WO2001066772A9 - Heparinase iii and uses thereof - Google Patents
Heparinase iii and uses thereofInfo
- Publication number
- WO2001066772A9 WO2001066772A9 PCT/US2001/007464 US0107464W WO0166772A9 WO 2001066772 A9 WO2001066772 A9 WO 2001066772A9 US 0107464 W US0107464 W US 0107464W WO 0166772 A9 WO0166772 A9 WO 0166772A9
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- Prior art keywords
- heparinase iii
- tumor
- ofthe
- heparinase
- hlgag
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
- A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/51—Lyases (4)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/06—Antipsoriatics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/02—Ophthalmic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/04—Antineoplastic agents specific for metastasis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
- C08B37/0075—Heparin; Heparan sulfate; Derivatives thereof, e.g. heparosan; Purification or extraction methods thereof
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/88—Lyases (4.)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/527—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving lyase
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/988—Lyases (4.), e.g. aldolases, heparinase, enolases, fumarase
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
- G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
- Y10S530/81—Carrier - bound or immobilized peptides or proteins and the preparation thereof, e.g. biological cell or cell fragment as carrier
- Y10S530/811—Peptides or proteins is immobilized on, or in, an inorganic carrier
Definitions
- the invention relates to heparinase III and mutants thereof.
- 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.
- HLGAGs Heparin like glycosaminoglycans
- 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 [ ⁇ -L-iduronic acid (I) or ⁇ - D-glucuiOnic acid (G)] linked 1, 4 to ⁇ -B-hexosamine (H).
- a uronic acid [ ⁇ -L-iduronic acid (I) or ⁇ - D-glucuiOnic acid (G)] linked 1, 4 to ⁇ -B-hexosamine (H).
- I ⁇ -L-iduronic acid
- G ⁇ - D-glucuiOnic acid
- H ⁇ -B-hexosamine
- 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.
- 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.
- Heparinase I primarily cleaves HLGAGs at the H NS,6 -i 2 s 2 -l n age found primarily in heparin-like regions (Ernst, S., et al., Crit, Rev. Biochem. Mol. Biol, 30, 387-444 (1995)). Desai, TJ., et al., Biochemistry, 32, 8140-8145 (1993)), and Jandik, K., et al, Glycobiology, 4, 289-296 (1994)).
- Heparinase III cleaves at the H N A C -I and HNY ;6 X-G 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 ofthe molecule. Cysteine 348 and histidines 238, 451, and 579 were determined to be crucial for heparinase II activity.
- Heparinase III is unique in that it is the only member ofthe heparinase family that recognizes and preferentially cleaves heparan sulfate. Heparinase III also contains no cysteines in its amino acid sequence. Tumor metastasis involves the spread of tumor cells primarily via the vasculature to remote sites in the body.
- 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 ofthe gene product for heparinase. . SUMMARY OF THE INVENTION It has been discovered, according to one aspect ofthe invention, that expression of heparinases does not necessarily represent a switch from a primary tumor to a metastatic diseased state.
- 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 ofthe tumor cells in order to prevent growth ofthe tumor.
- 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 ofthe tumor cell across a banier.
- the heparinase III may be a native heparinase III molecule or a modified heparinase III molecule. Native heparinase III may be synthesized or isolated. 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.
- the tumor cell can be exposed to the heparinase III by any method known in the art.
- heparinase III may be added to the in vitro culture.
- the heparinase III may be administered by any method for delivering the heparinase III to the tumor.
- 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.
- the heparinase III may be administered alone or in conjunction with other therapies such as an anti-cancer drag.
- the tumor is a prostate tumor or a melanoma.
- 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 ofthe tumor with heparinase III to produce HLGAG fragments, and isolating the HLGAG fragments, wherein the HLGAG fragments are the therapeutic agent.
- the method may also include the step of determining the sequence ofthe HLGAG fragments.
- 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.
- the method may involve identifying a therapeutic HLGAG fragment by identifying an HLGAG produced when the tumor is contacted with heparinase III.
- the therapeutic HLGAG fragment is a synthetic HLGAG fragment generated based on the sequence ofthe HLGAG fragment identified when the'tumor is contacted with heparinase III.
- the HLGAG fragment administered to the subject is an isolated HLGAG fragment produced when the tumor is contacted with the heparinase III.
- 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.
- the therapeutic HLGAG fragment is a composition of HLGAG fragments wherein at least 50%, 75%, or 90% ofthe HLGAG fragments are di- or tri- sulfated disaccharides. In other embodiments the therapeutic HLGAG fragment is free of mono- or un- sulfated disaccharides.
- 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 canier.
- 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.
- 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 canier.
- the invention in other aspects is based on the identification of important residues within the heparinase III molecule.
- the invention is a substantially pure heparinase III comprising a polypeptide having the amino acid sequence ofthe 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.
- the polypeptide has a substitution at His 110 or His 241.
- the invention is a substantially pure heparinase III comprising a polypeptide having the amino acid sequence ofthe 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.
- the invention is a substantially pure heparinase III which is a modified heparinase III having a modified product profile, wherein the modified product profile ofthe modified heparinase III is at least 10% different than a native product profile of a native heparinase III.
- 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 cat value, wherein the modified heparinase III k oat value is at least 10% different than a native heparinase III k Gat value.
- the invention also encompasses pharmaceutical preparations of any ofthe substantially pure heparinase III molecules with a pha ⁇ naceutically-acceptable canier.
- the invention also encompasses the modified heparinase III ofthe invention immobilized on a solid support membrane.
- a method of specifically cleaving a HLGAG is provided according to another aspect ofthe invention.
- the method of specifically cleaving a HLGAG includes the step of contacting an HLGAG with the modified heparinase III ofthe invention.
- the method is a method for preventing tumor cell proliferation or metastasis, as described above.
- the method is a method for sequencing HLGAGs.
- 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.
- the invention also includes a method for preparing LMWH by contacting an HLGAG sample with a modified heparinase III molecule to produce LMWH.
- the invention is a composition of the LMWH produced by this method.
- the invention is also a method for treating or preventing a disorder associated with coagulation, tumor, psoriasis, or neovascularization, by administering to a subject an effective amount of this composition to treat or prevent a disorder associated with coagulation, tumor, psoriasis, or neovascularization.
- a disorder associated with coagulation, tumor, psoriasis, or neovascularization by administering to a subject an effective amount of this composition to treat or prevent a disorder associated with coagulation, tumor, psoriasis, or neovascularization.
- Each ofthe limitations ofthe invention can encompass various embodiments of the invention. It is, therefore, anticipated that each ofthe limitations ofthe invention- involving any one element or combinations of elements can be included in each aspect of the invention.
- Figure 1 is a graph depicting the effect of DEPC inactivation of heparinase III on rate constant.
- Figure 2 is a graph depicting the pH dependence ofthe second order rate constant of inactivation upon incubation of heparinase III with varying concentrations of DEPC.
- Figure 3 is a graph depicting the quantification of DEPC-modified histidine residues in heparinase III over a period of time.
- Figure 4 is a graph depicting the substrate protection of heparinase III inactivation by DEPC III.
- Figure 5 is a reverse phase HPLC profile of a lys-C digest of heparinase III which was not exposed to DEPC (top panel) and a peptide profile of heparinase III labeled with DEPC (bottom panel).
- FIG 6 is a series of graphs depicting SAX analysis of exhaustive heparinase III digests of heparan sulfate. Heparan sulfate was digested with either heparinase HI from F. heparinum (panel A), recombinant heparinase III (panel B), H295 A mutant enzyme (panel C), H510A mutant enzyme (panel D), or the HI 05 A mutant enzyme (panel E).
- Figure 7 depicts a circular dichroism analysis of recombinant heparinase III and the H295A mutant enzyme, and the H510A mutant enzyme.
- Figure 8 is a graph depicting tumor volmne in mice, as well as mice treated with heparinase I.
- Figure 9 is a bar graph depicting number of lung nodules that developed 13 days after tail vein injection of B16 BL6 cells. The cells were either treated with PBS, heparinase I, or heparinase III.
- Figure 10, panel A depicts the tumor volume of mice that were treated with GAG fragments generated from treatment of B16 BL6 cells with either heparinase I, heparinase III, or PBS or fragments generated from heparinase I treatment of LLC cells. Tumor volume was measured over time between 7 and 15 days post-injection ofthe tumor cells.
- panel B is a bar graph which quantitates the number of lung nodules of the mice described in panel A.
- Figure 11 is a bar graph depicting the effect on B16 cellular migration and invasion of transfection with antisense 2OST in pcDNA3.1.
- Figure 12 shows bar graphs depicting the ability ofthe transfected cells of Figure 12 to develop into primary tumors as assessed by mean tumor volume (12a) and tumor weight (12b).
- Figure 13 depicts the results of compositional analysis of HLGAG saccharide fragments released from B 16BL6 cells.
- Figure 14 is a bar graph depicting FGF2 signaling modulated by HLGAG fragments
- Figure 15 is a table (15a) and a schematic depicting the modulation of FGF2 activity in vivo by B16BL6 fragments (15b).
- Sequence ID No. 1 is the nucleic acid sequence of heparinase III from F. bacterium.
- Sequence ID No. 2 is the amino acid sequence of heparinase III from F. bacterium.
- Sequence ID No. 3 is a peptide fragment.
- the invention in some aspects relates to heparinase III, modified forms thereof and uses thereof.
- the invention arose from several scientific findings which expand the field of heparinase biology.
- the invention is based in part on the discovery of new modified forms of heparinase that have varying enzymatic activity and produce differing product profiles.
- the invention is also based on the finding that native heparinase III, modified forms of heparinase III, and modified forms of heparinase II having heparinase III like activity are useful for the treatment and prevention of tumor cell growth and metastasis.
- the present invention provides a series of new modified heparinase III molecules.
- new heparinases with altered stability, activity and specificity are provided.
- the modified heparinases ofthe invention have many in vivo, in vitro and ex vivo utilities.
- Heparinase III is unique in that it is the only member ofthe heparinase family that recognizes and cleaves heparan sulfate as its only substrate.
- Heparinase III is also unique among its heparin-degrading family members in that it contains no cysteines in its primary amino acid sequence (Su, H., Blain, F., Musil, R.A., Zimmermann, J.J., Gu, K., and Bennett, D.C. (1996) Appl. Environ. Micro. 62, 2723-34 and Godavarti, R., Davis, M., Venkataraman, G., Cooney, C.L., Langer, R., and Sasisekharan, R. (1996) Biochem. and Biophys. Res. Comm. 225, 751-58).
- Heparinase III does contain thirteen histidines of which one or several might be involved in the activity ofthe enzyme.
- the nucleotide and amino acid sequences of heparinase III are provided in SEQ ID NO: 1 and SEQ ID NO: 2.
- the sequence of heparinase III has been reported in Su, H., Blain, F., Musil, R.A., Zimmermann, J.J., Gu, K., and Bennett, D.C. (1996) Appl. Environ. Micro.
- H295A, H510A, and recombinant heparinase III were nearly identical, strongly indicating that this is not the case. It is more likely that histidine 295 and histidine 510 play a direct role in the binding of HLGAGs to the enzyme or that histidine 295 and histidine 510 are critical active site residues directly involved in the catalytic degradation of HLGAGs.
- Modified heparinase III molecules having a change in amino acid at His 295 or 510 can be useful for a variety of purposes, e.g., as a competitive inhibitor to functional heparinase III.
- the studies described in the Examples section also identified several heparinase
- mutants which had altered levels of activity but which were still active. These mutants include heparinase III molecules having the following residues mutated or substituted: His36, Hisl05, HisllO, Hisl39, Hisl52, His225, His234, His241, His424, His469, and His539.
