WO2003006645A2 - Methode et composition permettant d'inhiber l'activite de l'heparanase - Google Patents

Methode et composition permettant d'inhiber l'activite de l'heparanase Download PDF

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WO2003006645A2
WO2003006645A2 PCT/US2002/021773 US0221773W WO03006645A2 WO 2003006645 A2 WO2003006645 A2 WO 2003006645A2 US 0221773 W US0221773 W US 0221773W WO 03006645 A2 WO03006645 A2 WO 03006645A2
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heparanase
animal
fragment
immunogen
polypeptide
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PCT/US2002/021773
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WO2003006645A3 (fr
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Peter Bohlen
Daniel Hicklin
Paul Kussie
Yiwen Li
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Imclone Systems Incorporated
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Priority to JP2003512403A priority Critical patent/JP2005521379A/ja
Priority to CA002453566A priority patent/CA2453566A1/fr
Priority to EP02763248A priority patent/EP1417304A4/fr
Priority to US10/483,858 priority patent/US20040247577A1/en
Publication of WO2003006645A2 publication Critical patent/WO2003006645A2/fr
Publication of WO2003006645A3 publication Critical patent/WO2003006645A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01166—Heparanase (3.2.1.166)
    • 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
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • 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
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)

Definitions

  • the present invention is directed to methods and compositions for inhibiting heparanase activity. More particularly, the present invention is directed to methods for treatment of conditions associated with heparanase activity.
  • Heparan sulfate proteoglycans are widely distributed in mammalian tissues and are involved in a number of processes related to malignancy. See generally Blackhall et al., Br. J. Cancer, 85(8): 1094-8 (Oct. 2001). Elevated levels of heparanase have been detected in sera from metastatic tumor-bearing animals and malignant melanoma patients, and a correlation exists between serum heparanase activity and the extent of tumor metastases. Cleavage of HSPGs by heparanase leads to disassembly of ECM and release of bioactive agents such as pro-angiogenic factors.
  • HSPGs are composed of a core protein to which chains of the glycosaminoglycan, heparan sulfate (HS), are attached.
  • the polysaccharide HS chains are typically composed of repeating hexuronic and D-glucosamine disaccharide units that are modified at various positions by sulfation, epimerization and N-acetylation, yielding clusters of sulfated disaccharides separated by low or non-sulfated regions.
  • HSPGs interact with many proteins, including growth factors, chemokines and structural proteins of the extracellular matrix (ECM), to influence cell growth, differentiation, and the cellular response to the environment. Specifically, interaction of T and B lymphocytes, platelets, granulocytes, macrophages and mast cells with the subendothelial ECM is associated with degradation of HS by a specific, endo- ⁇ -D-glucuronidase (heparanase) activity. See Nakajima et al., Science, 220: 611-613 (1983). Studies have also shown that HSPGs play an important role in the self-assembly and insolubility of ECM components, as well as in cell adhesion and locomotion.
  • ECM extracellular matrix
  • the heparanase enzyme that degrades HS is released from intracellular compartments, for example, from lysosomes and specific granules, in response to various activation signals, such as thrombin, calcium ionophore, immune complexes, antigens and mitogens, suggesting its regulated involvement in inflammation and cellular immunity.
  • Heparanase expressed by intact cells, platelets, mast cells, neutrophils and lymphoma cells was found to release active HS-bound basic fibroblast growth factor (bFGF) from ECM and basement membranes. Heparanase can thus elicit an indirect neovascular response in processes such as wound repair (resulting from injury) and inflammation.
  • bFGF basic fibroblast growth factor
  • the present invention is directed to methods of inhibiting heparanase activity and treating various conditions by administering to an animal an effective amount of an immunogen that elicits an immune response to heparanase.
  • the present invention provides methods whereby the immunogen is heparanase or a fragment thereof and, in a preferred embodiment, the immunogen is an antigen presenting cell (APC), such as a dendritic cell (DC), displaying heparanase or a fragment thereof on the surface.
  • APC antigen presenting cell
  • DC dendritic cell
  • compositions of the immunogen are also provided by the present invention.
  • the present invention is directed to an isolated heparanase mouse polypeptide, for example, SEQ ID NO:l or a fragment thereof.
  • the present invention is directed to an isolated heparanase polynucleotide encoding a mouse heparanase polynucleotide, for example, SEQ ID NO:2 or a fragment thereof, and a cloning (or expression) vector and a host cell having such a polynucleotide.
  • Figure 1 graphically depicts the concentration of heparanase specific antibodies at various dilutions induced in an animal as a result of in vivo administration to the animal of a control polypeptide.
  • Figure 2 graphically depicts the concentration of heparanase specific antibodies at various dilutions induced in an animal as a result of in vivo administration to the animal of DC pulsed with a control polypeptide.
  • Figure 3 graphically depicts the concentration of heparanase specific antibodies at various dilutions induced in an animal as a result of in vivo administration to the animal of DC pulsed with heparanase.
  • Figure 4 graphically depicts the number of Elispots producing IFN- ⁇ in an animal at different dilutions as a result of in vivo administration to the animal of a control polypeptide.
  • Figure 5 graphically depicts the number of Elispots producing IFN- ⁇ in an animal at different dilutions as a result of in vivo administration to the animal of DC pulsed with a control polypeptide.
  • Figure 6 graphically depicts the number of Elispots producing IFN- ⁇ in an animal at different dilutions as a result of in vivo administration to the animal of DC pulsed with heparanase.
  • Figure 7 graphically depicts the percent survival over time of an animal following in vivo administration to the animal of PBS, DC pulsed with a control polypeptide, or DC pulsed with heparanase.
  • Figure 8 graphically depicts the mean number of lung metastases in an animal following in vivo administration to the animal of PBS, DC pulsed with a control polypeptide, or DC pulsed with heparanase.
  • heparanase In the context of the present invention, heparanase, heparanase activity or heparanase catalytic activity refers to an animal endoglycosidase hydrolyzing activity that is specific for heparan or HS (including HSPG) substrates. This is in contrast to the activity of bacterial enzymes (heparinase I, II and III) that degrade heparan or HS by means of ⁇ -elimination.
  • Heparanase activity that is inhibited in the context of the present invention is preferably native. That is, the heparanase activity, while it may be altered relative to basal levels, is naturally occurring in the animal.
  • the present invention is directed to inhibiting heparanase activity by eliciting an immune response to heparanase.
