AU3085699A - Metastatin and hyaluronate binding proteins and methods of use - Google Patents

Description

WO 99/45942 PCT/US99/05498 5 10 METASTATIN AND HYALURONATE BINDING PROTEINS AND METHODS OF USE 15 Cross Reference to Prior Related Cases This application claims priority to provisional application Serial No. 60/077,898 filed March 13, 1998 and provisional application Serial No. 60/108,124 filed November 12, 1998. Each of the above-referenced applications is incorporated herein in its 20 entirety. Technical Field This application relates to the fields of immunology, oncology and angiogensis and more particularly to novel anti 25 tumor compositions comprising hyaluronate (HA) binding proteins and peptides, including HA link modules. The HA binding proteins are useful for treating metastatic cancers and angiogenesis-dependent diseases. In a preferred embodiment, the HA binding protein is metastatin protein. 30 Background of the Invention Several lines of direct evidence now suggest that the growth and persistence of solid tumors as well as their metastases to distant organs is critically dependent upon angiogenesis, or the 35 recruitment of new blood vessels (Folkman, 1989; Hori et al., WO 99/45942 PCT/US99/05498 2 1991; Kim et al., 1993; Millauer et al., 1994). 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 5 leave the primary site and enter the bloodstream (Zetter, 1998). In particular, 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 10 vessels (Zetter, 1998). It is postulated that the 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). Tumors themselves, 15 along with other accessory host cells such as macrophages, mast cells and lymphocytes, stimulate angiogenesis by up-regulating their production of a variety of angiogenic factors, including the fibroblast growth factors (FGF and BFGF) (Kandel et al., 1991) and vascular endothelial cell growth factor/vascular permeability 20 factor (VEGF/VPF) (Zetler, 1998). However, many malignant tumors also generate inhibitors of angiogenesis, including angiostatin and thrombospondin (Chen et al., 1995; Good et al., 1990; O'Reilly et al., 1994). Another factor that plays an important role in angiogenesis is 25 hyaluronate (HA). 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 30 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). On the contrary, in tissue development and remodeling, vascular growth requires degradation of high molecular weight HA into fragments in order WO 99/45942 PCT/US99/05498 3 to promote endothelial cell proliferation in vitro and facilitate angiogenesis in vivo (Deed et al.,1997; West et al., 1995). Prior to the present invention, the HA binding protein super family included CD44, cartilage link protein, hyaluronectin, 5 aggrecan, versican, receptor hyaluronan-mediated motility (RHAMM), inter-alpha trypsin inhibitor, intracellular hyaluronan binding protein (IHABP), I-CAM 1 and TSG-6. Interestingly, normal, quiescent endothelial cells do not express HA receptors such as CD44. Only endothelial cells that have been stimulated 10 with a pro-inflammatory stimulus or a growth factor such as bFGF (Griffioen et al., 1990; Mohamadzadeh et al., 1998), tumor associated endothelial cells, and endothelial cells from placental tissue express HA binding proteins. Because tumor cells express HA binding proteins such as 15 CD44, it has been postulated that the binding of CD44 and HA facilitate tumor angiogenesis and metastasis (Banerjee et al., 1992; Thomas et al., 1992; Goebele et al., 1996; Sy et al., 1991; Nakano et al., 1996; Hart et al., 1991; East et al., 1993; Culty et al., 1994). Indeed, it has been shown that increased CD44 expression often 20 correlates with increased metastatic invasiveness of breast, bladder, and melanoma cancer cells. Apparently, 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., 25 1992; Goebele et al., 1996). For example, after intravenous injection in C57BL/6J mice, B 16BL6 melanoma cells were shown to extravasate to the lungs via binding to pulmonary endothelium-associated HA. The importance of HA to the study of angiogenesis is also 30 evidenced in the prior investigation of cartilage, and more particularly, the lack of vascularization of cartilage. One of the most striking features of normal cartilage is the complete absence of blood vessels. In cartilaginous tissues, formation of blood vessels is observed only in embryogenesis and under pathological 35 conditions, such as rheumatoid arthritis (RA) and osteoarthritis WO 99/45942 PCT/US99/05498 4 (OA), usually, after extensive cartilage fragmentation (Culty et al., 1994). For example, in embryogenesis, avascular cartilage differentiates to hypertrophic cartilage that undergoes erosion and vascularization leading to bone formation. Presumably, 5 degradation of cartilage leads either to release and activation of pro-angiogenic factors stored away in the cartilage extracellular matrix or to neutralization of its inhibitory activity. Although poorly understood, the lack of vascularization has been attributed to the presence of angiogenic inhibitors that prevent endothelial 10 cell invasion of the cartilage extracellular matrix. The quest for the identification of proteins or small organic molecules responsible for the avascular character of cartilage started more than 20 years ago. Langer et al. first reported of a bovine cartilage fraction isolated by guanidine extraction and 15 purified by trypsin affinity chromatography that inhibited tumor-induced vascular proliferation (1976). Subsequently, Lee et al. described a guanidine extracted angiogenesis inhibitor of shark cartilage that suppressed tumor vascularization (1983). In addition, Moses et al. isolated an inhibitor of endothelial cell 20 proliferation and migration using NaCl extraction of bovine cartilage (1990). However, none of these inhibitors were identified and the mechanism of their antiangiogenic action remains unknown. 25 Summary of the Invention 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 30 activity. In particular, 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 WO 99/45942 PCT/US99/05498 5 animals. HA binding proteins can also comprise an HA binding link module. In a preferred embodiment of the present invention, the HA binding protein is metastatin protein. Metastatin protein is an 5 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. In 10 another preferred embodiment, 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 15 protein is provided in SEQ ID NO: 1. It is to be understood that the HA binding proteins and fragments thereof, including metastatin protein, can be animal or human in origin. The proteins, and fragments thereof, of the present invention can also be produced synthetically by chemical 20 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 25 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 30 techniques, including, but not limited to, positron emission tomography, autoradiography, flow cytometry, radioreceptor binding assays, and immunohistochemistry. These HA binding proteins also act as agonists and antagonists at the HA binding link module receptor, thereby enhancing or blocking the biological 35 activity of HA binding proteins.