- the present invention provides for novel modified heparinases rationally designed on the basis ofthe sequence ofthe heparinase III of F. heparinum and the structural and functional characterizations disclosed herein. In the description herein, reference is made to the amino acid residues and residue positions of native heparinase III disclosed in SEQ ID NO 2.
- residues and residue positions are refened to as "conesponding to" a particular residue or residue position of heparinase III.
- these positions are relative and, therefore, insertions or deletions of one or more residues would have the effect of altering the numbering of downstream residues.
- N-terminal insertions or deletions would alter the numbering of all subsequent residues. Therefore, as used herein, a residue in a recombinant modified heparinase will be refened to as "conesponding to" a residue ofthe full heparinase III if, using standard sequence comparison programs, they would be aligned.
- sequence alignment programs are now available to one of ordinary skill in the art and their use in sequence comparisons has become standard.
- this convention of referring to the positions of residues ofthe recombinant modified heparinases by their conesponding heparinase III residues shall extend not only to embodiments including N-terminal insertions or deletions but also to internal insertions or deletions (e.g., insertions or deletions in "loop" regions).
- a “conservative amino acid substitution” or “conservative substitution” refers to an amino acid substitution in which the substituted amino acid residue is of similar charge as the replaced residue and is of similar or smaller size than the replaced residue.
- Conservative substitutions of amino acids include substitutions made amongst amino acids within the following groups: (a) the small non-polar amino acids, A, M, I, L, and V; (b) the small polar amino acids, G, S, T and C; (c) the amido amino acids, Q and N; (d) the aromatic amino acids, F, Y and W; (e) the basic amino acids, K, R and H; and (f) the acidic amino acids, E and D. Substitutions which are charge neutral and which replace a residue with a smaller residue may also be considered "conservative substitutions" even if the residues are in different groups (e.g., replacement of phenylalanine with the smaller isoleucine).
- conservative amino acid substitution also refers to the use of amino acid analogs or variants. Methods for making amino acid substitutions, additions or deletions are well known in the art and are described in detail in the Examples below.
- the terms “conservative substitution”, “non-conservative substitutions”, “non-polar amino acids”, “polar amino acids”, and “acidic amino acids” are all used consistently with the prior art terminology. Each of these terms is well-known in the art and has been extensively described in numerous publications, including standard biochemistry text books, such as "Biochemistry” by Geoffrey Zubay, Addison- Wesley Publishing Co., 1986 edition, which describes conservative and non-conservative substitutions, and properties of amino acids which lead to their definition as polar, non-polar or acidic.
- some ofthe amino acid substitutions are non-conservative substitutions.
- the non-conservative substitutions are easily tolerated provided that they preserve the tertiary structure characteristic of native heparinase, thereby preserving the active and binding sites.
- Non-conservative substitutions such as between, rather than within, the above groups (or two other amino acid groups not shown above), which will differ more significantly in their effect on maintaining (a) the structure ofthe peptide backbone in the area ofthe substitution (b) the charge or hydrophobicity ofthe molecule at the target site, or (c) the bulk ofthe side chain.
- the invention is a substantially pure heparinase which is a modified heparinase III having a modified heparinase III k cat value, wherein the modified heparinase III k cat value is at least 10% different than a native heparinase III k cat value.
- the modified heparinase III k ca t value is at least 20% different than a native heparinase III k ca t value.
- the modified heparinase III k cat value is at least 50% different than a native heparinase III k cat value.
- a "modified heparinase III k cat value" as used herein is a measurement ofthe catalytic activity o the modified heparinase III enzyme with respect to a heparan sulfate-like glycosaminoglycan substrate.
- the k ca t value may be determined using any enzymatic activity assay which is useful for assessing the activity of a heparinase enzyme, such as the assays set forth in the Examples below.
- assays are well-known in the art. For instance, an assay for measuring k cat is described in (Ernst, S.
- the "native heparinase III k cat value” is the measure of enzymatic activity ofthe native heparinase III.
- the modified heparinase may have a reduced enzymatic activity with respect to HLGAGs.
- a "reduced enzymatic activity” is assessed by comparing the k cat value ofthe modified heparinase with that of native heparinase.
- the k ca t value ofthe modified heparinase III will be less than or equal to 75% ofthe native heparinase III k cat value.
- a modified heparinase having reduced enzymatic activity with respect to HLGAGs is one which has modifications in the residues essential for catalytic activity. For instance, mutation of His 110 or His 24 causes the heparinase III to have a reduced enzymatic activity.
- a modified heparinase III which has a increased enzymatic activity is one which has altered residues which produce an enzyme with greater enzymatic activity. For instance, mutation of His 13 produces modified heparinase III molecules having increased enzymatic activity.
- heparinases when His 225 is mutated in heparinase III, a modified heparinase III is produced which displays nearly the same enzymatic activity as native heparinase III. These enzymes are also useful.
- the term "substantially pure" means that the heparinases are essentially free of other substances with which they may be found in nature or in vivo systems to an extent practical and appropriate for their intended use. In particular, the heparinases are sufficiently free from other biological constituents of their hosts cells so as to be useful in, for example, producing pharmaceutical preparations or sequencing.
- the heparinases ofthe invention may be admixed with a pharmaceutically acceptable canier in a pharmaceutical preparation, the heparinase may comprise only a small percentage by weight ofthe preparation.
- the heparinase is nonetheless substantially pure in that it has been substantially separated from the substances with which it may be associated in living systems. Based on the disclosure provided herein, those of ordinary skill in the art will be able to identify other modified heparinase III molecules having altered enzymatic activity with respect to the native heparinase III molecule.
- the invention is a substantially pure heparinase which is a modified heparinase III having a modified product profile, wherein the modified product profile ofthe modified heparinase III is at least 10% different than a native product profile of a native heparinase III. Preferably it is at least 20%) or even at least 50%.
- a "modified product profile" as used herein is a set of degradation products produced by a modified heparinase which differ from the degradation products which are produced by a native heparinase under identical enzymatic conditions.
- the difference in the product profile may be due to the presence of different enzymatic products or simply in the number of enzymatic products formed by the modified heparinase compared to the native heparinase, or a combination ofthe two.
- the formation of different enzymatic products by a modified heparinase as opposed to the native heparinase would constitute a modified product profile.
- the production ofthe same types of enzymatic products but in a lesser or greater amount by the modified heparinase as opposed to the native heparinase would also constitute a modified product profile.
- the product profile produced by a modified heparinase or a native heparinase may be determined by any method known in the art for examining the type or quantity of degradation product produced by heparinase.
- One prefened method for determining the type and quantity of product is described in Rhomberg, A.J. et al., PNAS, v. 95, p. 4176- 4181 (April 1998), which is hereby incorporated in its entirety by reference.
- the method disclosed in the Rhomberg reference utilizes a combination of mass spectrometry and capillary electrophoretic techniques to identify the enzymatic products produced by heparinase.
- the Rhomberg study utilizes heparinase to degrade HLGAGs to produce HLGAG oligosaccharides.
- MALDI Microx-Assisted Laser Desorption Ionization
- mass spectrometry can be used for the identification and semiquantitative measurement of substrates, enzymes, and end products in the enzymatic reaction.
- the capillary electrophoresis technique separates the products to resolve even small differences amongst the products and is applied in combination with mass spectrometry to quantitate the products produced.
- Capillary electrophoresis may even resolve the difference between a disaccharide and its semicarbazone derivative.
- Detailed methods for sequencing polysaccharides and other polymers are disclosed in co-pending U.S. Patent Applications Serial Nos.
- the method is performed by enzymatic digestion, followed by mass spectrometry and capillary electrophoresis.
- the enzymatic assays can be performed in a variety of manners, as long as the assays are performed identically on the modified heparinase and the native heparinase, so that the results may be compared.
- enzymatic reactions are performed by adding 1 mL of enzyme solution to 5 mL of substrate solution.
- the digestion is then canied out at room temperature (22°C), and the reaction is stopped at various time points by removing 0.5 mL ofthe reaction mixture and adding it to 4.5 mL of a MALDI matrix solution, such as caffeic acid (approximately 12 mg/mL) and 70% acetonitrile/water.
- a MALDI matrix solution such as caffeic acid (approximately 12 mg/mL) and 70% acetonitrile/water.
- the reaction mixture is then subjected to MALDI mass spectrometry.
- the MALDI surface is prepared by the method of Xiang and Beavis (Xiang and Beavis (1994) Rapid. Commun. Mass. Spectrom. 8, 199-204). A two-fold lower access of basic peptide (Arg/Gly) !5 is premixed with matrix before being added to the oligosaccharide solution.
- sample/matrix mixture containing 1-3 picomoles of oligosaccharide is deposited on the surface. After crystallization occurs (typically within 60 seconds), excess liquid is rinsed off with water.
- MALDI mass spectrometry spectra is then acquired in the linear mode by using a PerSeptive Biosystems (Framingham, MA) Voyager Elite reflection time-of-flight instrument fitted with a 337 nanometer nitrogen laser. Delayed extraction is used to increase resolution (22 kV, grid at 93%, guidewire at 0.15%o, pulse delay 150 ns, low mass gate at 1,000, 128 shots averaged).
- Mass spectra are calibrated externally by using the signals for proteinated (Arg/Gly) 15 and its complex with the oligosaccharide.
- Capillary electrophoresis is then performed on a Hewlett-Packard CE unit by using uncoated fused silica capillaries (internal diameter 75 micrometers, outer diameter 363 micrometers, l det 72.1 cm, and 1 tot 85 cm). Analytes are monitored by using UV detection at 230 mn and an extended light path cell (Hewlett-Packard).
- the electrolyte is a solution of 10 mL dextran sulfate and 50 millimolar Tris/phosphoric acid (pH2.5).
- Dextran sulfate is used to suppress nonspecific interactions ofthe heparin oligosaccharides with a silica wall. Separations are ca ⁇ ied out at 30 kV with the anode at the detector side (reversed polarity). A mixture of a 1/5-naphtalenedisulfonic acid and 2-naphtalenesulfonic acid (10 micromolar each) is used as an internal standard. Other methods for assessing the product profile may also be utilized. For instance, other methods include methods which rely on parameters such as viscosity (Jandik, K.A., Gu, K.
- modified heparinases ofthe invention may be used for any ofthe same purposes as native heparinase III.
- the modified heparinase III molecules can be used to specifically cleave a HLGAG by contacting the HLGAG substrate with one ofthe modified heparinases ofthe invention.
- the invention is useful in a variety of in vitro, in vivo and ex vivo methods in which it is useful to cleave HLGAGs.
- the modified heparinase III may be used, for instance, in a method for inhibiting angiogenesis. In this method an effective amount for inhibiting angiogenesis ofthe heparinase III is administered to a subject in need of treatment thereof.
- Angiogenesis as used herein is the inappropriate formation of new blood vessels. "Angiogenesis" often occurs in tumors when endothelial cells secrete a group of growth factors that are mitogenic for endothelium causing the elongation and proliferation of endothelial cells which results in a generation of new blood vessels.
- heparin or heparan sulfate binding peptides which are related to endothelial cell growth factors.
- the modified heparinases are also useful for treating or preventing cancer cell growth or metastasis. This aspect ofthe invention is discussed in more detail below, with respect to both native and modified heparinase III.
- the modified heparinases are also useful for inhibiting neovascularization associated with disease such as eye disease.
- Neovascularization, or angiogenesis is the growth and development of new arteries. It is critical to the normal development ofthe vascular system, including injury-repair.
- diabetic retinopathy is a leading cause of blindness.
- proliferative retinopathy is characterized by neovascularization and scaning. About one-half of those patients with proliferative retinopathy progress to blindness within about five years.