  • One consequence of inhibition of heparanase activity is prevention of degradation of HSPG. It should be appreciated that this is but one consequence and is by no means the only consequence of inhibition of heparanase activity.
  • Other consequences include inhibition of degradation of the basement membrane, which prevents tumor metastasis, and prevention of invasion by endothelial cells, which is involved in angiogenesis.
  • inhibition of heparanase activity may prevent activated cells of the immune system from entering the circulatory system, thus inhibiting elicitation of both inflammatory-related conditions and autoimmune-related conditions.
  • Heparanase activity is inhibited by elicitation of an immune response to heparanase following administration of an effective amount of one or more immunogen(s).
  • an immunogen is effective to elicit an immune response, including a humoral or cell-mediated immune response, against native heparanase.
  • the immune response is preferably an active immunity that inhibits, that is, prevents, slows, or stops, heparanase activity. Therefore, in the context of the present inventive methods, heparanase activity need not be completely abrogated. It should be appreciated that the immune response against heparanase can be elicited either directly or indirectly.
  • an immunogen is not required by the present invention to inhibit heparanase activity directly. Rather, the immunogen can elicit the immune response indirectly by initiating a cascade through which heparanase activity is ultimately inhibited. Examples of such indirect inhibition include, but are not limited to, altering the rate or extent of transcription or degradation of one or more species of RNA relating to heparanase activity, translation or post-translational processing of the heparanase polypeptide and/or heparanase protein degradation.
  • the immunogen of the present invention can be a peptide, a DNA, an RNA, a small molecule, or any other suitable immunogenic molecule that inhibits heparanase activity.
  • the immunogen can be native to the animal; however, such an immunogen must be modified to provoke an immune response.
  • the term "native” as used herein means autologous or homologous to an animal, such that native antigens are "self polypeptides and are, absent modification, typically non-immunogenic in the animal from which they are derived.
  • the immunogen can be non-native to the animal, meaning foreign and not a "self polypeptide. As such, these immunogens can induce an immune response without additional modification.
  • the immunogen is a peptide or polypeptide.
  • Any suitable peptide or polypeptide can be used that inhibits heparanase activity, an example of which is heparanase or a fragment thereof. See U.S. Patent No. 5, 968,822.
  • a suitable heparanase may be a synthetically derived heparanase or a fragment thereof, a recombinantly derived heparanase or a fragment thereof, or a naturally derived heparanase or a fragment thereof.
  • a suitable heparanase can be native or foreign to the animal.
  • a suitable polypeptide immunogen is human heparanase, which is a 61.2 kDa polypeptide of 543 amino acids.
  • the mature 50 kDa enzyme, isolated from cells and tissues, has it N-terminus 157 amino acids downstream from the initiation codon, suggesting post-translational processing of the heparanase peptide.
  • Kussie et al. Biochem. Biophys. Res. Commun. 261: 183-7 (1999); Toyoshima & Nakajima, J. Biol. Chem. 274: 24153-60 (1999).
  • the amino acid sequence of heparanase contains a putative N-terminal signal peptide sequence (Met 1 to Ala 35 ) and a candidate transmembrane region (Pro 515 to He 534 ). See, e.g., Vlodavsky et al., Nat. Med. 5: 793-802 (1999); Hulett et al., Nat. Med. 5: 183-7 (1999). Site directed mutagenesis revealed that similar to other TIM-barrel glycosyl hydrolyses, heparanase has a common catalytic mechanism that involves two conserved acidic residues, a putative proton donor at Glu and a nucleophile at Glu .
  • a suitable foreign heparanase in the context of the present invention can be any heparanase that is non-native to the animal and that can induce an immune response without additional modification.
  • the heparanase can be derived from a mammal, such as a rabbit, rat, or mouse.
  • the heparanase or a fragment thereof is a mouse heparanase or a fragment thereof or a heparanase derived from a mouse heparanase or a fragment thereof.
  • a suitable polypeptide sequence for a mouse heparanase has been isolated and is set forth in SEQ ID NO: 1. Purification and polypeptide characterization of the mouse heparanase revealed a non-covalent heterodimer consisting of a 43-kDa polypeptide and a 7- kDa peptide, both of which are derived from a single precursor polypeptide. The enzymatic activity of the mouse heparanase was confirmed by its ability to degrade HSPG and inhibition with known heparanase inhibitors. Analysis of this full-length murine heparanase amino acid sequence also revealed approximately a 76% identity when compared with human heparanase.
  • the mouse heparanase polypeptides of the present invention have been isolated and/or purified.
  • isolated or purified means that a molecule, for example, the polypeptide, is separated from cellular material or other components that naturally accompany it.
  • it is substantially pure when it is at least 60% (by weight) free from the proteins and other naturally occurring organic molecules with which it is naturally associated.
  • the purity of the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99% (by weight) free.
  • a substantially pure polynucleotide or polypeptide can be obtained, for example, by extraction from a natural source, expression of a recombinant nucleic acid encoding the polypeptide, or chemical synthesis. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
  • a chemically synthesized polynucleotide or polypeptide or a recombinant polynucleotide or polypeptide produced in a cell type other than the cell type in which it naturally occurs is, by definition, substantially free from components that naturally accompany it. Accordingly, substantially pure polynucleotides or polypeptides include those having sequences derived from eukaryotic organisms that are produced in E. coli or other prokaryotes. Moreover, it should be appreciated that the term isolated or purified does not refer to a library-type preparation containing a myriad of other sequence fragments.
  • heparanase or a fragment thereof can also be used in the present invention.
  • Such equivalents include functional equivalents or derivatives, homologues, analogues (or fragments thereof), or mutant forms of the polynucleotide sequence that induce an immune response comparable to that of the heparanase polypeptide.
  • functional equivalent refers to alterations in the amino acid sequence, including additions, deletions, and substitutions, that do not substantially alter polypeptide characteristics, e.g., charge, IEF, affinity, avidity, conformation, solubility, and retain the specific function or immunological cross-reactivity of the polypeptide.
  • the term functional equivalents includes conservative amino acid substitutions, which involves a change in the amino acid sequence by way of substituting amino acids of the polypeptide with amino acids having generally similar properties, e.g., acidic, basic, aromatic, size, positively or negatively charged, polarity, non-polarity.