WO 99/45942 PCT/US99/05498 6 Additionally, 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 5 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 10 measure biosynthesis of HA binding proteins in tissues and cells. The 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 15 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 20 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. 25 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 30 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, 35 HA binding link module fragments, HA binding link module WO 99/45942 PCT/US99/05498 7 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 5 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 10 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 15 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 20 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 25 fluids. The diagnostic method and kit can be in any configuration well known to those of ordinary skill in the art. Accordingly, it is an object of the present invention to provide a composition comprising an isolated HA binding protein, which inhibits tumor growth and metastasis. 30 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. It is a further object of the present invention to provide a 35 composition comprising an active fragment of metastatin protein.

WO 99/45942 PCT/US99/05498 8 It is a further object of the present invention to provide a composition comprising at least a portion of an HA binding link module. It is a further object of the present invention to provide a 5 composition comprising at least a portion of a proteoglycan tandem repeat domain of a cartilage link protein. It is another object of the present invention to provide nucleic acids that correspond to the HA binding link proteins and peptides herein. 10 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. It is another object of the present invention to provide a method of treating diseases and processes that are mediated by 15 angiogenesis and/or endothelial cell migration. It is another object of the present invention to provide a composition for treating or preventing the metastasis or growth of a cancer. It is an object of present invention to provide a method for 20 detecting and quantifying the presence of an antibody specific for an HA binding link proteins and peptides in a body fluid or tissue. It is yet another object of the present invention to provide a diagnostic or prognostic method and kit for detecting the presence and amount of HA binding link proteins and peptides in a body 25 fluid or tissue. It is another object of the present invention to provide a method for the detection or prognosis of cancer. It is another object of the present invention to provide a composition for use in visualizing and quantitating sites of HA 30 binding link protein and peptide binding in vivo and in vitro. It is yet another object of the present invention to provide a composition for use in detection and quantification of HA binding link protein and peptide biosynthesis. These and other objects, features and advantages of the 35 present invention will become apparent after a review of the WO 99/45942 PCT/US99/05498 9 following detailed description of the disclosed embodiments and the appended claims. Brief Description of the Figures 5 Figure 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 10 inhibited HUVEC migration in a dose dependent manner. Each bar represents the mean + SEM. Figure 2 A-B: Inhibition of Pulmonary Metastases in the B16BL6 Melanoma Experimental Metastatic Tumor Model. 15 After injection of B16BL6 melanoma cells (5 x 104) 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 20 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 25 lung carcinoma experimental metastatic tumor model. After injection of Lewis lung carcinoma cells (5 x 104) into 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 30 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.

WO 99/45942 PCT/US99/05498 10 Heat-inactivation and pre-incubation with HA neutralized the metastatin anti-tumor activity. Detailed Description of the Invention 5 Applicants have discovered a new class of protein molecules that have the ability to inhibit endothelial cell migration in vitro as well as tumor growth and tumor metastasis in vivo. These protein molecules have been functionally defined as hyaluronate (HA) binding proteins and peptides, however, it is to be understood that 10 this functional definition in no way limits the bioactivity of HA binding proteins. Important terms that are used herein are defined as follows. The terms "a", "an" and "the" as used herein are defined to mean one or more and include the plural unless the context is 15 inappropriate. "Regression" refers to the reduction of tumor mass or size. Additionally, "substantial 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 20 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 25 from components which normally accompany it as found in its native state. Typically, 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. 30 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. 35 Although not wanting to be bound by this statement, it is WO 99/45942 PCT/US99/05498 11 hypothesized that 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 5 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 10 complete amino acid sequence of a human cartilage link protein is provided in SEQ ID NO: 1. 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. The invention further provides anti-tumor peptides 15 comprising at least a portion of a proteoglycan terminal repeat domain. In another preferred embodiment, 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 20 human cartilage link protein. It will also be appreciated that the term "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 25 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. 30 One example of such a modification is the addition of tyrosine to the first position. Tyrosine labeled molecules may be further labeled with 125iodine 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 35 metastatin receptors.