- Another example of abnormal neovascularization is that associated with solid tumors.
- an angiogenic condition means a disease or undesirable medical condition having a pathology including neovascularization.
- diseases or conditions include diabetic retinopathy, neovascular glaucoma and rheumatoid arthritis (non-cancer angiogenic conditions).
- Cancer angiogenic conditions are solid tumors and cancers or tumors otherwise associated with neovascularization such as hemangioendotheliomas, hemangiomas and Kaposi's sarcoma. Proliferation of endothelial and vascular smooth muscle cells is the main feature of neovascularization.
- the modified heparinase III ofthe invention is useful for preventing proliferation and, therefore, inhibiting or anesting altogether the progression ofthe angiogenic condition which depends in whole or in part upon such neovascularization.
- Neovascularization and angiogenesis are also important in a number of other pathological processes, including arthritis, psoriasis, diabetic retinopathy, chronic inflammation, scleroderma, hemangioma, retrolental fibroplasia and abnormal capillary proliferation in hemophiliac joints, prolonged menstruation and bleeding, and other disorders ofthe female reproductive system (J. Folkman, Nature Medicine, Vol 1, p. 27- 31, (1995); J. W. Miller, et al, J. Pathol., Vol. 145, pp. 574-584 (1994); A. P. Adamid, et al, Amer. J. Ophthal., Vol. 118, pp. 445-450 (1994); K.
- the modified heparinase is administered to treat diseases such as psoriasis.
- Psoriasis is a common dermatological disease caused by chronic inflammation.
- the H295A and H510A modified heparinases are also useful according to the invention as inhibitors of heparinase III activity. These modified heparinases have a minimum one base pair modification from native heparinase but have no enzymatic activity. Thus, modified heparinases having a H295 A or H510A modification can be used as competitive inhibitors of native or functional modified forms of heparinase III.
- modified heparinases ofthe invention are also useful as tools for sequencing HLGAGs. Detailed methods for sequencing polysaccharides and other polymers are disclosed in co-pending U.S. Patent Applications Serial Nos. 09/557,997 and 09/558,137, both filed on April 24, 2000 and having common inventorship. These methods utilize tools such as heparinases in the sequencing process.
- the modified heparinase III ofthe invention is useful as such a tool.
- the modified heparinases ofthe invention may also be used to remove active HLGAGs from a HLGAG containing fluid.
- a HLGAG containing fluid is contacted with the modified heparinase III ofthe invention to degrade the HLGAG.
- the method is particularly useful for the ex vivo removal of HLGAGs from blood.
- the modified heparinase is immobilized on a solid support as is conventional in the art.
- the solid support containing the immobilized modified heparinase may be used in extracorporeal medical devices (e.g. hemodialyzer, pump-oxygenator) for systemic heparinization to prevent the blood in the device from clotting.
- the support membrane containing immobilized heparinase III is positioned at the end ofthe device to neutralize the HLGAG before the blood is returned to the body.
- the invention is an immobilized substantially pure heparinase ofthe invention.
- the heparinase may be immobilized to any type of support but if the support is to be used in vivo or ex vivo it is desired that the support is sterile and biocompatible. A biocompatible support is one which would not cause an immune or other type of damaging reaction when used in a subject.
- the heparinase may be immobilized by any method known in the art. Many methods are known for immobilizing proteins to supports.
- the heparinase III is, in some embodiments, immobilized on a solid support.
- a "solid support” as used herein refers to any solid material to which a protein can be immobilized.
- Solid supports include but are not limited to membranes, e.g., natural and modified celluloses such as nitrocellulose or nylon, Sepharose, Agarose, glass, polystyrene, polypropylene, polyethylene, dextran, amylases, polyacrylamides, polyvinylidene difluoride, other agaroses, and magnetite, including magnetic beads.
- the canier can be totally insoluble or partially soluble and may have any possible stractural configuration.
- the support may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube or microplate well, or the external surface of a rod.
- the surface may be flat such as a sheet, test strip, bottom surface of a microplate well, etc.
- the modified heparinase III molecules are also useful for generating LMWHs which have many therapeutic utilities.
- the modified heparinase III molecules and LMWH can be used for the treatment of any type of condition in which LMWH therapy has been identified as a useful therapy, e.g., preventing coagulation, preventing psoriasis.
- the modified heparinase molecules are useful for treating or preventing disorders associated with coagulation.
- a "disease associated with coagulation” as used herein refers to a condition characterized by local inflammation resulting from an intenuption in the blood supply to a tissue due to a blockage of the blood vessel responsible for supplying blood to the tissue such as is seen for myocardial or cerebral infarction.
- a cerebral ischemic attack or cerebral ischemia is a form of ischemic condition in which the blood supply to the brain is blocked.
- This intenuption in the blood supply to the brain may result from a variety of causes, including an intrinsic blockage or occlusion ofthe blood vessel itself, a remotely originated source of occlusion, decreased perfusion pressure or increased blood viscosity resulting in inadequate cerebral blood flow, or a raptured blood vessel in the subarachnoid space or intracerebral tissue.
- the methods ofthe invention are useful also for treating cerebral ischemia. Cerebral ischemia may result in either transient or permanent deficits and the seriousness ofthe neurological damage in a patient who has experienced cerebral ischemia depends on the intensity and duration ofthe ischemic event.
- a transient ischemic attack is one in which the blood flow to the brain is intenupted only briefly and causes temporary neurological deficits, which often are clear in less than 24 hours.
- Symptoms of TIA include numbness or weakness of face or limbs, loss ofthe ability to speak clearly and/or to understand the speech of others, a loss of vision or dimness of vision, and a feeling of dizziness.
- Permanent cerebral ischemic attacks, also called stroke are caused by a longer intenuption in blood flow to the brain resulting from either a thromboembolism.
- a stroke causes a loss of neurons typically resulting in a neurologic deficit that may improve but that does not entirely resolve.
- Thromboembolic stroke is due to the occlusion of an extracranial or intracranial blood vessel by a thrombus or embolus. Because it is often difficult to discern whether a stroke is caused by a thrombosis or an embolism, the term "thromboembolism" is used to cover strokes caused by either of these mechanisms.
- the methods ofthe invention in some embodiments are directed to the treatment of acute thromboembolic stroke using modified heparinase III or the LMWHs generated therewith.
- An acute stroke is a medical syndrome involving neurological injury resulting from an ischemic event, which is an intenuption in the blood supply to the brain.
- An effective amount of a modified heparinase III or the LMWHs generated therewith alone or in combination with another therapeutic for the treatment of stroke is that amount sufficient to reduce in vivo brain injury resulting from the stroke.
- a reduction of brain injury is any prevention of injury to the brain which otherwise would have occurred in a subject experiencing a thromboembolic stroke absent the treatment of the invention.
- physiological parameters may be used to assess reduction of brain injury, including smaller infarct size, improved regional cerebral blood flow, and decreased intracranial pressure, for example, as compared to pretreatment patient parameters, untreated stroke patients or stroke patients treated with thrombolytic agents alone.
- the modified heparinase III or the LMWHs generated therewith may be used alone or in combination with a therapeutic agent for treating a disease associated with coagulation.
- therapeutics useful in the treatment of diseases associated with coagulation include anticoagulation agents, antiplatelet agents, and thrombolytic agents.
- Anticoagulation agents prevent the coagulation of blood components and thus prevent clot formation.
- Anticoagulants include, but are not limited to, heparin, warfarin, coumadin, dicumarol, phenprocoumon, acenocoumarol, ethyl biscoumacetate, and indandione derivatives.
- Antiplatelet agents inhibit platelet aggregation and are often used to prevent thromboembolic stroke in patients who have experienced a transient ischemic attack or stroke.
- Antiplatelet agents include, but are not limited to, aspirin, thienopyridine derivatives such as ticlopodine and clopidogrel, dipyridamole and sulfinpyrazone, as well as RGD mimetics and also antithrombin agents such as, but not limited to, l ⁇ rudin.
- Thrombolytic agents lyse clots which cause the thromboembolic stroke.
- Thrombolytic agents have been used in the treatment of acute venous thromboembolism and pulmonary emboli and are well known in the art (e.g.
- Thrombolytic agents include, but are not limited to, plasminogen, a 2 -antiplasmin, streptokinase, antistreplase, tissue plasminogen activator (tPA), and urokinase.
- tPA tissue plasminogen activator
- the enzymatic activity of tPA can be measured by assessing the ability of the molecule to convert plasminogen to plasmin.
- the fibrinolytic activity of tPA may be determined by any in vitro clot lysis activity known in the art, such as the purified clot lysis assay described by Carlson, et. al., Anal. Biochem. 168, 428-435 (1988) and its modified form described by Bennett, W. F. Et al., 1991, Supra, the entire contents of which are hereby incorporated by reference.
- the invention also relates to the discovery that heparinase III, modified forms thereof, modified forms of heparinase II and degradation products of heparinases (HLGAG fragments) actually are useful for treating and preventing cancer cell proliferation and metastasis.
- HLGAGs are linear polysaccharides characterized by a disaccharide-repeat unit of a uronic acid [ ⁇ -L-iduronic acid (I) or ⁇ -D-glucuronic acid (G)] linked 1,4 to -D-hexosamine (H).
- HLGAGs are the most acidic, heterogeneous and information dense biopolymer found in nature due to the highly variable chemical modification ofthe disaccharide repeat unit - primarily in the form of sulfation at the N-, 3O and 6O positions of H, and the 20 ofthe uiOnic acids.
- HLGAGs (along with collagen) are key components ofthe cell surface-extracellular matrix (ECM) interface. While collagen-like proteins provide the necessary extracellular scaffold for cells to attach and form tissues, the complex polysaccharides fill the space created by the scaffold and act as a molecular sponge by specifically binding and regulating the biological activities of numerous signaling molecules like growth factors, cytokines etc.
- Tu or metastasis involves the spread of tumor cells primarily via the vasculature following the disassembly of tumor cell-ECM interactions through the degradation o the ECM, and tumor cell extravasation through the capillary bed.
- collagen and related proteins
- enzymes that degrade the proteinaceous component ofthe ECM may play roles in the regulation of tumor angiogenesis or tumor cell invasion ofthe ECM.
- HLGAG degrading enzymes heparinases
- Heparinase I was unable to prevent cancer cell proliferation or metastasis, indicating that the effects are specific to heparinase III and functional variants thereof. These results are consistent with the unique specificities of heparinases, and hence the distinct oligosaccharide products they generate. Additionally, the data demonstrated that HLGAG fragments for one cell type were able to influence effects on another cell type, strongly indicating the involvement of specific sequences of HLGAG in modulating effects on tumor growth and metastasis. Thus, the invention includes methods for treating or preventing tumor formation and/or metastasis by administering to a subject a heparinase III molecule (native or modified) and/or therapeutic HLGAG fragments (including LMWH).
- a heparinase III molecule native or modified
- therapeutic HLGAG fragments including LMWH
- the heparinases useful in this aspect ofthe invention include native heparinase III, modified heparinase III and modified heparinases having the functional activity of heparinase III.
- Native heparinase III refers to the naturally occuning heparinase III molecule in an isolated form.
- the sequence ofthe naturally occuning molecule from F. heparinum is provided as SEQ ID NO.: 1 (nucleic acid sequence) and 2 (amino acid sequence), and has been extensively described in art including in issued patents.
- an isolated molecule is a molecule that is substantially pure and is free of other substances with which it is ordinarily found in nature or in vivo systems to an extent practical and appropriate for its intended use.
- the molecular species are sufficiently pure and are sufficiently free from other biological constituents of host cells so as to be useful in, for example, producing pharmaceutical preparations or sequencing if the molecular species is a nucleic acid, peptide, or polysaccharide.