  • the term homologue refers to a polypeptide sequence from a different species having equivalent characteristics and/or function. Mutant forms refer to alterations of the polypeptide sequence, arising due to splicing, polymorphisms, or other events and which may have been selected naturally.
  • equivalents are immunologically cross-reactive with their corresponding polypeptide; although it should be noted that there can be peptides that inhibit heparanase activity and function as an immunogen in the context of the present invention that do not have similar biological activity to that of native heparanase.
  • the equivalent can also be a fragment of the polypeptide, or a substitution, addition or deletion mutant of the polypeptide, for example.
  • Equivalent polypeptides have equivalent amino acid sequences. An amino acid sequence that is substantially the same as another sequence, but that differs from the other sequence by one or more substitutions, additions and/or deletions, is considered to be an equivalent sequence. Preferably, less than 25%, more preferably less than 10% and most preferably less than 5% of the number of amino acid residues in a sequence are substituted for, added to or deleted from the polypeptides of the invention.
  • a heparanase fragment of the present invention preferably contains sufficient amino acid residues to define an epitope of the antigen.
  • the fragment can be, for example, a minigene encoding only the epitope.
  • Methods for isolating and identifying immunogenic fragments from known immunogenic polypeptides are described, for example, by Salfeld et al. in J. Virol., 63: 798-808 (1989) and by Isola et al. in J. Virol., 63: 2325-34 (1989).
  • the fragment defines a suitable epitope, but is too short to be immunogenic, it can be conjugated to a carrier molecule.
  • Some suitable carrier molecules include keyhole limpet hemocyanin, Ig sequences, TrpE and human or bovine serum albumin. Conjugation can be carried out by methods known in the art (described in more detail below).
  • the immunogen of the present invention is an APC containing heparanase or a fragment thereof, which is displayed on the surface of the APC.
  • APCs are generally eukaryotic cells with major histocompatibihty complex (MHC), either class I or class II, gene products at their cell surface.
  • MHC major histocompatibihty complex
  • Some examples of APCs that can be used in the present invention include DC, as well as macrophages, preferably MHC class II positive macrophages, monocytes, preferably MHC class II positive monocytes, and lymphocytes. See generally U.S. Patent No. 5,597,563.
  • the APC of the present invention is a DC, which are widely considered the most potent APC and an efficient initiator of immune responses in vivo, including CD4 + T helper, CD8 + CTL and antibody responses.
  • DCs express high levels of MHC class II molecules and costimulatory molecules important for antigen presentation, such as CD80, CD86, CD40 ligand and ICAM-1. Inoculation of mice with small numbers of DC pulsed with peptide or polypeptide, whole protein or transfected with DNA or RNA has been shown to induce strong T cell-mediated responses in vivo and to elicit a strong immune response, overcoming tolerance to self-antigens.
  • Any suitable method can be used to introduce the heparanase or a fragment thereof into the APC.
  • One suitable method is to pulse the APC with the heparanase or a fragment thereof.
  • Another suitable method is to introduce a DNA or RNA encoding heparanase or a fragment thereof into the APC, which is then transcribed and/or translated into the heparanase or a fragment thereof within the APC.
  • This DNA or RNA can be introduced into the APC by any suitable method, such as through calcium phosphate transfection or insertion via a cloning or expression vector containing the DNA or RNA encoding heparanase or a fragment thereof, which is described in further detail below.
  • heparanase or a fragment thereof can be introduced into the APC, in the context of the present invention, there are several advantages when a full- length heparanase is used.
  • CTL epitopes due to intracellular processing of the antigen by the APC, multiple CTL epitopes can be expressed.
  • T helper epitopes can also exist within the entire heparanase polypeptide and such MHC class II determinants can be useful in establishing a sustained cellular response to the antigen.
  • B cell epitopes can also be present and lead to induction of a heparanase-specific antibody response.
  • the APC can be a bacterial cell or a eukaryotic cell, such as a peripheral blood cell, that expresses exogenous DNA or RNA encoding the heparanase or a fragment thereof.
  • a suitable bacterial cell is an avirulent strain of Mycobacterium bovis, such as bacille Calmette-Guerin (BCG), or an avirulent strain of Salmonella, such as S. typhimurium.
  • the bacterial cells can be prepared by cloning DNA having the active portion of the antigen (e.g., heparanase) in an avirulent strain, as is known in the art, see, e.g., Curtiss et al., Vaccine, 6: 155-60 (1988) and Galan et al., Gene, 94: 29-35 (1990) for preparing recombinant Salmonella and Stover et al., Vaccines, 91 : 393-8 (1991) for preparing recombinant BCG.
  • the antigen e.g., heparanase
  • the present invention also provides anti-idiotypic antibodies or fragments thereof that mimic heparanase molecules.
  • Anti-idiotypic antibodies are directed against the antigen specific part of the sequence of an antibody or T-cell receptor and thus recognize the binding sites of other antibodies.
  • an anti-idiotype antibody should inhibit a specific immune response and they are important to the regulation of the immune system.
  • Anti- idiotypic heparanase-specific antibodies can be obtained by methods known in the art. See generally Jerne et al., EMBO 1: 234 (1982); Jerne, Ann. Immunol. (Paris) 125C: 373 (1974).
  • peptidomimetics can function as immunogens in the context of the present invention.
  • a peptide mimetic is a molecule that mimics the biological activity of a peptide, yet is no longer peptidic in chemical nature.
  • a peptidomimetic is a molecule that no longer contains any peptide bonds, i.e., amide bonds between amino acids; however, in the context of the present invention, the term peptide mimetic is intended to include molecules that are no longer completely peptidic in nature, such as pseudo-peptides, semi-peptides and peptoids.
  • peptidomimetics provide a spatial arrangement of reactive chemical moieties that closely resembles the three-dimensional arrangement of active groups in the peptide on which the peptidomimetic is based.
  • the techniques of developing peptidomimetics are conventional.
  • non-peptide bonds that allow the peptidomimetic to adopt a similar structure to the original peptide can replace peptide bonds.
  • Replacing chemical groups of the amino acids with other chemical groups of similar structure can also be used to develop peptidomimetics.
  • the immunogen can be a DNA or RNA encoding the heparanase or a fragment thereof. Any DNA or RNA encoding a heparanase or a fragment thereof that elicits an immune response to heparanase and thereby inhibiting heparanase activity is suitable for use in the context of the present invention.