WO 99/45942 PCT/US99/05498 12 Additionally, 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 5 acid does not significantly alter the structure, conformation or activity of the protein. Also included in the definition of the term "hyaluronic acid binding link module" are modifications of the protein, its subunits and peptide fragments. Such modifications include substitutions of naturally occurring amino acids at specific 10 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. 15 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, 20 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 25 such as the polymerase chain reaction (PCR), and gene amplification from RNA sources using amplification techniques such as reverse transcriptase/PCR. The HA binding proteins described herein have a variety of uses. For example, the HA binding proteins may be employed to 30 treat metastatic and angiogenesis-dependent cancers and other angiogenesis-related diseases. One example of 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 35 tumor metastases; benign tumors, for example hemangiomas, WO 99/45942 PCT/US99/05498 13 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, 5 neovascular glaucoma, retrolental fibroplasia, rubeosis; Osler Webber Syndrome; myocardial angiogenesis; plaque neovascularization; telangiectasia; hemophiliac joints; angiofibroma; and wound granulation. The HA binding proteins of the present invention are useful in the treatment of disease of 10 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 15 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 20 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 25 group or label is commonly a fluorescent or radioactive group or an enzyme. Such applications provide important diagnostic and research tools. HA binding proteins and peptides can also be employed to develop affinity columns for isolation of HA binding link module 30 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.

WO 99/45942 PCT/US99/05498 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 5 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 10 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. 15 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 20 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 25 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 30 fragment. Alternatively, 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 35 the entire sequence may be synthesized or a series of smaller WO 99/45942 PCT/US99/05498 15 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 5 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. 10 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 15 isolation or purification techniques. The 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. 20 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 25 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 30 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 35 laboratory manuals such as "Molecular Cloning: A Laboratory WO 99/45942 PCT/US99/05498 16 Manual" Second Edition by Sambrook et al., Cold Spring Harbor Press, 1989. Another method of producing HA binding proteins, or biologically active fragments thereof, is by peptide synthesis. 5 Once the amino acid sequence of the peptide is known, 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. Similarly, multiple fragments can be synthesized which 10 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 15 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. 20 Results from these tests can be used to diagnose or predict the occurrence or recurrence of a cancer and other angiogenesis mediated diseases. Additionally, passive antibody therapy using antibodies that specifically bind hyaluronic binding link modules can be employed 25 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. Also, antisera directed to the Fab regions of hyaluronic acid binding link module antibodies can be 30 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 35 making monoclonal antibodies is a modified version of the method WO 99/45942 PCT/US99/05498 17 of Kearney et al. (1979), which is incorporated by reference herein. The monoclonal antibodies are utilized in well known immunoassay formats, such as competitive and non-competitive immunoassays, including ELISA, sandwich immunoassays and 5 radioimmunoassays (RIAs), to determine the presence or absence of the HA binding proteins of the present invention in body fluids and tissues. Examples of body fluids include, but are not limited to, blood, serum, synovial fluid, peritoneal fluid, pleural fluid, cerebrospinal fluid, uterine fluid, saliva, mucus and vitreous 10 humor. Examples of 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. For example, polyclonal antisera may be raised in mice, rabbits, rats, goats, 15 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 20 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 25 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 30 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 WO 99/45942 PCT/US99/05498 18 counter. The highest titer antisera are also purified on affinity columns which are commercially available. Techniques for the production of single chain antibodies are known to those skilled in the art and described in U.S. Patent No. 5 4,946,778 and can be used to produce single chain antibodies to the HA binding proteins described herein. 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 10 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. When labeled with a detectable biomolecule or chemical, the HA binding proteins and antibodies described above are useful for 15 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. 20 For example, the HA binding proteins and antibodies are conjugated to a radiolabel such as, but not restricted to, 32 p, 3 H, 14 C, 35S, 125I, or 131I. Detection of a label can be by methods such as scintillation counting, gamma ray spectrometry or autoradiography. 25 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 30 as luciferase, catalyzes a reaction with ATP causing the bioluminescent molecule to emit photons of light. Fluorogens may also be used as labels. Examples of fluorogens include fluorescein and derivatives, phycoerythrin, allo-phycocyanin, phycocyanin, rhodamine, and Texas Red. The 35 fluorogens are generally detected by a fluorescence detector.