- an isolated molecular species ofthe invention may be admixed with a pharmaceutically-acceptable canier in a phannaceutical preparation, the molecular species may comprise only a small percentage by weight ofthe preparation.
- a "modified heparinase III" as used herein is any heparinase III molecule which has at least one mutation, deletion or substitution, compared to native heparinase III but which retains the ability to enzymatically cleave heparan sulfate.
- modified heparinases described herein as well as any other modified heparinase having the appropriate function. These can be identified by those of ordinary skill in the art using the methods described above or in the examples section.
- the modified heparinase III may have a simple conservative substitution within a region ofthe molecule which is not critical for enzymatic activity or folding and thus which has no effect on the ability ofthe heparinase to cleave the substrate.
- substitutions such as the histidine substitutions described herein which influence the enzymatic activity or product profile ofthe heparinase but which still retain some enzymatic activity are also useful for this aspect ofthe invention because they are still able to cleave heparan sulfate.
- the two histidine mutations (His 295 and His 510) which lost all activity, however, are not useful in this aspect ofthe invention.
- modified heparinases having functional activity of heparinase III refers to heparinases other than heparinase III which have been modified such that they are enzymatically active towards heparan sulfate but only have minimal or no activity towards heparin. For instance, mutation of Cys 348 of heparinase II, a residue which is involved in heparin binding, causes the heparinase II to have a reduced enzymatic activity with respect to heparin.
- This modification produces a modified heparinase II which becomes, exclusively a heparan sulfate degrading enzyme. Additionally, when histidine 440 is mutated in heparinase III, a modified heparinase III is produced which has reduced enzymatic activity with respect to heparin but which displays nearly the same enzymatic activity as native heparinase III when heparan sulfate is used as the substrate. Mutation of histidines 451, 238, and 579 of heparinase II produces modified heparinase II molecules having reduced enzymatic activity with respect to heparan sulfate.
- modified heparinase II molecules in which the Cys or His 440 is mutated are "modified heparinases having functional activity of heparinase III" according to the invention, whereas heparinases in which histidines 451, 238, or 579 have been mutated are not within this class of molecules.
- the invention also contemplates the use of therapeutic HLGAGs for the treatment and prevention of tumor cell proliferation and metastasis.
- HLGAG fragment refers to a molecule or molecules which are pieces or fragments of an HLGAG that have been identified through the use ofthe native heparinase III, modified heparinase III and modified heparinases having the functional activity of heparinase III described above.
- HLGAG fragments also include low molecular weight heparins (LMWHs).
- LMWHs low molecular weight heparins
- the compositional analysis of some therapeutic HLGAGs is described below in the Examples section.
- the invention also encompasses screening assays for identifying therapeutic HLGAG fragments for the treatment of a tumor and for preventing metastasis.
- the assays are accomplished by treating a tumor or isolated tumor cells with heparinase III, native or modified and isolating the resultant HLGAG fragments.
- these HLGAG fragments have therapeutic activity in the prevention of tumor cell proliferation and metastasis.
- these HLGAG fragments are useful as therapeutic agents for the treatment ofthe tumor cells from which they were generated as well as other tumors.
- the invention encompasses individualized therapies, in which a tumor or portion of a tumor is isolated from a subject and used to prepare the therapeutic HLGAG fragments.
- therapeutic HLGAG fragment refers to an HLGAG which has therapeutic activity in that it prevents the proliferation and/or metastasis of a tumor cell.
- Such compounds ' can be generated using heparinase III to produce therapeutic fragments or they can be synthesized de novo. Putative HLGAG fragments can be tested for therapeutic activity using any ofthe assays described herein or known in the art.
- the therapeutic HLGAG fragment may be a synthetic HLGAG fragment generated based on the sequence ofthe HLGAG fragment identified when the tumor is contacted with heparinase III, or having minor variations which do not interfere with the activity ofthe compound.
- the therapeutic HLGAG fragment may be an isolated HLGAG fragment produced when the tumor is contacted with heparinase III.
- the invention is useful for treating and/or preventing tumor cell proliferation or metastasis in a subject.
- the terms "prevent” and "preventing” as used herein refer to inhibiting completely or partially the proliferation or metastasis of a cancer or tumor cell, as well as inhibiting any increase in the proliferation or metastasis of a cancer or tumor cell.
- a "subject having a cancer” is a subject that has detectable cancerous cells.
- the cancer may be a malignant or non-malignant cancer.
- Cancers or tumors include but are not limited to biliary tract cancer; brain cancer; breast cancer; cervical cancer; choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer; gastric cancer; intraepithelial neoplasms; lymphomas; liver cancer; lung cancer (e.g.
- a "subject at risk of having a cancer” as used herein is a subject who has a high probability of developing cancer. These subjects include, for instance, subjects having a genetic abnormality, the presence of which has been demonstrated to have a correlative relation to a higher likelihood of developing a cancer and subjects exposed to cancer causing agents such as tobacco, asbestos, or other chemical toxins, or a subject who has previously been treated for cancer and is in apparent remission.
- Effective amounts ofthe native heparinase III, modified heparinases, or therapeutic HLGAGs ofthe invention are administered to subjects in need of such treatment. Effective amounts are those amounts which will result in a desired reduction in cellular proliferation or metastasis without causing other medically unacceptable side effects. Such amounts can be determined with no more than routine experimentation. It is believed that doses ranging from 1 nanogram/kilogram to 100 milligrams/kilogram, depending upon the mode of administration, will be effective.
- the absolute amount will depend upon a variety of factors (including whether the administration is in conjunction with other methods of treatment, the number of doses and individual patient parameters including age, physical condition, size and weight) and can be determined with routine experimentation. It is preferred generally that a maximum dose be used, that is, the highest safe dose according to sound medical judgment.
- the mode of administration may be any medically acceptable mode including oral, subcutaneous, intravenous, etc.
- the effective amount of heparinase III is that amount effective to prevent invasion of a tumor cell across a banier.
- the invasion and metastasis of cancer is a complex process which involves changes in cell adhesion properties which allow a transformed cell to invade and migrate through the extracellular matrix (ECM) and acquire anchorage-independent growth properties.
- Metastatic disease occurs when the disseminated foci of tumor cells seed a tissue which supports their growth and propagation, and this secondary spread of tumor cells is responsible for the morbidity and mortality associated with the majority of cancers.
- metastasis refers to the invasion and migration of tumor cells away from the primary tumor site.
- the banier for the tumor cells may be an artificial barrier in vitro or a natural banier in vivo.
- In vitro banier s include e but are not limited to extracellular matrix coated membranes, such as Matrigel.
- extracellular matrix coated membranes such as Matrigel.
- the heparinase compositions can be tested for their ability to inhibit tumor cell invasion in a Matrigel invasion assay system as described in detail by Parish, C.R., et al., "A Basement-Membrane Permeability Assay which Conelates with the Metastatic Potential of Tumour Cells," Int. J. Cancer (1992) 52:378-383.
- Matrigel is a reconstituted basement membrane containing type IV collagen, laminin, heparan sulfate proteoglycans such as perlecan, which bind to and localize bFGF, vitronectin as well as transforming growth factor- ⁇ (TGF- ⁇ ), urokinase- type plasminogen activator (uPA), tissue plasminogen activator (tPA), and the serpin known as plasminogen activator inhibitor type 1 (PAI-1).
- TGF- ⁇ transforming growth factor- ⁇
- uPA urokinase- type plasminogen activator
- tPA tissue plasminogen activator
- PAI-1 plasminogen activator inhibitor type 1
- An in vivo banier refers to a cellular banier present in the body of a subject.
- the formulations ofthe invention are applied in pharmaceutically acceptable solutions. Such preparations may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible earners, adjuvants, and optionally other therapeutic ingredients.
- the compositions ofthe invention may be administered per se (neat) or in the form of a pharmaceutically acceptable salt. When used in medicine the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically acceptable salts thereof and are not excluded from the scope ofthe invention.
- Such pharmacologically and pharmaceutically acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulphuric, nitric, phosphoric, maleic, acetic, salicylic, p-toluene sulphonic, tartaric, citric, methane sulphonic, formic, malonic, succinic, naphthalene-2-sulphonic, and benzene sulphonic.
- pharmaceutically acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts ofthe carboxylic acid group.
- Suitable buffering agents include: acetic acid and a salt (1-2% W V); citric acid and a salt (1-3% W/V); boric acid and a salt (0.5-2.5%) W/V); and phosphoric acid and a salt (0.8-2% W/V).
- Suitable preservatives include benzalkonium chloride (0.003-0.03% W/V); chlorobutanol (0.3-0.9% W/V); parabens (0.01-0.25% W/V) and thimerosal (0.004-0.02% W/V).
- compositions for medical use, which comprise native heparinase III, modified heparinases ofthe invention, or therapeutic HLGAG fragments together with one or more pharmaceutically acceptable earners and optionally other therapeutic ingredients.
- pharmaceutically-acceptable carrier means one or more compatible solid or liquid filler, dilutants or encapsulating substances which are suitable for administration to a human or other animal.
- canier denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application.
- the components ofthe pharmaceutical compositions also are capable of being commingled with the modified heparinases ofthe present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficiency.
- a variety of administration routes are available. The particular mode selected will depend, of course, upon the particular modified heparinase selected, the particular condition being treated and the dosage required for therapeutic efficacy.
- the methods of this invention may be practiced using any mode of administration that is medically acceptable, meaning any mode that produces effective levels of an immune response without causing clinically unacceptable adverse effects.
- a prefened mode of administration is a parenteral route.
- parenteral includes subcutaneous injections, intravenous, intramuscular, intraperitoneal, intra sternal injection or infusion techniques.
- Other modes of administration include oral, mucosal, rectal, vaginal, sublingual, intranasal, intratracheal, inhalation, ocular, transdermal, etc.
- the compounds can be formulated readily by combining the active compound(s) with pharmaceutically acceptable earners well known in the art.
- Such ca ⁇ iers enable the compounds ofthe invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, shinies, suspensions and the like, for oral ingestion by a subject to be treated.
- compositions for oral use can be obtained as solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
- suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellul ⁇ se, sodium carboxymethylcellulose, and/or polyvinylpynolidone (PVP).
- fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol
- cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl
- disintegrating agents may be added, such as the cross-linked polyvinyl pynolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
- the oral formulations may also be formulated in saline or buffers for neutralizing internal acid conditions or may be administered without any earners.
- Dragee cores are provided with suitable coatings.
- concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pynolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
- Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
- Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
- the push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.
- the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
- microspheres formulated for oral administration may also be used. Such microspheres have been well defined in the art. All formulations for oral administration should be in dosages suitable for such administration.
- buccal administration the compositions may take the form of tablets or lozenges formulated in conventional manner.
- the compounds for use according to the present invention may be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g. , dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
- the dosage unit may be determined by providing a valve to deliver a metered amount.
- Capsules and cartridges of e.g. gelatin for use in an inhaler or insufflator may be formulated containing a powder mix ofthe compound and a suitable powder base such as lactose or starch.
- the compounds, when it is desirable to deliver them systemically, may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
- Formulations for injection may be presented in unit dosage form, e.g. , in ampoules or in multi-dose containers, with an added preservative.
- compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
- Pharmaceutical formulations for parenteral administration include aqueous solutions ofthe active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions.
- Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.
- Aqueous injection suspensions may contain substances which increase the viscosity ofthe suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
- the suspension may also contain suitable stabilizers or agents which increase the solubility ofthe compounds to allow for the preparation of highly concentrated solutions.
- the active compounds may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
- the compounds may also be formulated in rectal or vaginal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
- a subject is any human or non-human vertebrate, e.g., dog, cat, horse, cow, pig.