  • a suitable DNA encodes the human heparanase, which contains an open reading frame of 1629 bp. See, e.g., Kussie et al., 1999, supra; Toyoshima & Nakajima, 1999, supra.
  • a suitable DNA can be a plasmid having the DNA encoding heparanase or a fragment thereof. See, e.g., U.S. Patent Nos. 5,589,466 and 5,630,796.
  • a suitable DNA or RNA can encode a heparanase that is native or foreign to the animal.
  • a suitable DNA or RNA encoding a foreign heparanase can be any DNA or RNA that encodes a heparanase that is non-native to the animal and that can induce an immune response without additional modification.
  • the DNA or RNA can encode a heparanase that is derived from a mammal, such as a rabbit, rat, or mouse.
  • the DNA or RNA encoding heparanase or a fragment thereof is a DNA or RNA encoding a mouse heparanase or a fragment thereof.
  • Equivalents of DNA or RNA encoding heparanase or a fragment thereof, including DNA or RNA encoding a foreign heparanase can also be used in the present invention.
  • Such equivalents include DNA, RNA, DNA/RNA duplexes, polypeptide-nucleic acid (PNA), or derivatives thereof that encode functional derivatives or analogues (or fragments thereof) that induce an immune response comparable to that of the heparanase polypeptide.
  • the equivalent can be a fragment of the DNA or RNA, or a substitution, addition or deletion mutant of the DNA or RNA, for example.
  • Equivalent DNAs or RNAs have substantially equivalent nucleic acid sequences.
  • a nucleic acid sequence that is substantially the same as another sequence, but that differs from the other sequence by one or more substitutions, additions and/or deletions, is considered to be an equivalent sequence.
  • Preferably, less than 25%, more preferably less than 10% and most preferably less than 5% of the number of nucleic acid residues in a sequence are substituted for, added to or deleted from the DNA or RNA of the invention.
  • degenerate variant refers to changes in polynucleotide sequences, particularly in the third base of the codon, that do not affect the amino acid sequence encoded by the nucleotide sequences.
  • homologue refers to a polynucleotide sequence from a different species having equivalent structure and/or function.
  • Mutant forms refer to alterations of the polynucleotide sequence, such as addition, deletion, or substitution of one or more nucleotides using recombinant DNA techniques well known in the art, or which have been selected naturally. Kunkel et al. (1987) Meth. Enzymol. 154: 367-382-382.
  • the DNA or RNA polynucleotide sequence encoding heparanase or a fragment thereof of the present invention includes fragments or segments that are long enough to use in polymerase chain reaction (PCR) or various hybridization techniques well known in the art for identification, cloning and amplification of all or part of mRNA or DNA molecules.
  • hybridization under high stringency conditions means the following nucleic acid hybridization and wash conditions: hybridization at 42° C in the presence of 50% formamide; a first wash at 65° C with 2X SSC containing 1% SDS; followed by a second wash at 65° C with 0. IX SSC.
  • the polynucleotides of the present invention include complements of any of the nucleotide or peptides recited above, e.g., cDNA and mRNA.
  • the DNA or RNA encoding heparanase or a fragment thereof of the present invention can be introduced into mammalian cells, particularly endothelial cells, by methods known in the art. Such methods have been described, for example, in U.S. Patent No. 5,674,722. Suitable methods include calcium phosphate transfection or insertion via a cloning or expression vector containing the DNA or RNA encoding heparanase or a fragment thereof.
  • Cells both eukaryotic and prokaryotic
  • Methods for the production of these cells are well known in the art.
  • Suitable cloning or expression vectors for inserting DNA or RNA into eukaryotic cells include well-known derivatives of S V-40, adenovirus, cytomegalovirus (CMV) and retrovirus-derived DNA or RNA sequences. Any such vectors, when coupled with vectors derived from a combination of plasmids and phage DNA (shuttle vectors) allow for the cloning and/or expression of protein coding sequences in both prokaryotic and eukaryotic cells.
  • Other eukaryotic expression vectors are known in the art, see, e.g., Southern & Berg, J. Mol. Appl. Genet., 1: 327-41 (1982); Subramani et al., o/. Cell.
  • prokaryotic cloning vectors include plasmids from E. coli, such as colEl, pCRl, pBR322, pMB9, pUC, pKSM and RP4.
  • Prokaryotic vectors also include derivatives of phage DNA, such as Ml 3, fd and other filamentous single-stranded DNA phages.
  • vectors for expressing polypeptides in bacteria, especially E. coli are also known.
  • Such vectors include the pK233 (or any of the tac family of plasmids), T7 and lambda P L .
  • Examples of vectors that express fusion polypeptides are PATH vectors described by Dieckmann and Tzagoloff in J. Bioi.
  • TrpE anthranilate synthetase
  • Other expression vector systems are based on beta- galactosidase (pEX); lambda P L ; maltose binding protein (pMAL); glutathione S-transferase (pGST). See, e.g., Smith & Johnson, Gene, 61: 31-40 (1988); Abath & Simpson, Peptide Research, 3: 167-68 (1990).
  • Cloning vectors can have segments of chromosomal, non-chromosomal and synthetic DNA sequences.
  • the vectors useful in the present invention contain at least one expression control sequence that is operatively linked to the DNA or RNA sequence or fragment to be expressed, i.e., the DNA or RNA encoding heparanase or a fragment thereof.
  • the control sequence is inserted in the vector in order to control and to regulate the expression of the cloned DNA or RNA sequence.
  • useful expression control sequences are the lac system, the trp system, the tac system, the trc system, major operator and promoter regions of phage lambda, the control region of fd coat protein and promoters derived from polyoma, adenovirus, retrovirus and simian virus, for example, the early and late promoters of SV40 and other sequences known to control the expression of genes in prokaryotic or eukaryotic cells and their viruses or combinations thereof.
  • the immunogen of the present invention can be a small molecule.
  • Small molecules of the present invention are entities having carbon and hydrogen atoms, as well as heteroatoms, which include, but are not limited to, nitrogen, sulfur, oxygen, and phosphorus. Atoms in a small molecule are linked together via covalent and ionic bonds; the former is typical for small organic compounds, for example, small molecule tyrosine kinase inhibitors and the latter is typical of small inorganic compounds.
  • the arrangement of atoms in a small organic molecule may represent a chain, for example, a carbon-carbon chain or carbon-heteroatom chain, or ring containing carbon atoms, for example, benzene, or a combination of carbon and heteroatoms, i.e., heterocycles, for example, a pyrimidine or quinazoline.