WO 99/45942 PCT/US99/05498 19 The HA binding proteins and antibodies can alternatively be labeled with a chromogen to provide an enzyme or affinity label. For example, the hyaluronic acid binding link module or antibody can be biotinylated so that it can be utilized in a biotin-avidin 5 reaction, which may also be coupled to a label such as an enzyme or fluorogen. Alternatively, 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. Additives such as 5-amino-2,3-dihydro 10 1,4-phthalazinedione (also known as Luminol T M ) (Sigma Chemical Company, St. Louis, MO) and rate enhancers such as p-hydroxybiphenyl (also known as p-phenylphenol) (Sigma Chemical Company, St. Louis, MO) can be used to amplify enzymes such as horseradish peroxidase through a luminescent 15 reaction; and luminogeneic or fluorogenic dioxetane derivatives of enzyme substrates can also be used. Such labels can be detected using enzyme-linked immunoassays (ELISA) or by detecting a color change with the aid of a spectrophotometer. In addition, HA binding proteins or antibodies may be labeled with colloidal gold 20 for use in immunoelectron microscopy in accordance with methods well known to those skilled in the art. The proteins, nucleic acid sequences and antibodies of the present invention are useful for diagnosing and treating metastatic and angiogenesis-related diseases. One example of metastatic 25 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, acoustic neuromas, neurofibromas, trachomas, and 30 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; Osler Webber Syndrome; myocardial angiogenesis; plaque 35 neovascularization; telangiectasia; hemophiliac joints; WO 99/45942 PCT/US99/05498 20 angiofibroma; and wound granulation. 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, 5 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). A particularly important aspect of the present invention is 10 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 15 metastatin peptide having the amino acid sequence of SEQ ID NO:2 was found to inhibit HUVEC migration in vitro. Accordingly, 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 20 agent by reducing or preventing uterine vascularization required for embryo implantation. Thus, 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. In one aspect of the birth control 25 method, 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 30 of vascularization of the uterine endometrium interferes with implantation of the blastocyst. Similar inhibition of vascularization of the mucosa of the uterine tube interferes with implantation of the blastocyst, preventing occurrence of a tubal pregnancy. Administration methods may include, but are not 35 limited to, pills, injections (intravenous, subcutaneous, WO 99/45942 PCT/US99/05498 21 intramuscular), suppositories, vaginal sponges, vaginal tampons, and intrauterine devices. It is also believed that administration of HA binding proteins will interfere with normal enhanced vascularization of the placenta, and also with the development of 5 vessels within a successfully implanted blastocyst and developing embryo and fetus. Conversely, blockade of HA binding protein and peptide receptors with HA binding protein and peptide analogs which act as receptor antagonists may promote endothelialization and 10 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 15 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, 20 including but not limited to the cerebral cortex and spinal cord. According to the present invention, HA binding proteins may be used in combination with other compositions and procedures for the treatment of the above described diseases and conditions. For example, a tumor may be treated conventionally 25 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. The proteins and protein fragments with the HA binding 30 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, 35 the combinations may be administered by the topical, transdermal, WO 99/45942 PCT/US99/05498 22 intraperitoneal, intracranial, intracerebroventricular, intracerebral, intravaginal, intrauterine, oral, rectal or parenteral (e.g., intravenous, intraspinal, subcutaneous or intramuscular) route. In addition, the hyaluronic acid binding link module may be 5 incorporated 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 10 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 15 hereby incorporated by reference in its entirety. The dosage of 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 20 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 25 mg/kilogram. Depending upon the half-life of the HA binding protein and peptide in the particular animal or human, 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 30 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), 35 intrauterine, vaginal or parenteral (including subcutaneous, WO 99/45942 PCT/US99/05498 23 intraperitoneal, intramuscular, intravenous, intradermal, intracranial, intratracheal, and epidural) administration. The HA binding protein and peptide formulations may conveniently be presented in unit dosage form and may be prepared by 5 conventional pharmaceutical techniques. Such techniques include the step of bringing into association the active ingredient and the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely 10 divided solid carriers or both, and then, if necessary, shaping the product. Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which 15 render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials, and may be 20 stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described. 25 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 30 invention may include other agents conventional in the art having regard to the type of formulation in question. In addition to methods of treatment, the present invention also relates to methods of using HA binding proteins, and active fragments thereof, nucleic acid sequences corresponding to HA 35 binding proteins and active peptide fragments thereof, and WO 99/45942 PCT/US99/05498 24 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 5 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 10 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. These assay kits include but 15 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 20 dipsticks for rapid monitoring of urine or blood, and immunocytochemistry. For each kit, the range, sensitivity, precision, reliability, specificity and reproducibility of the assay is established. Intra-assay and inter-assay variation is established at 20%, 50% and 80% points on the standard curves of displacement 25 or activity. 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 30 proteins or peptides in biological fluids and tissue extracts of animals and humans with and without tumors or angiogenesis dependent diseases. Also included in the present invention are kits for the localization of HA binding proteins or peptides in tissues and cells. 35 This HA binding protein and peptide immunohistochemistry kit WO 99/45942 PCT/US99/05498 25 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. 5 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 10 purposes. 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 purposes in the detection and treatment of cancer. 15 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 20 techniques. These methods are important for the study of angiogenesis and metastasis in cancer and other angiogenesis related diseases. This invention is further illustrated by the following examples, which are not to be construed in any way as imposing 25 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 spirit of the present 30 invention and/or the scope of the appended claims.