- the compounds may also be formulated as a depot preparation.
- Such long acting formulations may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
- the pharmaceutical compositions also may comprise suitable solid or gel phase caniers or excipients.
- suitable solid or gel phase caniers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.
- Suitable liquid or solid pharmaceutical preparation forms are, for example, aqueous or saline solutions for inhalation, microencapsulated, encochleated, coated onto microscopic gold particles, contained in liposomes, nebulized, aerosols, pellets for implantation into the skin, or dried onto a sharp object to be scratched into the skin.
- the pharmaceutical compositions also include granules, powders, tablets, coated tablets, (micro)capsules, suppositories, syrups, emulsions, suspensions, creams, drops or preparations with protracted release of active compounds, in whose preparation excipients and additives and/or auxiliaries such as disintegrants, binders, coating agents, swelling agents, lubricants, flavorings, sweeteners or solubilizers are customarily used as described above.
- the pharmaceutical compositions are suitable for use in a variety of drug delivery systems. For a brief review of methods for drag delivery, see Langer, Science 249: 1527-1533, 1990," which is incorporated herein by reference.
- compositions may conveniently be presented in unit dosage form and may be prepared by any ofthe methods well known in the art of pharmacy. All methods include the step of bringing the active modified heparinase into association with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the polymer into association with a liquid canier, a finely divided solid canier, or both, and then, if necessary, shaping the product. The polymer may be stored lyophilized.
- Other delivery systems can include time-release, delayed release or sustained release delivery systems. Such systems can avoid repeated administrations ofthe heparinases ofthe invention, increasing convenience to the subject and the physician. Many types of release delivery systems are available and known to those of ordinary skill in the art.
- polymer based systems such as polylactic and polyglycolic acid, polyanhydrides and polycaprolactone; nonpolymer systems that are lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono-, di and triglycerides; hydrogel release systems; silastic systems; peptide based systems; wax coatings, compressed tablets using conventional binders and excipients, partially fused implants and the like.
- specific examples include, but are not limited to: (a) erosional systems in which the polysaccharide is contained in a form within a matrix, found in U.S. Patent Nos.
- the compounds may also be administered in cocktails containing agents that treat the side- ' effects of radiation therapy, such as anti-emetics, radiation protectants, etc.
- Anti-cancer drugs that can be co-administered with the compounds ofthe invention include, but are not limited to Acivicin; Aclarubicin; Acodazole Hydrochloride; Acronine; Adriamycin; Adozelesin; Aldesleukin; Altretamine; Ambomycin; Ametantrone Acetate; Aminoglutethimide; Amsacrine; Anastrozole;
- Irinotecan Hydrochloride Lanreotide Acetate; Letrozole; Leuprolide Acetate; Liarozole
- Mitogillin Mitomalcin; Mitomycin; Mitosper; Mitotane; Mitoxantrone Hydrochloride;
- Pipobroman Piposulfan; Piroxantrone Hydrochloride; Plicamycin; Plomestane; Porfimer Sodium; Porfiromycin; Prednimustine; Procarbazine Hydrochloride; Puromycin;
- Spirogermanium Hydrochloride Spiromustine; Spiroplatin; Streptonigrin; Streptozocin;
- Tubulozole Hydrochloride Uracil Mustard; Uredepa; Vapreotide; Verteporfin;
- Vinblastine Sulfate Vincristine Sulfate; Vindesine; Vindesine Sulfate; Vinepidine Sulfate; Vinglycinate Sulfate; Vinleursine Sulfate; Vinorelbine Tartrate; Vinrosidine
- the heparinase III compounds may also be linked to a targeting molecule.
- a targeting molecule is any molecule or compound which is specific for a particular cell or tissue and which can be used to direct the heparinase III to the cell or tissue.
- the targeting molecule is a molecule which specifically interacts with a cancer cell or a tumor.
- the targeting molecule may be a protein or other type of molecule that recognizes and specifically interacts with a tumor antigen.
- Tumor-antigens include Melan-A/M A RT-1, Dipeptidyl peptidase IV (DPPIV), adenosine deaminase-binding protein (ADAbp), cyclopbilin b, Colorectal associated antigen (CRC)--C017-1A/GA733, Carcinoembryonic Antigen (CEA) and its immunogenic epitopes CAP-1 and CAP-2, etv6, amll, Prostate Specific Antigen (PSA) and its immunogenic epitopes PSA-1, PSA-2, and PSA-3, prostate-specific membrane antigen (PSMA), T-cell receptor/CD3-zeta chain, MAGE-family of tumor antigens (e.g., MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12,
- One of ordinary skill in the art, in light ofthe present disclosure, is enabled to produce substantially pure preparations of any ofthe native or modified heparinases by standard technology, including recombinant technology, direct synthesis, mutagenesis, etc. For instance, using recombinant technology one may substitute appropriate codons in SEQ ID NO: 1 to produce the desired amino acid substitutions by standard site- directed mutagenesis techniques. Obviously, one may also use any sequence which differs from SEQ ID NO: 1 only due to the degeneracy ofthe genetic code as the starting point for site directed mutagenesis. The mutated nucleic acid sequence may then be ligated into an appropriate expression vector and expressed in a host such as F. heparinum or E. coli.
- the resultant modified heparinase may then be purified by techniques well known in the art, including those disclosed below and in Sasisekharan, et al. (1993).
- the term "substantially pure” means that the proteins are essentially free of other substances to an extent practical and appropriate for their intended use.
- the proteins are sufficiently pure and are sufficiently free from other biological constituents of their hosts cells so as to be useful in, for example, protein sequencing, or producing pharmaceutical preparations.
- an isolated nucleic acid encoding the substantially pure modified heparinase ofthe invention is provided.
- isolated means: (i) amplified in vitro by, for example, polymerase chain reaction (PCR); (ii) recombinantly produced by cloning; (iii) purified, as by cleavage and gel separation; or (iv) synthesized by, for example, chemical synthesis.
- An isolated nucleic acid is one which is readily manipulable by recombinant DNA techniques well known in the art.
- PCR polymerase chain reaction
- An isolated nucleic acid may be substantially purified, but need not be.
- a nucleic acid that is isolated within a cloning or expression vector is not pure in that it may comprise only a tiny percentage ofthe material in the cell in which it resides.
- Such a nucleic acid is isolated, however, as the term is used herein because it is readily mampulable by standard techniques known to those of ordinary skill in the art.
- a coding sequence and regulatory sequences are said to be
- operably joined when they are covalently linked in such a way as to place the expression or transcription ofthe coding sequence under the influence or control ofthe regulatory sequences. If it is desired that the coding sequences be translated into a functional protein the coding sequences are operably joined to regulatory sequences. Two DNA sequences are said to be operably joined if induction of a promoter in the 5' regulatory sequences results in the transcription ofthe coding sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability ofthe promoter region to direct the transcription ofthe coding sequences, or (3) interfere with the ability ofthe conesponding RNA transcript to be translated into a protein.
- a promoter region would be operably joined to a coding sequence if the promoter region were capable of effecting transcription of that D A sequence such that the resulting transcript might be translated into the desired protein or polypeptide.
- the precise nature ofthe regulatory sequences needed for gene expression may vary between species or cell types, but shall in general include, as necessary, 5' non-transcribing and 5' non-translating sequences involved with initiation of transcription and translation respectively, such as a TATA box, capping sequence,
- a "vector" may be any of a number of nucleic acids into which a desired sequence may be inserted by restriction and ligation for transport between different genetic environments or for expression in a host cell. Vectors are typically composed of DNA although RNA vectors are also available. Vectors include, but are not limited to, plasmids and phagemids.
- a cloning vector is one which is able to replicate in a host cell, and which is further characterized by one or more endonuclease restriction sites at which the vector may be cut in a determinable fashion and into which a desired DNA sequence may be ligated such that the new recombinant vector retains its ability to replicate in the host cell.
- replication ofthe desired sequence may occur many times as the plasmid increases in copy number within the host bacterium, or just a single time per host as the host reproduces by mitosis.
- replication may occur actively during a lytic phase or passively during a lysogenic phase.
- An expression vector is one into which a desired DNA sequence may be inserted by restriction and ligation such that it is operably joined to regulatory sequences and may be expressed as an RNA transcript.
- Vectors may further contain one or more marker sequences suitable for use in the identification of cells which have or have not been transformed or transfected with the vector. Markers include, for example, genes encoding proteins which increase or decrease either resistance or sensitivity to antibiotics or other compounds, genes which encode enzymes whose activities are detectable by standard assays known in the art (e.g., ⁇ -galactosidase or alkaline phosphatase), and genes which visibly affect the phenotype of transformed or transfected cells, hosts, colonies or plaques.
- Preferred vectors are those capable of autonomous replication and expression ofthe stractural gene products present in the DNA segments to which they are operably joined.
- stringent conditions refers to parameters known to those skilled in the art.
- One example of stringent conditions is hybridization at 65°C in hybridization buffer (3.5 x SSC, 0.02% Ficoll, 0.02% polyvinyl pyrolidone, 0.02% bovine serum albumin (BSA), 25mM NaH 2 PO 4 (pH7), 0.5% SDS, 2mM EDTA).
- SSC 0.15M sodium chloride/0.15M sodium citrate, pH7;
- SDS is sodium dodecylsulphate; and
- EDTA is ethylene diamine terra acetic acid.
- suitable plasmid vectors include pBR322, pUC18, pUC19 and the like; suitable phage or bacteriophage vectors include ⁇ gtlO, ⁇ gtl 1 and the like; and suitable virus vectors include pMAM-neo, pKRC and the like.
- the selected vector ofthe present invention has the capacity to autonomously replicate in the selected host cell.
- Useful prokaryotic hosts include bacteria such as E. coli, Flavobacterium heparinum, Bacillus, Streptomyces, Pseudomonas, Salmonella, Serratia, and the like.
- a substantially pure modified heparinase of the invention to a functional prokaryotic promoter.
- Such promoter may be either constitutive or, more preferably, regulatable (i.e., inducible or derepressible).
- constitutive promoters include the int promoter of bacteriophage ⁇ , the bla promoter ofthe ⁇ -lactamase gene sequence of pBR322, and the CAT promoter ofthe chloramphenicol acetyl transferase gene sequence of pPR325, and the like.
- inducible prokaryotic promoters examples include the major right and left promoters of bacteriophage ⁇ (P L and P R ), the tip, recA, lacZ, lad, and gal promoters of E. coli, the -amylase (Ulmanen et al., J Bacteriol. 162:176-182 (1985)) and the ⁇ -28-specific promoters of B.
- subtilis (Gilman et al., Gene sequence 32:11-20 (1984)), the promoters ofthe bacteriophages of Bacillus (Gryczan, In: 77ze Molecular Biology ofthe Bacilli, Academic Press, Inc., NY (1982)), and Streptomyces promoters (Ward et al, Mol. Gen. Genet. 203:468-478 (1986)).
- Prokaryotic promoters are reviewed by Glick (J. bid. Microbiol. 1 :277-282 (1987)); Cenatiempo (Biochimie 68:505-516 (1986)); and Gottesman (Ann. Rev. Genet. 18:415-442 (1984)). Proper expression in a.
- prokaryotic cell also requires the presence of a ribosome binding site upstream ofthe encoding sequence.
- ribosome binding sites are disclosed, for example, by Gold et al. (Ann. Rev. Microbiol. 35:365-404 (1981)).
- eukaryotic hosts include, for example, yeast, fungi, insect cells, and mammalian cells, either in vivo or in tissue culture.