  • a combination of one or more chains in a small organic molecule attached to a ring system constitutes a substituted ring system and fusion of two rings constitutes a fused policyclic system, which can be referred to as simply a policyclic system.
  • Small molecules include both compounds found in nature, such as hormones, neurotransmitters, nucleotides, amino acids, sugars, lipids and their derivatives, and those compounds made synthetically, either by traditional organic synthesis, bio-mediated synthesis, or a combination thereof. See, e.g., Ganesan, Drug Discov. Today, 7(1): 47-55 (2002); Lou, Drug Discov. Today, 6(24): 1288-1294 (2001). Any suitable small molecule that inhibits heparanase can be used in the context of the present invention, including lipids and polymers of polysaccharides, as well as derivatives thereof, such as, for example, lipopolysaccharides.
  • the immunogen of the present invention can be modified in various ways known to one of skill in the art, for example, by co-administering with or conjugating or genetically fusing it to an immunogenic reagent.
  • Conjugation or fusion to an immunogenic reagent can stimulate an immune response or augment the existing immune response elicited by the immunogen.
  • conjugates and fused molecules can be prepared by any of the known methods for coupling or fusing antigens to carriers or fusion molecules.
  • the conjugates can also be prepared recombinantly as fusion polypeptides by methods well known in the art.
  • the preferred method of conjugation is covalent coupling, whereby the antigen is bound directly to the immunogenic reagent.
  • co- administration can be such that the immunogenic reagent is administered prior to, concurrently with, or subsequent to the immunogen.
  • Preferred immunogenic reagents include polysaccharides, see generally U.S. Patent No. 5,623,057, and peptidoglycans, see generally U.S. Patent No. 5,153,173.
  • Other immunogenic reagents include, for example, cytokines, lymphokines, hormones or growth factors.
  • chemokines include, but are not limited to, chemokines, interferons, granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), vascular endothelial growth factor (VEGF), stem cell factor (SCF), bFGF and interleukins (IL), such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6 and IL-7.
  • G-CSF granulocyte colony stimulating factor
  • GM-CSF granulocyte macrophage colony-stimulating factor
  • VEGF vascular endothelial growth factor
  • SCF stem cell factor
  • IL interleukins
  • the immunogenic reagent can be any suitable medication or therapy conventionally used to prophylactically and/or therapeutically treatment any of the various conditions described herein.
  • the immunogenic reagent when treating a tumor with the present inventive methods, can be a chemotherapeutic agent, radiation, or a receptor antagonist.
  • the immunogen can also be modified by co-administration with or binding to MHC antigen, such as class I and class II restricted antigens, so as to form a complex with the MHC.
  • MHC antigen such as class I and class II restricted antigens
  • the immunogen can be modified by haptenization (chemically linking) of the immunogen. See generally U.S. Patent Nos. 4,778,752 and 5,290,551.
  • a hapten is a substance having the ability to, when coupled with a polypeptide, elicit an immune response.
  • the immunogen of the invention can itself be haptenized, or can be bound to hapten-modified proteins. See generally U.S. Patent Nos. 4,778,752 and 5,290,551.
  • An additional method of modifying the immunogen of the present invention is glycosylation or pegylation of the heparanase or a fragment thereof or glycosylation or pegylation of the carrier molecules of the immunogen, which is described generally in U.S. Patent Nos. 5,484,735 and 4,629,692.
  • the immunogen can also be bound to an adjuvant or the adjuvant can be administered with the immunogen.
  • Pharmaceutically acceptable adjuvants that can be useful in the context of the present invention to enhance the anti-heparanase immune response elicited by the immunogen, include, but are not limited to, muramyl peptides, lymphokines, such as interferon, interleukin-1 and interleukin-6, saponins and CpG oligonucleotides.
  • the adjuvant can also be suitable particles onto which the immunogen is adsorbed, such as aluminum oxide particles.
  • Other examples of pharmaceutically acceptable adjuvants that can be useful in the context of the present invention to enhance the anti-heparanase immune response are bacterial adjuvants.
  • BCG Bactetrachloro-1 (described above)
  • recombinant BCG can additionally act as its own adjuvant.
  • an additional adjuvant may not be needed, although one or more additional adjuvants can optionally be present.
  • BCG acts solely as an adjuvant by being combined with the immunogen, resulting in a form that induces an effective immune response.
  • the induced immune response against heparanase can induce production in the animal of either an antibody that specifically binds heparanase, CD4+ T helper cells or cytotoxic lymphocytes against the heparanase.
  • T helper cells are triggered by antibodies to seek and attack invading organisms. Cells called macrophages summon T-helper cells to the site of the infection and present a protruding antigen onto which the t-helper cell locks, thus recognizing the invading substance.
  • the T4-helper cell then reproduces and secretes its potent lymphokine hormones that stimulate B-cell production of antibodies; signal natural killer or cytotoxic (cell-killing) T-cells; and summon more macrophanges to the site of the infection.
  • Thl which is an acquired immune response whose most prominent feature is high cytotoxic T lymphocyte activity relative to the amount of antibody production, is promoted by CD4+ Thl T-helper cells.
  • Th2 which is an acquired immune response whose most prominent feature is high antibody production relative to the amount of cytotoxic T lymphocyte activity, is promoted by CD4+ Th2 T-helper cells.
  • CD4 is a 55-kD glycoproteins originally defined as differentiation antigens on T-lymphocytes, but also found on other cells including monocytes/macrophages.
  • CD4 antigens are members of the immunoglobulin supergene family and are implicated as associative recognition elements in MHS class Il-restricted immune responses.
  • T-lymphocytes they define the helper/inducer subset.
  • CD4 receptors are present on CD4 cells (helper T cells), macrophages and DC, among others.
  • CD4 acts as an accessory molecule, forming part of larger structures (such as the T-cell receptor) through which T cells and other cells signal each other.
  • Cytotoxic lymphocytes are immunized T lymphocytes that can directly destroy appropriate target cells.
  • cytotoxic lymphocytes may be generated in vitro in mixed lymphocyte cultures, in vivo during a graft-versus-host reaction, or after immunization with an allograft, tumor cell, or virally transformed or chemically modified target cell.
  • the lytic phenomenon is sometimes referred to as cell-mediated lympholysis.