WO 99/45942 PCT/US99/05498 26 Example 1 Isolation of a cartilage-derived inhibitor of endothelial cell migration Bovine nasal cartilage shredded with a superform blade was 5 disassociated with 4M guanidinium HC1 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 10 HUVECs in a migration assay. In this 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 15 (Fig. lA). 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. Example 2 20 Identification of cartilage-derived endothelial cell migration inhibitor HA-affinity chromatography was employed to purify a cartilage-derived preparation with the inhibitory activity. The trypsonized material was loaded onto an HA affinity 25 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 30 protein was responsible for the inhibitory effect observed in the migration assay described as follows. 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 35 containing either bFGF (5 ng/ml) alone or bFGF (5 ng/ml) and WO 99/45942 PCT/US99/05498 27 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 5 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. Amino-terminal analysis of the purified product gave an 10 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). Indeed, purified metastatin protein was immunoreactive toward an antibody raised 15 against LP (Fig. IB). Metastatin protein inhibited bFGF-induced migration of HUVECs with an ICs 0 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). 20 Example 3 Inhibition of pulmonary metastases in the B16BL6 melanoma experimental metastatic tumor model by Metatstatin The anti-metastatic potential of metastatin was tested in the 25 B16BL6 experimental metastatic tumor model. Bl6BL6 melanoma cells (5x 104) 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. 30 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 35 neutralized the anti-metastatic activity of the protein.

WO 99/45942 PCT/US99/05498 28 Neutralization was also observed after pre-incubation of approximately 20 nmol/kg or 25 kg/animal of metastatin with a 100-fold excess of HA (Fig. 2A). Metastatin had no effect on serum driven proliferation of B16BL6 cells in vitro (data not 5 shown) and the anti-metastatic effect was not due to a toxic effect. Macropathology of the lungs of animals that received either metastatin or vehicle control are shown in Figure 2B. Example 4 10 Inhibition of pulmonary metastases in the Lewis lung carcinoma experimental metastatic tumor model by Metatstatin Lewis lung carcinoma cells (5 x 104) were injected into the tail vein of C57BL/6J mice. The animals were then treated intraperitoneally with increasing doses of metastatin. Treatment of 15 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 20 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. 25 Example 5 Inhibition of Bl6 tumor growth in a chick CAM assay by Metastatin synthetic peptides Metastatin was used in a chick CAM assay (Brooks et al. 30 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.

WO 99/45942 PCT/US99/05498 29 Example 6 Endothelial cell migration inhibition of HA Binding Peptide A peptide of selected from the proteoglycan terminal repeat domain of the HA link protein, having the sequence 5 QYPITKPREP (SEQ ID NO:2) was tested for endothelial cell migration inhibition activity. 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 10 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 15 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. Example 7 20 Inhibition of pulmonary metastases in the B16BL6 melanoma experimental metastatic tumor model by an HA Binding Peptide The anti-metastatic potential of the peptide of Example 6 was tested in the B 16BL6 experimental metastatic tumor model. B 16BL6 melanoma cells (5 x 104) were injected into the tail vein of 25 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 30 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. 35 WO 99/45942 PCT/US99/05498 30 References The following references are hereby incorporated by reference herein in their entirety. 5 Banerjee, S.D. & Toole, B.P. (1992). Hyaluronan-binding protein in endothelial cell morphogenesis. J. Cell Biol. 119, 643-652. Brem et al. (1991) J. Neurosurg. 74:441-446. 10 Chen, C., Parangi, S., Tolentino, M. J., and Folkman, J. (1995). A strategy to discover circulating angiogenesis inhibitors generated by human tumors. Cancer Res. 55, 4230-4233. Culty, M., Shizari, M., Thompson, E.W., & Underhill, C.B. 15 (1994). Binding and degradation of hyaluronan by human breast cancer cell lines expressing different forms of CD44: correlation with invasive potential. J Cell. Physiol. 160, 275-286. Deed, R. et al. (1997). Early-response gene signaling is induced by 20 angiogenic oligosaccharides of hyaluronan in endothelial cells. Inhibition by non-angiogenic, high-molecular-weight hyaluronan. Int. J. Cancer 71, 251-256. East, J.A., Mitchell, S.D., & Hart, I.R. (1993). Expression and 25 function of the CD44 glycoprotein in melanoma cell lines. Melanoma Res. 3, 341-346. Folkman, J. (1996). Tumor angiogenesis and tissue factor. Nature Med. 2, 167-168. 30 Folkman, J. (1989). What is the evidence that tumors are angiogenesis dependent?. J. Natl. Cancer Inst. 82, 4-6. Folkman, J. (1985). Angiogenesis and its inhibitors. In Important 35 Advances in Oncology 1985, V. T. DeVita, S. Hellman, and S.