- Mammalian cells which may be useful as hosts include HeLa cells, cells of fibroblast origin such as VERO or CHO-K1, or cells of lymphoid origin, such as the hybridoma SP2/0-AG14 or the myeloma P3x63Sg8, and their derivatives.
- Prefened mammalian host cells include SP2/0 and J558L, as well as neuroblastoma cell lines such as IMR 332 that may provide better capacities for conect post-translational processing.
- Embryonic cells and mature cells of a transplantable organ also are useful according to some aspects ofthe invention.
- plant cells are also available as hosts, and control sequences compatible with plant cells are available, such as the nopaline synthase promoter and polyadenylation signal sequences.
- Another preferred host is an insect cell, for example in Drosophila larvae. Using insect cells as hosts, the Drosophila alcohol dehydrogenase promoter can be used (Rubin, Science 240:1453-1459 (1988)).
- baculoviras vectors can be engineered to express large amounts of the modified heparinases ofthe invention in insects cells (Jasny, Science 238:1653 (1987); Miller et al., In: Genetic Engineering (1986), Setlow, J.K., et al., eds., Plenum, Vol.
- Any of a series of yeast gene sequence expression systems which incorporate promoter and termination elements from the genes coding for glycolytic enzymes and which are produced in large quantities when the yeast are grown in media rich in glucose may also be utilized.
- glycolytic gene sequences can also provide very efficient transcriptional control signals.
- Yeast provide substantial advantages in that they can also cany out post-translational peptide modifications.
- a number of recombinant DNA strategies exist which utilize strong promoter sequences and high copy number plasmids which can be utilized for production ofthe desired proteins in yeast.
- Yeast recognize leader sequences on cloned mammalian gene sequence products and secrete peptides
- ⁇ bearing leader sequences i.e., pre-peptides.
- the transcriptional and translational regulatory signals may be derived from viral sources, such as adenoviras, bovine papilloma virus, simian virus, or the like, where the regulatory signals are associated with a particular gene sequence which has a high level of expression.
- promoters from mammalian expression products such as actin, collagen, myosin, and the like, may be employed.
- Transcriptional initiation regulatory signals may be selected which allow for repression or activation, so that expression ofthe gene sequences can be modulated.
- eukaryotic regulatory regions will, in general, include a promoter region sufficient to direct the initiation of RNA synthesis.
- Preferred eukaryotic promoters include, for example, the promoter ofthe mouse metallothionein I gene sequence (Hamer et al., J. Mol. Appl. Gen.
- the linkage between a eukaryotic promoter and a DNA sequence which encodes the modified heparinases ofthe invention does not contain any intervening codons which are capable of encoding a methionine (i.e., AUG).
- AUG methionine
- the presence of such codons results either in the formation of a fusion protein (if the AUG codon is in the same reading frame as the modified heparinases ofthe invention coding sequence) or a frame-shift mutation (if the AUG codon is not in the same reading frame as the modified heparinases ofthe invention coding sequence).
- a vector is employed which is capable of integrating the desired gene sequences into the host cell chromosome.
- Cells which have stably integrated the introduced DNA into their chromosomes can be selected by also introducing one or more markers which allow for selection of host cells which contain the expression vector.
- the marker may, for example, provide for prototrophy to an auxotrophic host or may confer biocide resistance to, e.g., antibiotics, heavy metals, or the like.
- the selectable marker gene sequence can either be directly linked to the DNA gene sequences to be expressed, or introduced into the same cell by co-transfection. Additional elements may also be needed for optimal synthesis ofthe modified heparinases ofthe invention mRNA. These elements may include splice signals, as well as transcription promoters, enhancers, and termination signals.
- cDNA expression vectors incorporating such elements include those described by Okayama, Molec. Cell. Biol. 3:280 (1983).
- the introduced sequence will be incorporated into a plasmid or viral vector capable of autonomous replication in the recipient host. Any of a wide variety of vectors may be employed for this purpose. Factors of importance in selecting a particular plasmid or viral vector include: the ease with which recipient cells that contain the vector may be recognized and selected from those recipient cells which do not contain the vector; the number of copies ofthe vector which are desired in a particular host; and whether it is desirable to be able to "shuttle" the vector between host cells of different species.
- Prefened prokaryotic vectors include plasmids such as those capable of replication in E. coli (such as, for example, pBR322, Col ⁇ l, pSClOl, pACYC 184, and ⁇ VX. Such plasmids are, for example, disclosed by Sambrook, et al.
- Bacillus plasmids include pC194, pC221, pT127, and the like. Such plasmids are disclosed by Gryczan (In: 77ze Molecular Biology ofthe Bacilli, Academic Press, NY (1982), pp. 307-329). Suitable Streptomyces plasmids include pIJlOl (Kendall et al., J. Bacterial.
- Pseudomonas plasmids are reviewed by John et al. (Rev. Infect. Dis. 8:693- 704 (1986)), and Izaki (Jpn. J. Bacteriol. 33:729-742 (1978)).
- Preferred eukaryotic plasmids include, for example, BPV, ⁇ BV, SV40, 2-micron circle, and the like, or their derivatives.
- Such plasmids are well known in the art (Botstein et al, Miami Wntr. Symp. 19:265-274 (1982); Broach, In: The Molecular Biology ofthe Yeast Saccharomyces: Life Cycle and Inheritance, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, p. 445-470 (1981); Broach, Cell 28:203-204 (1982); Bollon et al, J. Clin. Hematol. Oncol. 10:39-48 (1980); Maniatis, In: Cell Biology: A Comprehensive Treatise, Vol. 3, Gene Sequence Expression, Academic Press, NY, pp. 563-608 (1980)).
- Other preferred eukaryotic vectors are viral vectors.
- the pox virus, herpes virus, adenovirus and various retrovirases may be employed.
- the viral vectors may include either DNA or RNA vimses to cause expression ofthe insert DNA or insert RNA.
- DNA or RNA encoding the modified heparinases ofthe invention polypeptides may be directly injected into cells or may be impelled through cell membranes after being adhered to microparticles.
- the DNA constract(s) may be introduced into an appropriate host cell by any of a variety of suitable means, i.e., transformation, transfection, conjugation, protoplast fusion, electroporation, calcium phosphate-precipitation, direct microinjection, and the like.
- recipient cells are grown in a selective medium, which selects for the growth of vector-containing cells. Expression ofthe cloned gene sequence(s) results in the production ofthe modified heparinases of the invention.
- the chemical modification reagent diethylpyrocarbonate (DEPC) was purchased from Sigma and used as received (Milwaukee, WI). Heparan sulfate was purchased from Celsus Laboratories (Cincinnati, OH). Lys-C from Lysobacter enzymogenes (EC 3.4.21.50) was from Roche Molecular Biochemicals (Indianapolis, IN). Heparinase III from Flavobacterium heparinum (EC 4.2.2.8) was purified as described previously (Godavarti, R., Cooney, C.L., Langer, R., and Sasisekharan, R.
- Heparinase III Activity Assay The activity of heparinase III was measured using a UV 232 nm assay similar to those reported for heparinase I and heparinase II (Godavarti, R., Cooney, C.L., Langer, R., and Sasisekharan, R. (1996) Biochemistry 35, 6846-52-shriver, Z., Hu, Y., and Sasisekharan, R.
- heparinase III 50 ⁇ g/mL was incubated with DEPC in 50 mM sodium phosphate buffer at 25°C.
- the DEPC stock solution (6.9 M) was diluted with ethanol.
- Control reactions contained an equivalent amount of ethanol instead of DEPC and were found to not affect enzymatic activity over the experimental time range.
- three reactions were run using different concentrations of DEPC, ranging from 50 ⁇ M to 2.5 mM. At fixed time intervals, 25 ⁇ L aliquots were withdrawn from the reaction mixtures for the UV 232 nm activity assay.
- k' is the first order rate constant for DEPC hydrolysis and t is the measured time after addition of DEPC to the heparinase III solution.
- t is the measured time after addition of DEPC to the heparinase III solution.
- the order ofthe reaction in DEPC was determined by plotting the observed rate constants of inactivation at each pH vs. log [DEPC]. The slope of this graph is n, the order ofthe reaction with respect to DEPC (Lundblad, R. (1995) Techniques in Protein Modification,
- Peptides derived from heparinase III digestion were separated by reverse phase high performance liquid chromatography (RPHPLC) and monitored at 210, 277, and 240 nm. Peptide peaks not present in the control digest were collected and sequenced using an Applied Biosystems Sequencer model 477 with an on-line model 120 PTH amino acid analyzer (Biopolymers Laboratory, MIT). Site-directed Mutagenesis Each ofthe thirteen histidine residues of heparinase III was mutated to alanine using overlap extension PCR for 15 cycles (Higuchi, R.
- the thirteen mutant heparinase III sequences were prepared in ⁇ CR2.1 using a Miniprep kit (QIAGEN, Valencia, CA) and cloned using Nde I/BamH I (New England Biolabs, Beverly, MA) into pET-15b (Novagen, Madison, WI) for expression.
- the pET-15b plasmid contains a NH 2 -terminal His-Tag for Ni 2+ -column purification.
- Recombinant heparinase III was also expressed and compared to the native heparinase III isolated directly from Flavobacterium heparinum. Expression, Isolation, and Purification of r-heparinase III and Mutants in E.
- the cell pellet was re-suspended in 20 mM Tris, 500 mM NaCl, 5mM immidazole-HCl, pH 7.9 (1/50 of the initial culture volume).
- the re-suspended cells were placed on ice and sonicated as described previously (Ernst, S., Venkataraman, G., Winkler, S., Godavarti, R., Langer, R., Cooney, C.L., and Sasisekharan, R. (1996) Biochem. J. 315, 589-97).
- the soluble protein ofthe cell lysate was isolated by centrifugation at 12,000 rpm for 20 min. at 4°C.
- the supemant was filtered through a 0.45 ⁇ m filter and loaded onto a nickel column using a Biocad Perfusion Chromatography system (PerSeptive Biosystems, Framingham, MA). The column was washed and the protein was subsequently eluted in 20 mM Tris, 500 mM NaCl, 500 mM immidazole-HCl, pH 7.9. SDS-polyacrylamide gel electrophoresis analysis using precast 12% gels, the Mini-Protean II apparatus, and the Silver Stain Pus kit (Bio-Rad, Hercules, CA) was performed to determine the concentration and purity ofthe individual proteins.
- Circular Dichroism Recombinantly expressed heparinase III and the heparinase III mutants, H295A and H510A were concentrated and buffer-exchanged into 50 mM sodium phosphate, pH 7.0 using a Centricon 30 Filter (Millipore, Watertown, Massachusetts).
- CD spectra were collected on an Aviv 62DS spectropolarimeter equipped with a thermostatic temperature controller and interfaced to an IBM microcomputer. Measurements were performed in a quartz cell with a 1 mm path length. Spectra were recorded at 25°C, in an average of 10 scans between 205 and 260 nm, with a 1.0 nm bandwidth and a scan rate of 3 nm/min.
- CD band intensities are expressed as molar ellipticities, ⁇ M , in degrees-cm 2 -dmol "1 .
- Transfection of B16 cells B16BL6 melanoma cells were transfected with antisense 2OST in pcDNA3.1. Stable transfectant clones were selected with G418 and propagated. The success of transfection was confirmed with PCR screening of transfected cells.
- In vitro invasion assay 10 5 of B16BL6 and B16BL6 transfectant were loaded onto inserts coated with 15 ug of Matrigel. MEM- ⁇ with 40 ng/ml of bFGF was used as chemoattractant. After 20 hours incubation at 37° C, inserts were fixed and stained.
