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  Compositions of low molecular weight heparin produced with modified heparinase iiiUSPTO Application #: 20060183713Title: Compositions of low molecular weight heparin produced with modified heparinase iii Abstract: The invention relates to heparinase III and mutants thereof. Modified forms of heparinase III having reduced enzymatic activity which are useful for a variety of purposes, including sequencing of heparin-like glycosaminoglycans (HLGAGs), removing active heparan sulfate from a solution, inhibition of angiogenesis, etc. have been discovered according to the invention. The invention in other aspects relates to methods of treating cancer and inhibiting tumor cell growth and/or metastasis using heparinase III, or products produced by enzymatic cleavage by heparinase III of HLGAGs. (end of abstract)
Agent: Wolf Greenfield & Sacks, PC - Boston, MA, US Inventors: Dongfang Liu, Kevin Pojasek, Zachary Shriver, Kristine Holley, Yosuf El-Shabrawi, Ganesh Venkataraman, Ram Sasisekharan USPTO Applicaton #: 20060183713 - Class: 514056000 (USPTO) Related Patent Categories: Drug, Bio-affecting And Body Treating Compositions, Designated Organic Active Ingredient Containing (doai), O-glycoside, Polysaccharide, Heparin Or Derivative The Patent Description & Claims data below is from USPTO Patent Application 20060183713. Brief Patent Description - Full Patent Description - Patent Application Claims
RELATED APPLICATIONS [0001] This application is a divisional application of U.S. Non-Provisional application Ser. No. 10/291,337, filed Nov. 8, 2002, which is a divisional application of U.S. Non-Provisional application Ser. No. 09/802,285, filed Mar. 8, 2001, now issued as U.S. Pat. No. 6,869,789, which claims priority to U.S. Provisional Patent Application Ser. No. 60/187,846, filed Mar. 8, 2000, the entire contents of each of which are incorporated herein by reference in their entirety. FIELD OF THE INVENTION [0003] The invention relates to heparinase III and mutants thereof. In particular, the invention relates to modified forms of heparinase III having reduced enzymatic activity which are useful for a variety of purposes, including sequencing of heparin-like glycosaminoglycans (HLGAGs), removing HLGAGs from a solution, inhibition of angiogenesis, inhibiting coagulation, etc. The invention in other aspects relates to methods of treating cancer and inhibiting tumor cell growth and/or metastasis using heparinase III, or HLGAG products produced by enzymatic cleavage with heparinase III. BACKGROUND OF THE INVENTION [0004] Heparin like glycosaminoglycans (HLGAGs) are important components of the extracellular matrix that are believed to regulate a wide variety of cellular activities including invasion, migration, proliferation and adhesion. Khodapkar, et al. 1998; Woods, et al., 1998) HLGAGs accomplish some of these functions by binding to and regulating the biological activities of diverse molecules, including growth factors, morphogens, enzymes, extracellular proteins. HLGAGs are linear polysaccharides characterized by a disaccharide-repeat unit of a uronic acid [.alpha.-L-iduronic acid (I) or .beta.-D-glucuronic acid (G)] linked 1, 4 to .alpha.-B-hexosamine (H). (1) These polymers of 20-100 disaccharide units can be additionally modified through N-- and O-sulfation, epimerization at the C5 position of the uronic acid moiety, adding an additional micro-heterogenecity to these information dense molecules. (1.5). [0005] Although the structure and chemistry of HLGAGs are fairly well understood, information on how specific HLGAG sequences modulate different biological processes has proven harder to obtain. The inventors have recently developed a rapid sequencing methodology for polysaccharides using chemical and enzymatic tools to modify or degrade an unknown HLGAG polymer in a sequence-specific manner. (Venkataraman, G., et al., Science, 286, 537-542 (1999), and U.S. patent applications Ser. Nos. 09/557,997 and 09/558,137, both filed on Apr. 24, 2000). An important enzymatic tool in this sequencing process is the heparinases, including heparinases I, II and III. The three heparinases are HLGAG degrading enzymes which can be produced by Flavobacterium heparinum. Each of the heparinases has its own unique HLGAG sequence at which it cleaves, making these enzymes valuable tools in obtaining sequence specific information. Heparinase I primarily cleaves HLGAGs at the H.sub.NS,6XI.sub.2S.sup.2-linkage found primarily in heparin-like regions (Ernst, S., et al., Crit, Rev. Biochem. Mol. Biol., 30, 387-444 (1995)). Desai, U., et al., Biochemistry, 32, 8140-8145 (1993)), and Jandik, K., et al., Glycobiology, 4, 289-296 (1994)). Heparinase III cleaves at the H.sub.NAC-I and H.sub.NY,6X-G.sup.2 linkages which are the major disaccharides found in heparan sulfate (Ernst, et al., (1995), supra, and Linhardt, R., et al., Biochemistry, 29, 2611-2617 (1990)). Heparinase II is capable of recognizing and cleaving both sets of substrate linkages (Ernst, et al., (1995), supra). We have recently identified several residues which are critical to the activity of heparinase I and heparinase II. Cysteine 135 and histidine 203, as well as lysines 198, 199, and 132 of heparinase I were found to be critical to the enzymatic activity of the molecule. Cysteine 348 and histidines 