WO 99/45942 PCT/US99/05498 31 Rosenberg, eds. (Philadelphia: J.B. Lippincott Company), pp. 42-62. Folkman, J., Haundenschild, C. C., and Zetter, B. R. (1979). 5 Long-term culture of capillary endothelial cells. Proc. Natl. Acad. Sci. USA 76, 5217-5221. Garfinkel, S., Hu, X., Prudovsky, I. A., McMahon, G. A., Kapnik, E. M., McDowell, S. D., and Maciag, T. (1996). FGF-1 dependent 10 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. 15 Goebele, M., Kaufinann, D., Brocker, E.-B., & Klein, C.E. (1996). Migration of highly aggressive melanoma cells on hyaluronic acid is associated with functional changes, increased turnover and shedding of CD44 receptors. J. Cell Science 109, 1957-1964. 20 Good, D. J., Polverini, P. J., Rastinejad, F., Le Beau, M. M., Lemons, R. S., Frazier, W.A., and Bouck, N. P. (1990). 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. 25 Griffioen, A.W. et al. (1990). CD44 is involved in tumor angiogenesis; an activation antigen on human endothelial cells. Blood. 90, 1150-1159. 30 Hart, I.R., Birch, M., & Marshall, J.F. (1991). Cell adhesion receptor expression during melanoma progression and metastasis. Cancer Metastasis Rev. 10, 115-28. Hori, A., Sasada, R., Matsutani, E., Naito, K., Sakura, Y., Fujita, 35 T., and Kozai, Y. (1991). Suppression of solid tumor growth by WO 99/45942 PCT/US99/05498 32 immunoneutralizing monoclonal antibody against human basic fibroblast growth factor. Cancer Res. 51, 6180-6184. Kandel, J., Bossy-Wetzel, E., Radvany, F., Klagsburn, M., 5 Folkman, J., and Hanahan, D. (1991). Neovascularization is associated with a switch to the export of bFGF in the multistep development of fibrosarcoma. Cell 66, 1095-1104. Kearney et al. (1979). J. Immunol. 123:1548-1558. 10 Kim, K. J., Li, B., Winer, J., Armanini, M., Gillett, N., Phillips, H. S., and Ferrara, N. (1993). Inhibition of vascular endothelial growth factor-induced angiogenesis suppresses tumor growth in vivo. Nature 362, 841-844. 15 Langer, R., Brem, H., Falterman, K., Klein, M., and Folkman, J. (1976). Inhibition of a cartilage factor that inhibits tumor neovascularization. Science 193, 70-71. 20 Lee, A. and Langer, R. (1983). Shark cartilage contains inhibitors of tumor angiogenesis. Science 221, 1185-1187. Millauer, B., Shawver, L. K., Plate, K. H., Risau, W., and Ullrich, A. (1994). Glioblastoma growth inhibited in vivo by a 25 dominant-negative Flk-1 mutant. Nature 367, 576-579. Mohamadzadeh, M. et al. (1998). Proinflammatory stimuli regulate endothelial hyaluronan expression and CD44/HA-dependent primary adhesion. J. Clin. Invest. 101, 30 97-108. Moses, A.M., Sudhalter, J., and Langer, R. (1990). Identification of an inhibitor of neovascularization from cartilage. 248, 1388-1410. 35 WO 99/45942 PCT/US99/05498 33 Nakano, T., Matsui, T., & Ota, T. (1996). Benzyl-alpha-GalNAc inhibits sialylation of Oglycosidic sugar chains on CD44 and enhances experimental metastatic capacity in B I 6BL6 melanoma cells. Anticancer Res. 16, 3577-3784. 5 O'Reilly, M. S., Holmgren, L., Chen, C. C., and Folkman, J. (1996). Angiostatin induces and sustains dormancy of human primary tumors in mice. Nature Med. 2, 689-692. 10 O'Reilly, M. S., Holmgren, L., Shing, Y., Chen, C., Rosenthal, R. A., Moses, M., Lane, W. S., Cao, Y., Sage, E. H., and Folkman, J. (1994). Angiostatin: A novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis lung carcinoma. Cell 79, 315-328. 15 Parangi, S., O'Reilly, M., Christofori, G., Holmgren, L., Grosfeld, J., Folkman, J., and Hanahan, D. (1996). Antiangiogenic therapy of transgenic mice impairs de novo tumor growth. Proc. Natl. Acad. Sci. USA 93, 2002-2007. 20 Rastinejad, F., Polverini, P. J., and Bouck, N. P. (1989). Regulation of the activity of a new inhibitor of angiogenesis by a cancer suppressor gene. Cell 56, 345-355. 25 Sy, M.S., Guo, Y.-J., & Stamenkovic, I. (1991). Distinct effects of two CD44 isoforms on tumor growth in vivo. J. Exp. Med. 174, 859-866. Thomas, L., Byers, H.R., Vink, J., & Starnenkovic, I. (1992). CD44H regulates tumor cell migration on HA-coated substrate. J 30 Cell Biology 118, 971-977. West, D.C., Hampson, I.N., & Kunar, S.S. (1985). Angiogenesis induced by degradation products of hyaluronic acid. Science 228, 1324-1326. Zetter, B.C. (1998). Angiogenesis and Tumor Metastasis. Annu. 35 Rev. Med. 49, 387-424.