- Compositional Analysis of HLGAGs resulting from heparinase treatment of B16 cells B16 cells were treated with either heparinase I, III or PBS. The supernatant was collected, boiled, and filtered through a 0.45 ⁇ m filter. This sample was then subjected to fractionation using a centricon spin column with a nominal molecular weight cutoff of 5 kDa. The retentate was exchanged into water and concentrated 50-fold by lyophilization. Compositional analysis of oligosaccharides was completed by exhaustive digest ofthe high molecular weight fraction with heparinases I-III.
- aqueous oligosaccharide was added 1 mU of heparinases I-III in 25 mM sodium acetate, 2 mM calcium chloride buffer at pH 7.0. The reaction was allowed to proceed at 37°C overnight after which CE analysis was completed. Compositional analysis was completed on a Hewlett Packard 3D CE unit by using uncoated fused silica capillaries (i.d. 75 ⁇ M). Analytes were measured using an extended path length capillary. The electrolyte was 50 mM tris/phosphate pH 2.5. Separations were canied out at 30 kV with reverse polarity. Assignments and quantification of disaccharides were made by comparison with known standards.
- Example 1 DEPC inactivates heparinase III.
- DEPC is a common reagent used for the determination of catalytically critical histidines in enzymes.
- DEPC is useful for the determination of catalytically critical histidines, however care needs to be taken to ensure that other nucleophilic residues, namely tyrosines, lysines, and cysteines are not modified.
- Heparinase III was incubated with 0.31 (D), 0.54 (•), 0.97 (O), 1.5 (A), 1.9 ( ⁇ ) mM DEPC at pH 6.5 and at 25°C (shown in inset of Figure 1).
- the natural log ofthe percent activity remaining was plotted versus an adjusted time term (f) to account for the decomposition of DEPC.
- the slope of each ofthe lines at the various DEPC concentrations represents the pseudo-first order rate constants of inactivation.
- heparinase III contains no cysteines in its primary amino acid sequence. Furthermore, there was no loss of absorbance at 278 mn upon incubation of DEPC with heparinase III as would be expected if tyrosines were modified. Finally, addition of hydroxylamine to DEPC-modified heparinase III reversed most ofthe inactivation indicating that strongly nucleophilic residues, such as lysine, were not modified by DEPC (Table 1).
- Heparinase III (50 ⁇ g/mL) was incubated with 2 mg/mL heparan sulfate for 30 min. 1.5 mM DEPC was added to the reaction and time course of inactivation was completed using the heparinase III activity assay (O). A control reaction without pre- incubation with heparan sulfate was also done (D).
- Example 2 Peptide Mapping ofthe Histidine Modified by DEPC.
- DEPC-modified heparinase III was digested with Lys-C.
- Peptides that had altered retention times and an increased in absorbance at 240 nm as compared to a control digest were collected and sequenced ( Figure 5).
- Three peptides had altered retention times and increased absorbance at 240 nm were isolated and sequenced.
- Two ofthe peptides contained histidine 295 and one contained no modified histidine residues.
- each ofthe thirteen histidine residues present in heparinase III was mutated to alanine.
- the recombinant heparinase III mutant proteins were expressed, purified, and assessed for enzymatic activity towards heparan sulfate (Table 2).
- the r-heparinase III construct without its putative signal sequence was expressed.
- concentration and purity of all recombinant enzyme preparations were determined using SDS-PAGE.
- the recombinantly expressed heparinase III was also compared to the heparinase III isolated from F. heparinum to ensure that they were the same molecular weight.
- SAX analysis of exhaustive heparinase III digests of heparan sulfate is shown in Figure 6. Heparinase III (20 ⁇ g/mL) was incubated with a 4 mg/mL of heparan sulfate overnight at 37°C.
- the reaction was loaded onto a SAX column and the saccharide products were eluted using a gradient of 0.2-1.0 M NaCl, pH 3.5 over 30 min. and monitored at 232 nm.
- A Heparan sulfate digested with heparinase III from F. heparinum.
- B Heparan sulfate digested with recombinant heparinase III.
- C Heparan sulfate digested with the H295A mutant enzyme.
- D Heparan sulfate digested with the H510A mutant enzyme.
- E Heparan sulfate digested with the H105A mutant enzyme.
- the recombinantly expressed heparinase III, the H295A mutant, and the H510A mutant were compared using circular dichroism (CD). Circular dichroism analysis of recombinant heparinase III and the H295A mutant enzyme is shown in Figure 7.
- the recombinant heparinase III (•), the H295A mutant enzyme (O), and the H510A mutant enzyme (El) were concentrated and buffer exchanged into 50 M sodium phosphate buffer, pH 7.0. Readings were taken using a quartz cell with a 1 mm path length at 25°C.
- heparinase I cleaves at the highly sulfated regions of HLGAGs
- heparinase III only cleaves at the under-sulfated regions ofthe polysaccharide chain, thereby rendering these enzymes powerful tools to investigate in vivo and in vitro roles of HLGAGs, in development, morphogenesis, angiogenesis etc.
- B16BL6 melanoma was used as a model system and treated tumor-bearing mice with either heparinases I or III to investigate both primary tumor growth as well as tumor metastasis. Consistent with the cunent paradigm, heparinase I accelerated tumor growth (Figure 8).
- heparinase III inhibited primary tumor growth ( Figure 8).
- the inhibition of melanoma growth by heparinase III was shown to be dose dependent. Inhibition of primary tumor growth by heparinase III was first observed at 2 mg/kg per day. Tumor growth was inliibited by 73% at 12 mg/kg per day, the maximum dosage tested in the study ( Figure 8).
- mice treated with heat inactivated heparinase III exhibited comparable growth curves with that of mice treated with PBS ( Figure 8), suggesting that the catalytic activity of heparinase III was responsible for heparinase Ill's ability to inhibit primary tumor growth. Histological examination of tumor samples revealed increased apoptosis in heparinase III treated tumors, while heparinase I treated tumors revealed reduced apoptosis. Mice, 15 days after tumor implantation with B16BL6 melanoma, were examined. 4 x 10 5 log growth phase B16BL6 melanoma cells in 0.1 ml PBS were injected to the flank of C57BL/6 mice on day 1.
- the data was depicted as growth curves of mice bearing melanoma treated with PBS, inactive hep III and active hep III. To ensure that these observations were not limited to the tumor model chosen, hep III was used to treat mice bearing Lewis lung carcinoma (LLC) tumors. Growth curves of primary tumor growth for LLC tumor in C57BL/6 mice treated with either PBS or heparinase III were plotted. 4 x 10 5 log phase LLC cells were injected subcutaneously to the flank of mice on day 1.
- heparinase III Daily injection of 0.1 ml of either PBS or 2 mg/ml recombinantly expressed heparinase III started at the day 4 and continued throughout the experiment. At day 8, osmotic pumps (100 ⁇ l capacity delivering at 0.5 ⁇ l per hour) containing PBS or 3 mg/ml hep III were implanted subcutaneously at a place remote from the tumor site. Mice were sacrificed at day 20. Lung metastasis of LLC cells injected through tail vein were quantitated as number of lung nodules. Log growth LLC cells were trypsinized for 30 seconds and resuspended in PBS to a final concentration of 1 x 10 6 per ml.
- mice were incubated with 200 nm hep III for 30 minutes at 37 °C before injecting 0.2 ml of cell suspension via tail vein.
- Mice were euthanized 12 days after tail vein injections, lungs were harvested, rinsed in tap water and fixed overnight in Bouin's solution. The number of nodules on lung surface was counted with the aid of a dissection microscope. Similar to the B16BL6, heparinase III treatment of mice-bearing LLC tumor at 12 mg/kg per day showed inhibition in tumor growth.
- HLGAG fragments derived from LLC cells were isolated, harvested in PBS. Consistent with the B16BL6 results, heparinase I generated LLC HLGAG fragments promoted growth of B16BL6 tumor cells, while heparinase III generated LLC HLGAG fragments showed minimal effect on growth of B16BL6 cells.
- heparinase III derived LLC HLGAG fragments inhibited B16BL6 metastasis to the lungs.
- the in vivo studies, along with in vitro cell culture experiments points to the enzymatic action of hep III reducing the tumorigenicity of a variety of tumor cell types.
- heparinase III treatment of cells may result in cells losing their unique surface HLGAG coat and this directly or indirectly may impinge on their ability to grow or metastasize.
- heparinase III treatment of cells may also result in the generation of distinct HLGAG fragments, and these fragments could then directly or indirectly modulate tumor cell function. It was thought that heparinase III may function though either one of these mechanisms or through some combination of these mechanisms.
- B16BL6 cells with either heparinases I or III, to remove the HLGAG coat on the tumor cell surface.
- the removal ofthe HLGAG coat either by heparinase I or III, had no effect on the ability of these cells to grow in mice compared to untreated cells.
- mice Prior to injection, cells were treated with either PBS, hep I (200 nm) or hep III (200 nm) for 30 min. at 37 °C. 0.2 ml cell suspensions (2 x 10 5 cells) were injected slowly via tail vein. Mice were sacrificed 13 days later and lungs were harvested, rinsed with tap water. The number of nodules on lung surface were counted with the assistance of a dissection microscope. * indicates p ⁇ 0.05 (Mann- Whitney test). B16BL6 cells were treated with either heparinases I or III and then injected via tail vein of syngeneic mice.
- heparinase III treated B16BL6 cells were significantly less able to metastasize to the lungs, while heparinase I treated cells were marginally effected, if any, in their ability to metastasize to the lungs when compared to the control PBS treated cells (Figure 9).
- the removal of specific HLGAG coat present on the B 16BL6 tumor cells significantly affected the ability of the tumor cells to metastasize but had no effect on the growth of B16BL6 tumor cells. It should be pointed out that heparinase treatment of cells would generate HLGAG fragments that might still bind to specific proteins on the B16BL6 surface to inhibit tumor metastasis.
- both the heparinase I and III generated HLGAG fragments from the B16BL6 cells were isolated, harvested in PBS and tested ( Figure 10).
- B16BL6 melanoma were treated with GAG fragments generated from treatment of B16BL6 cells with hep I and III. Briefly, 80-90%o confluent cells were washed with PBS once. 1.5 ml of PBS containing 200 nm of heparinase I or III were added to the flasks, incubated at 37 °C on a shaker for 2 h.
- HLGAG fragments generated from B16BL6 cells by heparinase treatment also appear to play a role in tumor growth and metastasis.
- tumor tissues were fixed in either 4% (vol/vol) formaldehyde overnight for von Willebrand factor (vWF) staining and terminal deoxynucleotide transferase (TdT) labeling or in Glyo-Fixx solution overnight for Ki-67 nuclear antigen staining.
- Tissues were embedded in paraffin according to standard histological procedures.
- vWF staining sections (5 ⁇ m thick) were incubated with 0.2 N HCl for 10 min. autoclaved in a Coplin jar immersed with Target retrieval Solution (Dako) for 15 min. and permeabilized with 2 ⁇ g/ml proteinase K at 37 °C for 15 min.
- Sections were incubated with rabbit anti-human vWF antibody coupled with horseradish peroxidase (HRP) (Dako). Positive staining was detected by substrate reaction with diaminobenzidine. Sections were counterstaiiied with Gill's hematoxylin and mounted in Permount (Fisher). Ki-67 antigen staining (rabbit anti-human Ki-67 antigen antibody coupled with HRP, Dako) and TdT labeling (DeadEnd Colorimetric Apoptosis Detection System, Promega) were done essentially according to manufacture's protocol. Capillary density was determined by counting the number of vWF-positive capillaries per high power field (HPF, x200).
- the proliferative and apoptotic indices of tumor cells within areas of viable tumor were estimated from the percentage of cells scored under a light microscope at 400-fold magnification. A minimum of 2000 cells were counted in each animal. # indicates standard e ⁇ or.