238, 451, and 579 were determined to be crucial for heparinase II activity. (Pending U.S. patent application Ser. No. 09/384,959; Sasisekharan, R., et al., Biochemistry, 34, 14441-14448 (1995); Godavarti, R., et al., Biochemistry, 35, 6846-6852 (1996); Godavarti, R., and Sasisekharan, R., J. Biol. Chem. 273, 248-255 (1998); Shriver, Z., et al., J. Biol. Chem., 273, 22904-22912 (1998); and Shriver, Z., J. Biol. Chem., 273, 10160-10167 (1998)). [0006] Heparinase III is unique in that it is the only member of the heparinase family that recognizes and preferentially cleaves heparan sulfate. Heparinase III also contains no cysteines in its amino acid sequence. [0007] Tumor metastasis involves the spread of tumor cells primarily via the vasculature to remote sites in the body. It is believed that as the extracellular matrix is degraded, the tumor cell-extracellular matrix interactions are disassembled, freeing the tumor cell to extravagate through the capillary bed. Extraordinary progress has been made to elucidate the roles of collagen and related proteins, enzymes (collagenases and others) that degrade the extracellular matrix proteins to regulate tumor angiogenesis and/or tumor cell invasion. It has also recently been hypothesized that HLGAG degrading enzymes, heparinases, assist in the breakdown of the extracellular matrix to regulate tumor growth, angiogenesis and metastasis. It has been suggested that the expression of heparinases in association with tumor development, represents a switch from a metastatic tumor to a non-metastatic tumor and plays a role in initiating the process of metastasis. The hypothesis was reaffirmed by recent cloning of a human heparinase gene and by the demonstration of enhanced malignancy of cancer cells by over-expression of the gene product for heparinase. (Hulett, et al., 1999 and Vlodavsky, et al., 1999). SUMMARY OF THE INVENTION [0008] It has been discovered, according to one aspect of the invention, that expression of heparinases does not necessarily represent a switch from a primary tumor to a metastatic diseased state. Consistent with the current paradigm, heparinase I activity was found to accelerate tumor growth and correlate with increased metastasis. Surprisingly, heparinase III, however, was found to inhibit primary tumor growth and significantly reduce metastasis. Thus, in one aspect the invention is a method for preventing growth of a tumor by exposing a tumor cell to an effective amount of heparinase III for preventing proliferation of the tumor cells in order to prevent growth of the tumor. In other aspects, the invention is a method for preventing tumor cell metastasis by exposing a tumor cell to an effective amount of heparinase III for preventing invasion of the tumor cell across a barrier. The heparinase III may be a native heparinase III molecule or a modified heparinase III molecule. Native heparinase III may be synthesized or isolated. [0009] Additionally, it has been discovered according to the invention that therapeutic HLGAG fragments can be used to treat cancer. These fragments are useful for preventing the growth of a tumor as well as preventing metastasis. These fragments can be generated by heparinase III treatment of cancer cells. The fragments generated from the heparinase III treatment of a cancer cell can be used to prevent or treat cancer from the same or different cancer cells than are used to generate the fragments. Additionally, they can be used to treat or prevent cancer in the same or a different subject than was used to generate the fragments. [0010] The tumor cell can be exposed to the heparinase III by any method known in the art. For instance, when the tumor cell is a tumor cell in vitro, heparinase III may be added to the in vitro culture. When the tumor cell is in vivo, the heparinase III may be administered by any method for delivering the heparinase III to the tumor. For instance, in some embodiments the heparinase III may be administered systemically, such as by oral delivery, injection, etc. or locally, such as by direct injection into the tumor or tumor site or by direct application during surgical manipulation, etc. [0011] The heparinase III may be administered alone or in conjunction with other therapies such as an anti-cancer drug. In some embodiments, the tumor is a prostate tumor or a melanoma. [0012] In other aspects, the invention is a method for preparing therapeutic agents for the treatment of a tumor. The method involves isolating at least a portion of a tumor, treating the portion of the tumor with heparinase III to produce HLGAG fragments, and isolating the HLGAG fragments, wherein the HLGAG fragments are the therapeutic agent. In some embodiments, the method may also include the step of determining the sequence of the HLGAG fragments. [0013] In other aspects of the invention, a method for treating a subject having a tumor is provided. The method involves administering to the subject therapeutic HLGAG fragments to treat the tumor. Optionally the method may involve identifying a therapeutic HLGAG fragment by identifying an HLGAG produced when the tumor is contacted with heparinase III. In some embodiments, the therapeutic HLGAG fragment is a synthetic HLGAG fragment generated based on the sequence of the HLGAG fragment identified when the tumor is contacted with heparinase III. In other