Claims (49)

1. An isolated hyaluronate (HA) binding protein, wherein the protein comprises at least a portion of an amino acid 5 sequence of a proteoglycan tandem repeat domain, and is further characterized by its anti-tumor activity.

2. The protein of Claim 1, wherein the protein is metastatin protein having a molecular weight of approximately 38 10 kDa as determined by non-reducing gel electrophoresis.

3. The protein of Claim 1, further comprising at least a portion of a cartilage link protein. 15

4. The protein of Claim 1, wherein the protein is a peptide having an amino acid sequence of QYPITKPREP (SEQ ID NO:2).

5. The protein of Claim 1, wherein the protein further 20 has an endothelial cell migration inhibitory activity.

6. A composition comprising the protein of Claim 1 and a pharmaceutically acceptable excipient. 25

7. A method of inhibiting the growth of a tumor comprising administering to the tumor a growth inhibiting amount of a hyaluronate (HA) binding protein, wherein the protein comprises at least a portion of an amino acid sequence of a proteoglycan tandem repeat domain, and is further characterized 30 by its anti-tumor activity.

8. The method of Claim 7, wherein the protein is metastatin protein having a molecular weight of approximately 38 kDa as determined by non-reducing gel electrophoresis. 35 WO 99/45942 PCT/US99/05498 35

9. The method of Claim 7, wherein the protein comprises at least a portion of a cartilage link protein.

10. The method of Claim 7, wherein the protein is a 5 peptide having an amino acid sequence of QYPITKPREP (SEQ ID NO:2).

11. The method of Claim 7, wherein the protein further has an endothelial cell migration inhibitory activity. 10

12. The method of Claim 7, wherein the tumor is metastatic. WO 99/45942 36 PCT/US99/05498 AMENDED CLAIMS [received by the International Bureau on 16 August 1999 (16.08.99); original claims 1-12 amended; new claims 13-49 added (6 pages)] 5 1. A method of inhibiting the growth of a tumor comprising administering to the tumor a growth inhibiting amount of a hyaluronate (HA) binding protein, wherein the protein has an amino acid sequence of at least a portion of a naturally occurring HA binding protein and has a HA binding activity and an anti 10 tumor activity. 2. The method of Claim 1, wherein the protein comprises at least a portion of an amino acid sequence of a proteoglycan tandem repeat domain. 15 3. The method of Claim 1, wherein the protein is metastatin protein having a molecular weight of approximately 38 kDa as determined by non-reducing gel electrophoresis. 20 4. The method of Claim 1, wherein the protein comprises at least a portion of a cartilage link protein. 5. The method of Claim 1, wherein the protein comprises at least a portion of an aggregan protein. 25 6. The method of Claim 1, wherein the protein comprises at least a portion of a naturally occurring HA binding protein selected from CD44, hyaluronectin, versican, receptor hyaluronan-mediated motility (RHAMM), inter-alpha trypsin 30 inhibitor, intracellular hyaluronan binding protein (IHABP), I CAM 1 and TSG-6. 7. The method of Claim 1, wherein the protein is recombinantly expressed. 35 8. The method of Claim 1, wherein the protein is recombinantly expressed in vivo. 9. The method of Claim 1, wherein the protein has an 40 amino acid sequence of QYPITKPREP (SEQ ID NO:2). AMENDED SHEET (ARTICLE 19) WO 99/45942 37 PCT/US99/05498 5 10. The method of Claim 1, wherein the protein further has an endothelial cell migration inhibitory activity. 11. A method of inhibiting angiogenesis comprising 10 administering to an endothelial cell an angiogensis inhibiting amount of a hyaluronate (HA) binding protein, wherein the protein has an amino acid sequence of at least a portion of a naturally occurring HA binding protein and has a HA binding activity and an anti-angiogenic activity. 15 12. The method of Claim 11, wherein the protein comprises at least a portion of an amino acid sequence of a proteoglycan tandem repeat domain. 20

13. The method of Claim 11, wherein the protein is metastatin protein having a molecular weight of approximately 38 kDa as determined by non-reducing gel electrophoresis.

14. The method of Claim 11, wherein the protein 25 comprises at least a portion of a cartilage link protein.

15. The method of Claim 11, wherein the protein comprises at least a portion of an aggregan protein. 30

16. The method of Claim 11, wherein the protein comprises at least a portion of a naturally occurring HA binding protein selected from CD44, hyaluronectin, versican, receptor hyaluronan-mediated motility (RHAMM), inter-alpha trypsin inhibitor, intracellular hyaluronan binding protein (IHABP), I 35 CAM 1 and TSG-6.