  • These cells are distinct from natural killer cells and from killer cells mediating antibody-dependent cell cytotoxicity.
  • the present inventive immunogen can be administered for prophylactic and/or therapeutic treatments of various conditions.
  • Treatment in the context of the present invention, is intended to encompass inhibiting, slowing, or reversing the progress of the underlying condition, ameliorating clinical symptoms of a condition or preventing the appearance of clinical symptoms of the condition.
  • the methods and compositions of the present invention can be used to treat any condition associated with heparanase activity in the animal and, thus, can be useful in treating conditions generally associated with excess heparanase activity.
  • the methods of the present invention can be used to treat conditions relating to injury, inflammation, diabetes or auto immunity.
  • the methods of the present invention can be used to treat an angiogenic condition, such as atherosclerosis, arthritis, macular degeneration and psoriasis.
  • an angiogenic condition such as atherosclerosis, arthritis, macular degeneration and psoriasis.
  • suitable animals including mammals such as rabbits, rats, mice, or, preferably, humans, that have conditions for which treatment with an immunogen that elicits an immune response to heparanase is well within the ability and knowledge of one skilled in the art.
  • any of the methods described herein for determining heparanase activity can be useful to determine animals having conditions for which treatment according to the present inventive methods is suitable.
  • compositions containing the present immunogens are administered to a patient not presently actively suffering from the condition, but rather, have not yet exhibited symptoms or are in a symptom- free period of the condition, in an amount sufficient to at least partially reduce the future effects of the condition.
  • Such an amount is also defined to be an "effective amount.”
  • the precise amounts again depend upon the patient's state of health and general level of immunity, as well as dosing schedules, which are described below.
  • compositions are administered to a patient already suffering from the condition in an amount sufficient to cure or at least partially arrest the condition.
  • An amount adequate to accomplish this is defined as an "effective amount.” Amounts effective for this use will depend upon the severity of the condition and the general state of the patient's own immune system. Dosing schedules will also vary with the disease state and status of the patient and will typically range from a single bolus dosage or continuous infusion to multiple administrations per day, for example, every 4-6 hours, or as indicated by the treating physician and the patient's condition.
  • the methods of the present invention can be used to inhibit tumor growth or prevent metastasis of a tumor, which is the growth of secondary tumors at sites different from the primary tumor.
  • suitable tumors include the following: brain tumors, such as astrocytoma, oligodendroglioma, ependymoma, medulloblastomas and PNET (Primitive Neural Ectodermal Tumor); pancreatic tumors, such as pancreatic ductal adenocarcinomas; lung tumors, such as small and large cell adenocarcinomas, squamous cell carcinoma and bronchoalveolarcarcinoma; colon tumors, such as epithelial adenocarcinoma and liver metastases of these tumors; liver tumors, such as hepatoma and cholangiocarcinoma; breast tumors, such as ductal and lobular adenocarcinoma; gy
  • the immunogens of the present invention where used in an animal for the purpose of prophylaxis or treatment, can be administered in the form of a composition additionally having a carrier. Therefore, the present invention includes compositions for inhibiting heparanase activity in an animal of an immunogen and a carrier. Any suitable immunogen, examples of which are described above, can be used in the context of the present inventive composition.
  • Suitable carriers which can, for example, be in the form of a capsule, sachet, paper or other container, include, for example, one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof.
  • Carriers can further have minor amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the binding polypeptides.
  • auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the binding polypeptides.
  • the carrier can be a solid, semi-solid, or liquid material, which acts as a vehicle, excipient or medium for the active ingredient.
  • Such compositions of the present invention are prepared in a manner well known in the pharmaceutical art.
  • compositions of this invention can be in a variety of forms. These include, for example, solid, semi-solid and liquid dosage forms, such as tablets, lozenges, sachets, cachets, elixirs, suspensions, aerosols (as a solid or in a liquid medium), ointments containing, for example, up to 10% by weight of the active compound, soft and hard gelatin capsules, suppositories, suspensions, pills, powders, liquid solutions, dispersions, lyopholyzed forms, liposomes, injectable and infusible solutions, and sterile packaged powders and as a topical patch.
  • solid, semi-solid and liquid dosage forms such as tablets, lozenges, sachets, cachets, elixirs, suspensions, aerosols (as a solid or in a liquid medium), ointments containing, for example, up to 10% by weight of the active compound, soft and hard gelatin capsules, suppositories,
  • the immunogen can also be administered by various routes, for example, by the oral or rectal route, topically or parenterally, for example by injection or infusion (intravenous, intraperitoneal, subcutaneous, intramuscular, intradermal).
  • routes for example, by the oral or rectal route, topically or parenterally, for example by injection or infusion (intravenous, intraperitoneal, subcutaneous, intramuscular, intradermal).
  • the compositions of the invention can, as is well known in the art, be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the mammal.
  • the present invention can be used in vivo and in vitro for investigative, diagnostic, prophylactic, or treatment methods, which are well known in the art.
  • the cDNA and polypeptide sequence of full-length mouse heparanase may enable large-scale production of the polypeptide, benefiting heparanase protein structure studies, as well as screening and validation of heparanase inhibitors involved in the development of novel anti-cancer and anti-inflammation drugs.
  • DC were isolated from the bone marrow of C57BL/6 syngeneic mice using the following protocol. Bone marrow cells were harvested from tibia and femurs, which cells were subsequently depleted of existing T cells, B cells, macrophages and granulocytes by incubating with an antibody cocktail (anti-CD4 (clone GK1.5), anti-CD8 (2.43), anti-la (B21- 2), anti-B220 (RA3-3A1/6.1) and anti-Gr-1 (RB6-8C5/1)) for 30 min at 4° C and then with rabbit complement for additional 30 min at 37° C.
  • an antibody cocktail anti-CD4 (clone GK1.5), anti-CD8 (2.43), anti-la (B21- 2), anti-B220 (RA3-3A1/6.1) and anti-Gr-1 (RB6-8C5/1)
  • the remaining cells were cultured in 10% fetal calf serum (FCS) Dulbecco's Modified Eagles Medium (DMEM) in the presence of GM-CSF (20 ng/ml) and IL-4 (50 ng/ml) at 37° C, 5% CO2, for 3 days. Non-adherent cells were discarded and the remaining cells were cultured for an additional 3 days. The resulting non-adherent cells were transferred to new plates with fresh media and cultured for another 3 days. These mature DC were harvested and cryopreserved for later use as APCs for immunization experiments. Morphology and phenotypic analysis for the DC was verified by flow cytometry.