embodiments, the HLGAG fragment administered to the subject is an isolated HLGAG fragment produced when the tumor is contacted with the heparinase III. [0014] In another aspect the invention is a method for treating or preventing a subject having a cancer or at risk of developing a cancer by administering to the subject a therapeutic HLGAG fragment. In some embodiments the therapeutic HLGAG fragment is a composition of HLGAG fragments wherein at least 50%, 75%, or 90% of the HLGAG fragments are di- or tri-sulfated disaccharides. In other embodiments the therapeutic HLGAG fragment is free of mono- or un-sulfated disaccharides. [0015] According to another aspect of the invention, a composition is provided. The composition includes heparinase III or a therapeutic HLGAG fragment in an effective amount for preventing metastasis of a tumor cell, and a targeting molecule for targeting the heparinase III to the tumor, in a pharmaceutically-acceptable carrier. In some embodiments the heparinase III is a modified heparinase III and in other embodiments it is a native heparinase III. The targeting molecule may be, for instance, a compound which binds specifically to an antigen on the surface of a tumor cell. [0016] The invention in another aspect is a composition of a heparinase III or a therapeutic HLGAG fragment in an effective amount for preventing metastasis of a tumor cell, and an anti-cancer compound in a pharmaceutically-acceptable carrier. [0017] The invention in other aspects is based on the identification of important residues within the heparinase III molecule. It has been discovered according to the invention that modification of certain histidine residues within the heparinase III molecule causes changes in the enzymatic rate of the enzyme as well as the product profile produced by the enzyme. In particular, it was discovered that histidine 295 and histidine 510 are important for enzymatic degradation of heparan sulfate by heparinase III. When these two histidines were changed to other amino acids, all of the activity of the enzyme was lost. Modification of the other histidine residues resulted in changes in kinetic constants of the enzyme, but the enzyme still retained activity. Thus, in another aspect the invention is a substantially pure heparinase III comprising a polypeptide having the amino acid sequence of the mature peptide of SEQ ID NO:2 or having conservative substitutions thereof within residues non-essential to enzymatic function, wherein at least one histidine residue selected from the group consisting of His 36, His 105, His 110, His 139, His 152, His 225, His 234, His 241, His 424, His 469, and His 539 has been substituted with a residue selected from the group consisting of alanine, serine, tyrosine, threonine, and lysine. In some embodiments the polypeptide has a substitution at His 110 or His 241. In other aspects, the invention is a substantially pure heparinase III comprising a polypeptide having the amino acid sequence of the mature peptide of SEQ ID NO:2 or having conservative substitutions thereof within residues non-essential to enzymatic function, wherein at least one histidine residue selected from the group consisting of His 295 and His 510 has been substituted with any other amino acid. [0018] In another aspect, the invention is a substantially pure heparinase III which is a modified heparinase III having a modified product profile, wherein the modified product profile of the modified heparinase III is at least 10% different than a native product profile of a native heparinase III. [0019] In another aspect, the invention is a substantially pure heparinase III that is a modified heparinase III that can cleave a HLGAG substrate having a modified heparinase III k.sub.cat value, wherein the modified heparinase III k.sub.cat value is at least 10% different than a native heparinase III k.sub.cat value. The invention also encompasses pharmaceutical preparations of any of the substantially pure heparinase III molecules with a pharmaceutically-acceptable carrier. The invention also encompasses the modified heparinase III of the invention immobilized on a solid support membrane. [0020] A method of specifically cleaving a HLGAG is provided according to another aspect of the invention. The method of specifically cleaving a HLGAG includes the step of contacting an HLGAG with the modified heparinase III of the invention. In some embodiments, the method is a method for preventing tumor cell proliferation or metastasis, as described above. In other embodiments, the method is a method for sequencing HLGAGs. In yet other embodiments, the method is a method for removing active HLGAGs from an HLGAG-containing fluid, a method for inhibiting angiogenesis, a method for inhibiting neovascularization, e.g., such as that associated with eye disease, a method for treating psoriasis, or a method for inhibiting coagulation. Continue reading... Full patent description for Compositions of low molecular weight heparin produced with modified heparinase iii Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Compositions of low molecular weight heparin produced with modified heparinase iii patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. Each week you receive an email with patent applications related to your keywords. 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