17. The method of Claim 11, wherein the protein is recombinantly expressed. AMENDED SHEET (ARTICLE 19) WO 99/45942 38 PCT/US99/05498 5

18. The method of Claim 11, wherein the protein is recombinantly expressed in vivo.

19. The method of Claim 11, wherein the protein has an amino acid sequence of QYPITKPREP (SEQ ID NO:2). 10

20. The method of Claim 11, wherein the protein further has an endothelial cell migration inhibitory activity.

21. An isolated hyaluronate (HA) binding protein, 15 wherein the protein has an amino acid sequence of a portion of a naturally occurring HA binding protein and has a HA binding activity and an anti-tumor activity.

22. The protein of Claim 21, wherein the protein 20 comprises at least a portion of an amino acid sequence of a proteoglycan tandem repeat domain.

23. The protein of Claim 21, wherein the protein is metastatin protein having a molecular weight of approximately 38 25 kDa as determined by non-reducing gel electrophoresis.

24. The protein of Claim 21, wherein the protein has an amino acid sequence of a portion of a cartilage link protein. 30

25. The protein of Claim 21, wherein the protein has an amino acid sequence of a portion of an aggregan protein.

26. The protein of Claim 21, wherein the protein comprises a portion of a naturally occurring HA binding protein 35 selected from CD44, hyaluronectin, versican, receptor hyaluronan mediated motility (RHAMM), inter-alpha trypsin inhibitor, intracellular hyaluronan binding protein (IHABP), I-CAM 1 and TSG-6. AMENDED SHEET (ARTICLE 19) WO 99/45942 39 PCT/US99/05498 5

27. The protein of Claim 21, wherein the protein is recombinantly expressed.

28. The protein of Claim 21, wherein the protein has an amino acid sequence of QYPITKPREP (SEQ ID NO:2). 10

29. The protein of Claim 21, wherein the protein further has an endothelial cell migration inhibitory activity.

30. A composition comprising a pharmaceutically 15 acceptable excipient and a first hyaluronate (HA) binding protein, wherein the first protein has an amino acid sequence of a portion of a first naturally occurring HA binding protein that has a HA binding activity and an anti-tumor activity. 20

31. The composition of Claim 30, further comprising at least a portion of a second hyaluronate (HA) binding protein, wherein the second protein has an amino acid sequence of a portion of a second naturally occurring HA binding protein that has a HA binding activity and an anti-tumor activity. 25

32. The composition of Claim 30, wherein the protein comprises at least a portion of an amino acid sequence of a proteoglycan tandem repeat domain. 30

33. The composition of Claim 30, wherein the protein is metastatin protein having a molecular weight of approximately 38 kDa as determined by non-reducing gel electrophoresis.

34. The composition of Claim 30, wherein the protein 35 comprises a portion of a cartilage link protein.

35. The composition of Claim 30, wherein the protein comprises a portion of an aggregan protein. ARAI"klirr1 ni irr-r /Arrr.I"- - - WO 99/45942 40 PCT/US99/05498 5

36. The composition of Claim 30, wherein the protein comprises a portion of a naturally occurring HA binding protein selected from CD44, hyaluronectin, versican, receptor hyaluronan mediated motility (RHAMM), inter-alpha trypsin inhibitor, intracellular hyaluronan binding protein (IHABP), I-CAM 1 and 10 TSG-6.

37. The composition of Claim 30, wherein the protein is recombinantly expressed. 15

38. The composition of Claim 30, wherein the protein has an amino acid sequence of QYPITKPREP (SEQ ID NO:2).

39. The composition of Claim 30, wherein the protein further has an endothelial cell migration inhibitory activity. 20

40. A nucleic acid coding for a hyaluronate (HA) binding protein, wherein the protein has an amino acid sequence of a portion of a naturally occurring HA binding protein and has a HA binding activity and an anti-tumor activity. 25

41. The nucleic acid of Claim 40, wherein the protein comprises at least a portion of an amino acid sequence of a proteoglycan tandem repeat domain. 30

42. The nucleic acid of Claim 40, wherein the protein is metastatin protein having a molecular weight of approximately 38 kDa as determined by non-reducing gel electrophoresis.

43. The nucleic acid of Claim 40, wherein the protein has 35 an amino acid sequence of a portion of a cartilage link protein.

44. The nucleic acid of Claim 40, wherein the protein has an amino acid sequence of a portion of an aggregan protein. WO 99/45942 41 PCT/US99/05498 5

45. The nucleic acid of Claim 40, wherein the protein comprises a portion of a naturally occurring HA binding protein selected from CD44, hyaluronectin, versican, receptor hyaluronan mediated motility (RHAMM), inter-alpha trypsin inhibitor, intracellular hyaluronan binding protein (IHABP), I-CAM 1 and 10 TSG-6.

46. The nucleic acid of Claim 40, wherein the protein is recombinantly expressed. 15

47. The nucleic acid of Claim 40, wherein the protein is recombinantly expressed in vivo..

48. The nucleic acid of Claim 40, wherein the protein has an amino acid sequence of QYPITKPREP (SEQ ID NO:2). 20

49. The nucleic acid of Claim 40, wherein the protein further has an endothelial cell migration inhibitory activity. AMENDED SHEET (ARTICLE 19)