  • FCS fetal calf serum
  • DMEM Dulbecco's Modified Eagles Medium
  • DC typically have extensive dendrites, form cell clumps and express high levels of MHC class II, co-stimulatory molecules, such as B7.1/CD80 and B7.2/CD86, yet are negative for other cell lineage markers, such as the monocyte/macrophage marker CD 14.
  • DC were washed twice in serum-free AIMV media and incubated with heparanase protein (100 ⁇ g/ml) in AIMV for 6-10 hours. The cells were washed twice in AIMV before being used.
  • the present example demonstrates administration to an animal of an effective amount of an immunogen that elicits an immune response to heparanase.
  • DC which are an example of an APC
  • DC-heparanase were pulsed with heparanase
  • C57BL/6 mice were administered the DC-heparanase intravenously (i.v.) at a concentration of 5 x 10 4 cells per mouse.
  • mice were administered i.v. either alkaline phosphatase (AP) pulsed DC (DC- AP or DC-control) or PBS.
  • AP alkaline phosphatase
  • DC- AP alkaline phosphatase
  • DC-control DC-control
  • the present example demonstrates administration to an animal of an effective amount of an immunogen that elicits an immune response to heparanase.
  • DC which are an example of an APC
  • DC-heparanase were pulsed with heparanase
  • C57BL/6 mice were administered the DC-heparanase i.v. at a concentration of 5 x 10 4 cells per mouse.
  • mice were administered i.v. either chick ovalbumin (OVA) pulsed DC (DC-OVA or DC-control) or PBS.
  • OVA chick ovalbumin
  • DC-OVA or DC-control DC-control
  • spleen cells from the three experimental groups of mice were added at 2 x 10 5 per well in 96-well flat-bottomed plates. Stimulated cells received 2 x 10 DC-heparanase, DC-control, or PBS . Cells were cultured in RPMI 1640 with 10% fetal calf serum (FCS). After 4 days of in vitro culture, blue stained spots (due to antibody staining of IFN- ⁇ released by single antigen-activated T cells) were counted and compared with plates set up with spleen cells from non-immunized or control-immunized mice.
  • FCS fetal calf serum
  • the present example demonstrates inhibition of tumor growth, including growth of tumor metastases, in an animal after administration to the animal of an effective amount of an immunogen.
  • DC which are an example of an APC
  • DC- heparanase were pulsed with heparanase
  • C57BL/6 mice were administered the DC-heparanase i.v. at a concentration of 5 x 10 4 cells per mouse.
  • mice were administered i.v. either DC pulsed with AP (DC-AP or DC-control) or PBS. There were a total of three administrations given at intervals of 10 days.
  • mice Seven days later after the last immunization, mice were challenged by injecting 1 x 10 6 Lewis lung carcinoma cells (a syngeneic tumor line) intrafootpad. When the tumor grew to approximately 5mm in diameter, the tumor bearing leg was surgically removed. Mice were monitored daily for survival.
  • 1 x 10 6 Lewis lung carcinoma cells a syngeneic tumor line
  • the present example demonstrates inhibition of tumor growth, including growth of tumor metastases, in an animal after administration to the animal of an effective amount of an immunogen.
  • DC which are an example of an APC
  • DC- heparanase were pulsed with heparanase (DC- heparanase).
  • C57BL/6 mice were administered the DC-heparanase i.v. at a concentration of 5 10 cells per mouse.
  • mice were administered i.v. either DC pulsed with OVA (DC-OVA or DC-control) or PBS.
  • OVA DC-OVA or DC-control
  • mice were challenged by injecting 1 x 10 6 B16 Melanoma cells.
  • mice were sacrificed, the lungs removed and tumor growth was measured by counting the number of tumor nodules on the lungs.
  • Example 5 The present example demonstrates identification, expression, purification, and characterization of a polypeptide sequence of a mouse heparanase, which is set forth in SEQ ID NO:l, and a cDNA sequence of the mouse heparanase, which is set forth in SEQ ID NO:l, and a cDNA sequence of the mouse heparanase, which is set forth in SEQ ID NO:l
  • heparanase gene a mouse (strain FVB) embryo (day 12.5) cDNA library was screened.
  • Two primers derived from the human gene sequence forward 5'-CAAGAACAGC ACCTACTCAA GAAGC-3', reverse 5'-GCCACATAAA GCCAGCTGCA AAGG-3'
  • Positive subplate 10A was ordered and screened by PCR to identify positive subwells.
  • the positive subwell stock was plated out onto LB-Ampicillin plates and colonies were screened to identify heparanase cDNA clones.
  • a single clone with an insert of approximately 1800 bp was isolated and sequenced.
  • NS0 cells (Lonza Biologies) were transfected with linearized plasmid DNA by electroporation and cultured in glutamme-free DMEM with dialysed fetal calf serum and glutamine synthetase supplement (JRH Biosciences, Lenexa, KA).
  • Several clones were screened for polypeptide expression by western blot using polyclonal anti-heparanase antibody and the highest producer was selected for culture in roller bottles.
  • NS0 cells (8xl0 9 ) expressing mouse heparanase were harvested and treated with a buffer containing 1% Triton X-100, 500 mM NaCl, 15 mM sodium dimethyl glutarate, pH6.0 (30 min, 4° C). After centrifugation, the extract was incubated with 20 ml Con-A beads (Amersham Pharmacia Biotech, Piscataway, NJ) at 4° C, overnight, with gentle rocking. The bound material was eluted with 200 ml of 20% ⁇ -methyl mannoside, 500 mM NaCl, 15 mM sodium dimethyl glutarate, pH 6.0.
  • the Con-A eluate was diluted with 1800 ml of 15 mM sodium dimethyl glutarate, pH 6.0, and loaded onto a Hi-Trap heparin- Sepharose column (Amersham Pharmacia).
  • the column was eluted with a gradient of NaCl (0.025-1.5 M) in 15 mM sodium dimethly glutarate, pH 6.0, and all the fractions were tested for heparanase activity.
  • the active fractions were pooled, concentrated with an Ultrafree concentrator (NMWL 30 K, Millipore, Bedford, MA), and subjected to size exclusion chromatography using a Superdex 75 column (Amersham Pharmacia). All the fractions were monitored by UV absorbance at 280 nm and tested for heparanase activity. Enzymatically active fractions were pooled for further characterization.
