sacrificed, the lungs were removed, and the pulmonary surface metastases were counted. Administration of metastatin reduced the number of pulmonary metastases in a dose-dependent manner (Fig. 2A). Heat-inactivation of the metastatin preparation neutralized the anti-metastatic activity of the protein. 28
  • Lewis lung carcinoma cells (5 x 10 4 ) were injected into the tail vein of C57BL/6J mice. The animals were then treated intraperitoneally with increasing doses of metastatin. Treatment of the mice was initiated three days after tumor cell inoculation and continued every day for 10 days. At the end of the experiment, the mice were sacrificed and the lung weights were measured as a reflection of tumor mass and progression of the disease. Metastatin protein suppressed pulmonary metastases in a dose-dependent manner (Fig. 3A). Metastatin protein had no effect on serum-driven proliferation of Lewis lung carcinoma cells in vitro (data not shown) and a toxic effect was excluded. Macropathology of the lungs of animals treated with metastatin protein or vehicle control is shown in Figure 3B.
  • Metastatin was used in a chick CAM assay (Brooks et al. Cell 1994, 79(7): 1157- 1164). As shown in Figure 4, the metastatin reduced tumor weight by roughly 70% as compared to the PBS control. Heat-inactivation of the metastatin preparation neutralized the anti-tumor activity of the peptide. 29
  • a peptide of selected from the proteoglycan terminal repeat domain of the HA link protein, having the sequence QYPITKPREP (SEQ ID NO: 2) was tested for endothelial cell migration inhibition activity.
  • HUVECs Confluent monolayers of HUVECs were wounded with a razor blade and cellular debris was removed by washing with PBS. Subsequently, the cells were incubated overnight in EBM containing either bFGF (5 ng/ml) alone or bFGF (5 ng/ml) and various concentrations of the peptide. Controls were BSA and the presence or absence of bFGF. The cells were then fixed in 25% acetic acid/75% methanol and stained with hematoxylin (Sigma, St. Louis, MO). The number of cells migrating from the wound origin was counted with a light microscope at 100 times magnification using a grid. The peptide inhibited bFGF-induced migration of HUVECs with an IC 50 value of about 25 uM.
  • the anti-metastatic potential of the peptide of Example 6 was tested in the B 16BL6 experimental metastatic tumor model.
  • B16BL6 melanoma cells (5 x 10 4 ) were injected into the tail vein of C57BL/6J mice. Subsequently, the animals were treated intraperitoneally with increasing doses of the peptide QYPITKPREP (SEQ ID NO:2). PBS was used as a control. The treatment was initiated three days after the injection of tumor cells. Fourteen days after tumor cell inoculation, the animals were sacrificed, the lungs were removed, and the pulmonary surface metastases were counted. Administration of the peptide reduced the number of pulmonary metastases in a dose-dependent manner. At a dose of 50 ug, the T/C value was 0.15, and at a dose of 20 ug the T/C was 0.37. 30
  • FGF-1 dependent proliferative and migratory responses are impaired in senescent human umbilical vein endothelial cells and correlate with the inability to signal tyrosine phosphorylation of fibroblast growth factor receptor- 1 substrates. J. Cell Biol. 134, 783-91.
  • a tumor suppressor-dependent inhibitor of angiogenesis is immunologically and functionally indistinguishable from a fragment of thrombospondin. Proc. Nat. Acad. Sci. USA. 87, 6624-6628.
  • CD44 is involved in tumor angiogenesis; an activation antigen on human endothelial cells. Blood. 90, 1150-1159.
  • Neovascularization is associated with a switch to the export of bFGF in the multistep development of fibrosarcoma. Cell 66, 1095-1104.
  • Angiostatin A novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis lung carcinoma. Cell 79,
  • CD44H regulates tumor cell migration on HA-coated substrate. J Cell Biology 118, 971-977.

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Abstract

La présente invention concerne des compositions renfermant des peptides et des protéines de fixation d'hyaluronate (HA) ainsi que leurs procédés d'utilisations dans l'inhibition du cancer et des maladies liées à l'angiogenèse. Dans un mode préféré de réalisation, le module de liaison fixant l'acide hyaluronique est une protéine métastatine d'environ 38 kDA, inhibant la croissance tumorale et les métastases tumorales.
EP99912488A 1998-03-13 1999-03-12 Proteines de fixation de metastatine et d'hyaluronate et procedes d'utilisation Withdrawn EP1064011A1 (fr)

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US6358735B1 (en) 1995-06-30 2002-03-19 University Of Kansas Medical Center Method for inhibiting angiogenesis and tumors with the isolated NC1 α3 chain monomer of type IV collagen
US6440729B1 (en) 1995-06-30 2002-08-27 University Of Kansas Medical Center Treating angiogenesis-mediated diseases with the α2 monomer of type IV collagen
KR20020065510A (ko) 1999-12-17 2002-08-13 다케다 야쿠힌 고교 가부시키가이샤 ＫｉＳＳ-1 펩티드의 제조 방법
SE0003260D0 (sv) * 2000-09-14 2000-09-14 Anders Uhlin New use of hyaluronan
WO2003018044A1 (fr) * 2001-08-24 2003-03-06 Stroemblad Staffan Nouveau medicament
AUPR856501A0 (en) * 2001-10-30 2001-11-29 Peter Maccallum Cancer Institute, The Detection of haematopoietic stem cells and progeny and uses thereof
WO2008100789A2 (fr) 2007-02-16 2008-08-21 Benaroya Research Institute At Virginia Mason Compositions et procédés pour modifier l'élastogenèse
WO2009033047A2 (fr) * 2007-09-07 2009-03-12 University Of Chicago Procédés et compositions pour traiter des maladies et affections impliquant de l'hyaluronane de masse moléculaire supérieure
US20150017091A1 (en) * 2011-08-18 2015-01-15 Cornell University Detection and treatment of metastatic disease
CA2853358A1 (fr) 2011-10-24 2013-05-02 Halozyme, Inc. Diagnostic compagnon pour le traitement avec un agent anti-hyaluronane et procedes d'utilisation dudit diagnostic
WO2014062856A1 (fr) 2012-10-16 2014-04-24 Halozyme, Inc. Hypoxie et hyaluronane et leurs marqueurs pour le diagnostic et la surveillance de maladies et de pathologies, et méthodes associées
WO2015021993A1 (fr) 2013-08-13 2015-02-19 Ibcc Holding As Molécules sb101 modifiées pharmacologiquement actives et leurs utilisations
US9572832B2 (en) 2013-08-29 2017-02-21 Holy Stone Healthcare Co., Ltd. Compound of glycosaminoglycan and its fabrication method as well as application
CN104988561A (zh) * 2015-07-20 2015-10-21 江南大学 一种基于透明质酸复合胶束的生物涂层制备方法
US20220016203A1 (en) * 2018-10-02 2022-01-20 The Wistar Institute Of Anatomy And Biology Compositions and methods for prevention and reduction of metastasis

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