- the overall similarity of data for the B16BL6 and the LLC animal models suggests an important role for HLGAGs in tumor growth and metastasis.
- the differential effects of heparinases I and III, and the HLGAG fragments generated by heparinases are consistent with the unique specificities of heparinases, and hence the distinct oligosaccharide products they generate.
- HLGAG fragments for one cell type is able to influence effects on another cell type, strongly suggesting the involvement of specific sequences of HLGAG in modulating effects on tumor growth and metastasis.
- B16BL6 melanoma cells were transfected with antisense 2OST in pcDNA3.1 and tested in an in vitro invasion assay. The cells that migrated were removed and counted. The number of cells migrated per high power field (x 400) for antisense 2OST transfected cells was twice as much as that of unfransfected B16BL6 cells. The results are shown in the bar graph of Figure 11.
- the ability ofthe transfected cells to develop into primary tumors was assessed by subcutaneous inoculation of 4 x 10 5 B16BL6, transfected and unfransfected respectively into the flank of nude mice.
- the mean tumor volume and tumor weight of transfected group was more than two fold greater than that ofthe unfransfected control group, as shown in Figure 12 a and b respectively.
- the ability ofthe transfected cells to metastasize was determined by injection of 2 x 10 5 B16BL6 in 0.2 ml PBS, of transfected and unfransfected cells via tail vein of C57BL6 mice.
- the number of metastatic nodules on the lung surface for the transfected group was more than three fold greater than that ofthe unfransfected control.
- Example 5 HLGAG fragments with distinct composition are potent inhibitors of tumor growth and metastasis.
- B16BL6 cells were treated with either a, hep I; b, hep III; or c, a PBS control and the released HLGAG fragments harvested. Saccharide fragments were collected in PBS, and subjected to purification and fractionation. First, samples were bound to an Ultrafree-DEAE membrane, wliich had been equilibrated with pH 6.0 sodium phosphate, 0.15 M NaCl.
- Hep I or hep Ill-derived HLGAG saccharide fragments were subjected to partial enzymatic cleavage by 100 nM (8 ⁇ g/ml) heparinase II in 10 mM ethylenediamine, 10 ⁇ M ovalbumin, 1 ⁇ M dextran sulfate pH 7.0 for one hour. Resulting digests were complexed with the basic peptide (RG) ⁇ 9 R and subjected to matrix-assisted laser desorption ionization mass spectrometry. The HLGAG fragment fingerprint is different for the hep I vs hep III generated fragments consistent with each being stmcturally distinct.
- HLGAG binding proteins are growth factors and cytokines
- FGF2 signaling has been shown to be a prerequisite for melanoma progression promoting tumor growth in an autocrine fashion, and the interruption ofthe FGF2 autocrine loop by interfering with either FGF2 or FGF receptor (FGFR) activity results in inhibition of melanoma progression (Rodeck, U. et al Constitutive expression of multiple growth factor genes by melanoma cells but not normal melanocytes. J Invest Dermatol 97, 20-6 (1991). Becker, D., Meier, C. B.
- FGF2 is a potent and essential angiogenic factor regulating melanoma neovascularization (Wang, Y. & Becker, D. Antisense targeting of basic fibroblast growth factor and fibroblast growth factor receptor- 1 in human melanomas blocks intratumoral angiogenesis and tumor growth. Nat Med 3, 887-93 (1997).
- Protein concentration in the lysate was determined using the Bio-Rad protein assay kit (BioRad) and adjusted accordingly for electrophoresis analysis.
- BioRad Bio-Rad protein assay kit
- For the heparinase treated groups cells were treated with either hep I or hep III (200 nM) for 30 min at 37 °C prior to addition of FGF2.
- the immunoblot was probed with anti-Erk-1, 2 or anti-phospho-Erk-1, 2 antibody (New England Biolabs; MA) and detected by anti-rabbit IgG conjugated to HRP using SuperSignal West Pico Chemiluminescent substrate (Pierce, IL).
- BaF3 cells expressing FGFR were grown in the following fashion. The initial cell number was counted by Coulter counter, and resuspended to a density of 1 x 10 5 cells/ml into 12 samples of 6 ml. Each sample of cells was centrifuged 3 min at room temperature at 1085 x g, and resuspended in HLGAG preparations in PBS, producing two sets of cells in the same media. One of each set was supplemented with 50 ng/ml FGF2 (HH) while the other was unsupplemented (GJ).
- HH ng/ml FGF2
- GJ unsupplemented
- the index is defined as the increase in cell number for the experimental case divided by the increase in cell number for the positive control.
- the positive control was cells in DMEM with 10% BCS, 2 mM L-glutamine, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin, 500 ng/ml heparin, and 50 ng/ml FGF2.
- the negative control was cells in DMEM with 10%» BCS, 2 mM L-glutamine, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin, and 500 ng/ml heparin.
- c Effect of treatment of tumor with hep I and hep III in FGFR1 activation compared to PBS.
- phosphorylated FGFR1 in tumor samples was assessed by standard immunoprecipitation followed by Westemblotting with phosphotyrosine specific antibody.
- Primary B16BL6 tumors were grown and treated as described earlier and at day 15 the tumor was harvested in cold modified RIPA buffer containing enzyme inhibitors and homogenized. The homogenates were past through 25 -gauge needle 3 times and centrifuged. The supernatant was adjusted for protein concentration using the Bio-Rad protein assay kit (Bio-Rad).
- FGFR1 was immunoprecipitated with poly-clonal anti-FGFRl antibody (Santa Cruz Biotechnology, Inc., CA).
- the immunoblot was probed with phosphotyrosine specific antibody conjugated to HRP (RC20; Transduction Laboratories, Lexington, KY) and developed with SuperSignal West Pico Chemiluminescent substrate.
- the molecular weight of FGFR1 is 120 KDa.
- d Effect of heparinase treatment on activation of FAK in B16BL6 tumor.
- the FAK protein was immunoprecipitated with mouse anti-FAK monoclonal antibody (Transduction Laboratories, Lexington, KY) according to the procedures described above.
- the phosphorylated FAK was detected using phosphotyrosine specific antibody RC20.
- e Level of total and phosphorylated Erk-1, 2 in heparinase-treated B16BL6 tumor.
- Tumor homogenates were prepared and processed as described in c. The supernatant was used for total protein concentration assay and immunoblotting. The immunoblot was detected as described in a. f, Effect of heparinase treatment on Akt activation. The primary tumor was treated and processed as described above. Akt antibody and phospho-Akt antibody from New England Biolabs were used to probe the immunoblot. Results: Upon FGF2 stimulation, decreased Erk-1, 2 activation was seen in hep III treated cells while increased Erk-1, 2 activation was seen with cells treated with hep I.
- F32 cells a pre-lymphocyte cell line that has been transfected with FGFR, and that often has been used as a model system to study FGF-mediated signaling in cell culture unfettered by complications associated with signaling events initiated by other growth factors and/or receptors (Ornitz, D. M. et al. Receptor specificity of the fibroblast growth factor family. J Biol Chem 271, 15292-7 (1996).). Similar to what was observed in B16BL6 cells, hep I fragments promote, whereas hep III fragments inhibited FGF2-mediated cellular proliferation in these cells (Fig. 14).
- HLGAG fragments derived from the cell surface can substantially and specifically affect FGF2 signaling.
- hep I and hep Ill-derived B16BL6 fragments significantly affect FGF signaling pathways in vivo.
- FGFR phosphorylation in hep I and hep Ill- treated animals as well as Erk-1 and 2 signaling.
- Treatment of the primary tumor with hep III (or its generated fragments) inhibited phosphorylation of FGFR1 while hep I treatment had the opposite effect on the phosphorylation of FGFR1.
- FAK focal adhesion kinase
- Example 7 Modulation ofFGF2 activity in vivo by B16BL6 fragments
- Methods a-c, Assessment of FGF2 signaling in vivo with the rat corneal pocket assay. Representative slit lamp photographs of rat corneas on day 6 after implantation with Hydron pellets containing FGF2, hep I fragments with FGF2, and hep III fragments with FGF2. The amount of FGF2 loaded into each pellet was ⁇ 120 ng, and the amount of HLGAG fragments was approximately 1 ng. The pellets were prepared and implanted essentially as described (Kenyon, B. M. et al. A model of angiogenesis in the mouse cornea. Invest Ophthalmol Vis Sci 37, 1625-32 (1996)).
- HLGAGs as part of proteoglycans
- heparin-binding domains of FGF2 and FGFR allows the formation of a ternary complex at the cell surface that forms the basis of FGF2 signaling.
- Digestion ofthe cell HLGAG coat with hep I releases fragments with an appropriate spatial display of 2 O-, 6 O- and N-sulfated groups that would allow an optimal "fit" to both FGF2 and FGFR, leading to signaling through tyrosine kinase activation.
- hep Ill-generated HLGAG fragments display another pattern of sulfated groups are still able to bind FGF2 but fail to form a constmctive signaling complex at the cell surface, thus inhibiting FGF2 activity.
- Results To demonstrate a direct interaction between B16BL6 HLGAG fragments and their immediate target FGF2 in vivo, we evaluated the ability of B16BL6 HLGAG fragments to modulate FGF2-induced responses leading to cell migration, proliferation, and differentiation in vivo using corneal neovascularization assay (Fig. 15a table). In this model, hep I-generated fragments mixed with FGF2 bound to the growth factor and promoted the in vivo neovascularization response to FGF2 (Fig.
- the polysaccharide compartment exhibits a similar phenomenon.
- distinct HLGAG oligosaccharides upon release by enzymatic cleavage from the tumor cell surface can serve as potent inhibitors of tumor progression.
- the present study not only allows a new paradigm of how the polysaccharides modulate tumor growth and metastasis, but it identifies a novel therapeutic target by providing a framework towards the development of HLGAG-based novel anti-cancer molecules.
- HLGAG fragments may exert their effects through many pathways including autocrine growth and angiogenic factors, or through interactions with ECM molecules. Additionally , the sources of the endogenous HLGAG-degrading enzymes and it substrate specificity also become important. Production of HLGAG-degrading enzyme, presumably by the tumor cells, with substrate specificity similar to heparinase I will be advantageous to tumor cells.
- HLGAG-degrading enzyme secretion of HLGAG-degrading enzyme by a tumor cell would lead to the production of specific HLGAG sequences (from it own coat or the tumor bed ECM) which might exert effects via autocrine and angiogenic growth factors, or through other signaling pathways to support tumor growth and metastasis.
- HLGAG-degrading enzyme with substrate specificity similar to heparinase III would be extremely beneficial to the host.
- the endothelial cells in the vicinity of a tumor or macrophages can secrete an enzyme with substrate specificity of heparinase III leading to the production of specific HLGAG
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AU2001243512B2 (en) | 2007-05-10 |
EP1266013A2 (en) | 2002-12-18 |
US20030099628A1 (en) | 2003-05-29 |
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WO2001066772A3 (en) | 2002-05-02 |
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US20060183713A1 (en) | 2006-08-17 |
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US7939292B2 (en) | 2011-05-10 |
US20120027744A1 (en) | 2012-02-02 |
US20060067928A1 (en) | 2006-03-30 |
AU4351201A (en) | 2001-09-17 |
US20090081635A1 (en) | 2009-03-26 |
US20050233402A1 (en) | 2005-10-20 |
AU2001243512C1 (en) | 2008-04-17 |
WO2001066772A2 (en) | 2001-09-13 |
CA2402160A1 (en) | 2001-09-13 |
JP2003525946A (en) | 2003-09-02 |
CA2402160C (en) | 2012-02-14 |
EP1266013B1 (en) | 2014-10-15 |
US20020122793A1 (en) | 2002-09-05 |
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