  • Polypeptides were resolved by SDS-PAGE under reducing conditions using 4-20 % gradient polyacrylamide gels. After electrophoresis, the gels were either stained with Coomassie blue or transferred to polyvinyldene difluoride membrane (Millipore). The membrane was probed with polyclonal antibody raised against human heparanase. After incubation with a goat anti-rabbit antibody conjugated to horseradish peroxidase, the blot was developed using a chemiluminescence substrate (Amersham Pharmacia).
  • Polypeptides transferred onto polyvinyldene difluoride membrane were stained with Coomassie blue. Individual bands were cut out and polypeptide sequences were obtained by automated Edman degradation in an Applied Biosystems Procise Model 492 protein sequencer (Applied Biosystems, Norwalk, CT). Purifed polypeptide was directly analyzed by liquid chromatography-mass spectrometry (LC-MS) analysis using an Agilent 1100 HPLC with a Poros Reverse Phase Rl/10 column (Agilent Technologies, Palo Alto, CA). The column was coupled to a Thermo-Finnigan LCQ Deca XP ion trap mass spectrometer.
  • LC-MS liquid chromatography-mass spectrometry
  • mice gene was transfected into a mouse myeloma cell line (NS0) and stable clones were isolated using the glutamine synthetase selectable marker. Heparanase activity was detected in cell culture supernatants and detergent treated cell extracts, and expression was confirmed by western blot analysis.
  • NS0 cells were treated with detergent and the extracts were incubated with Con-A sepharose beads overnight in a batch mode. The beads were washed and polypeptide was eluted with ⁇ - methyl mannoside.
  • Enzymatically active fragments were loaded onto a heparin-sepharose affinity column, the column was washed, and polypeptide was eluted using a salt gradient. Active fractions from this step were concentrated and the polypeptides were resolved by size exclusion chromatography. When the active fractions were run on a SDS-PAGE gel, the major bands detected were at 50 and 8 kDa, which is similar to the human polypeptide.
  • Mature human heparanase polypeptide may exist as a heterodimer of 50 kDa and 8 kDa peptides.
  • the data reported here for the mouse enzyme are consistent with this idea.
  • the 535 amino acid mouse pre-proheparanase is first processed into a 60-kDa proheparanase polypeptide by cleavage of the signal peptide.
  • an internal 49-residue peptide (Glu 01 -Gln 149 ) is removed proteolytically, resulting in the mature enzyme that exists as a non-covalently bound heterodimer of the 50 kDa polypeptide (Lys 150 -Ile 535 ) and the 8 kDa peptide (Asp 28 -Lys 100 ).
  • mouse and human heparanases were determined to be 77% identical at the amino acid level.
  • the mouse polypeptide is eight residues shorter than the human heparanase (535 vs 543) and this difference could be attributed to the smaller mouse signal sequence.
  • the smaller 8-kDa fragment of mouse heparanase is 2 amino acids shorter than the human polypeptide, while the larger polypeptide is the same length, 385 amino acids.
  • Mouse heparanase has two fewer potential N-linked glycosylation sites than the human, but the four present in the mouse are conserved in human heparanase.
  • Laminarin sulfate which has been shown to inhibit human heparanase activity in vitro and tumor metastasis in vivo, completely inhibited mouse heparanase activity at 1 ⁇ M, with an IC 50 of 50 nM (Fig. 6). The enzyme activity was also inhibited by heparin and other heparanase inhibitors.

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Abstract

L'invention concerne des méthodes permettant d'inhiber l'activité de l'héparanase et de traiter différents états par administration, à un animal, d'une quantité efficace d'un immunogène sollicitant une réponse immunitaire contre l'héparanase. Selon l'invention, l'immunogène est l'héparanase ou un fragment de celle-ci, cet immunogène étant, de préférence, une cellule présentant un antigène (APC), telle qu'une cellule dendritique (DC) affichant l'héparanase ou un fragment de celle-ci sur sa surface. L'invention concerne également des compositions contenant ledit immunogène.
PCT/US2002/021773 2001-07-13 2002-07-10 Methode et composition permettant d'inhiber l'activite de l'heparanase WO2003006645A2 (fr)

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JP2003512403A JP2005521379A (ja) 2001-07-13 2002-07-10 ヘパラナーゼ活性を阻害するための方法及び組成物
CA002453566A CA2453566A1 (fr) 2001-07-13 2002-07-10 Methode et composition permettant d'inhiber l'activite de l'heparanase
EP02763248A EP1417304A4 (fr) 2001-07-13 2002-07-10 Methode et composition permettant d'inhiber l'activite de l'heparanase
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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1479764A1 (fr) * 2003-05-19 2004-11-24 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Peptides derives de Heparanas pour la vaccination des patients cancereux
WO2004101780A2 (fr) * 2003-05-19 2004-11-25 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Peptides derives d'heparanase pour la vaccination de patients ateints de tumeurs
WO2004101780A3 (fr) * 2003-05-19 2005-03-31 Deutsches Krebsforsch Peptides derives d'heparanase pour la vaccination de patients ateints de tumeurs
US7842782B2 (en) 2003-05-19 2010-11-30 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Heparanase-derived peptides for vaccination of tumor patients
WO2005084610A2 (fr) 2004-02-27 2005-09-15 Bioiberica, S.A. Nouvelle application therapeutique d'un groupe de polysaccharides sulfates
WO2005084610A3 (fr) * 2004-02-27 2005-12-08 Bioiberica Nouvelle application therapeutique d'un groupe de polysaccharides sulfates
ES2251289A1 (es) * 2004-02-27 2006-04-16 Bioiberica, S.A. Nuevo uso terapeutico de un grupo de polisacaridos sulfatados.
US7816329B2 (en) 2004-02-27 2010-10-19 Bioiberica, S.A. Therapeutic use for a group of sulphated polysaccharides
EP3188738A4 (fr) * 2014-09-02 2018-01-24 The Children's Hospital of Philadelphia Compositions et procédés permettant l'inhibition de la chondrogenèse

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EP1417304A2 (fr) 2004-05-12
WO2003006645A3 (fr) 2003-07-24
EP1417304A4 (fr) 2005-11-23
JP2005521379A (ja) 2005-07-21
CA2453566A1 (fr) 2003-01-23

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