Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Reexamination Certificate
1999-02-18
2002-05-14
McGarry, Sean (Department: 1635)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
C435S004000, C435S183000, C435S195000, C435S200000, C435S201000, C530S350000
Reexamination Certificate
active
06387643
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention provides isolated human heparanase polypeptides, and the isolated polynucleotide molecules that encode them, as well as vectors and host cells comprising such polynucleotide molecules. The invention also provides a method for the identification of an agent that alters heparanase activity.
2. Related Art
Heparanase is a human enzyme that can degrade both heparin proteoglycans (HPG) and heparan sulfate proteoglycans (HSPG). Heparanase activity in mammalian cells is well known. The activity has been identified in various melanoma cells (Nakajima, et al.,
Cancer Letters
31: 277-283, 1986), mammary adenocarcinoma cells (Parish, et al., Int.
J. Cancer
, 40: 511-518, 1987), leukemic cells (Yahalom, et al.,
Leukemia Research
12: 711-717, 1988), prostate carcinoma cells (Kosir, et al.,
J. Surg. Res
. 67: 98-105, 1997), mast cells (Ogren and Lindahl,
J. Biol. Chem
. 250: 2690-2697, 1975), macrophages (Savion, et al.,
J. Cell. Physiol
, 130: 85-92, 1987), mononuclear cells (Sewell, et al.,
Biochem. J
. 264: 777-783, 1989), neutrophils (Matzner, et al. 51: 519-524, 1992, T-cells (Vettel et al.,
Eur J. Immunol
. 21: 2247-2251, 1991), platelets (Haimovitz-Friedman, et al.,
Blood
78: 789-796, 1991), endothelial cells (Godder, et al.,
J. Cell Physiol
. 148: 274-280, 1991), and placenta (Klein and von Figura,
BBRC
73: 569, 1976). An earlier report that human platelet heparanase is a member of the CXC chemokine family (Hoogewerf et al.,
J.Biol. Chem
. 270: 3268-3277) is in error.
Elevated heparanase activity has been documented in mobile, invasive cells. Examples include invasive melanoma, lymphoma, mastocytoma, mammary adenocarcinoma, leukemia, and rheumatoid fibroblasts. Heparanase activity has also been documented in non-pathologic situations involving the migration of lymphocytes, neutrophils, macrophages, eosinophils and platelets (Vlodavsky et al., Invasion Metastasis 12: 112-127, 1992).
A number of uses have been proposed for bacterial heparanases. One such use is described in Freed et al. (
Ann. Biomed. Eng
. 21: 67-76 (1993)), wherein purified bacterial heparanase is immobilized onto filters and connected to extracorporeal devices for use in the degradation of heparin and the neutralization of its anticoagulant properties post surgery.
Other proposed uses for bacterial heparanases include the use of heparanase in a method for inhibiting angiogenesis (U.S. Pat. No. 5,567,417), an application of the enzyme as a means of decreasing inflammatory responses (WO 97/11684), and the use of heparanase-inhibiting compositions for preventing tumor metastasis (U.S. Pat. No. 4,882,318).
In view of the observation that heparanase activity is present in mobile, invasive cells associated with pathologic states, it may be hypothesized that an inhibitor of heparanase would broadly influence the invasive potential of these diverse cells. Further, inhibition of heparan sulfate degradation would inhibit the release of bound growth factors and other biologic response modifiers that would, if released, fuel the growth of adjacent tissues and provide a supportive environment for cell growth (Rapraeger et al.,
Science
252: 1705-1708, 11991). Inhibitors of heparanase activity would also be of value in the treatment of arthritis, asthma, and other inflammatory diseases, vascular restenosis, atherosclerosis, tumor growth and progression, and fibro-proliferative disorders.
A major obstacle to designing a screening assay for the identification of inhibitors of mammalian heparanase activity has been the difficulty of purifying any mammalian heparanase to homogeneity so as to determine its structure, including its amino acid sequence. For this reason, therapeutic applications of mammalian heparanase, or of inhibitors of mammalian heparanase, have been based on research carried out using bacterial heparanase.
WO 91/02977 describes a substantially, but partially, purified heparanase produced by cation exchange resin chromatography and the affinity absorbent purification of heparanase-containing extract from the human SK-HEP-1 cell line. WO 91/02977 also describes a method of promoting wound healing utilizing compositions comprising a “purified” form of heparanase. This enzyme was not thoroughly characterized, and its amino acid sequence was not determined. WO 98/03638 describes a method for the pourification of mammalian heparanase from a heparanase-containing material, such as human platelets. However, the amino acid sequence of this heparanase, and the sequence of the polynucleotide molecule that encodes it, are not disclosed in this reference. Furthermore, this heparanase is characterized only as having a native molecular mass of about 50 kDa, and as degrading both heparin and heparan sulfate.
Although a number of assays for heparanase have been described, the complexity of the HSPG substrate has caused methods for assay of heparanase activity to be rudimentary and lacking in kinetic sophistication. Haimovitz-Friedman et al. (
Blood
78: 789-796, 1991) describe an assay for heparanase activity that involves the culturing of endothelial cells in radiolabeled
35
SO
4
to produce radiolabeled heparan sulfate proteoglycans, the removal of the cells which leaves the deposited extracellular matrix that contains the
35
S-HSPG, the addition of potential sources of heparanase activity, and the detection of possible activity by passing the supernatant from the radiolabeled extracellular matrix over a gel filtration column and monitoring for changes of the size of the radiolabeled material that would indicate that HSPG degradation had taken place. However, this assay cannot be used in a high-throughput screening format.
Nakajima et al. (
Anal. Biochem
. 196: 162-171, 1986) describe a solid-phase substrate for the assay of melanoma heparanase activity. Heparan sulfate from bovine lung is chemically radiolabeled by reacting it with [
14
C]-acetic anhydride. Free amino groups of the [
14
C]-heparan sulfate were acetylated and the reducing termini were aminated. The [
14
C]-heparan sulfate was chemically coupled to an agarose support via the introduced amine groups on the reducing termini. However, the usefulness of the Nakajima et al. assay is limited by the fact that the substrate is an extensively chemically modified form of naturally occurring heparan sulfate.
Khan and Newman (
Anal. Biochem
. 196: 373-376, 1991) describe an indirect assay for heparanase activity. In this assay, heparin is quantitated by its ability to interfere with the color development between a protein and the dye Coomassie brilliant blue. Heparanase activity is detected by the loss of this interference. This assay is limited in use for screening because it is so indirect that other non-heparin compounds could also interfere with the protein-dye reaction.
In view of the foregoing, it will be clear that there is a need in the art for recombinantly produced human heparanase.
SUMMARY OF THE INVENTION
The present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding human heparanase polypeptides. Unless otherwise indicated, any reference herein to a “human heparanase polypeptide” will be understood to encompass human pre-pro-heparanase, pro-heparanase, and both the 8 kDa and the 56 kDa subunits of the human heparanase enzyme. Pre-pro-heparanase refers to an amino acid sequence which includes a leader sequence, and which can be processed to remove 48 amino acids yielding both the 8 kDa and the 56 kDa subunits of the human heparanase enzyme; pro-heparanase refers to the enzymatically inactive, full-length molecule from which the signal peptide has been removed and which can be processed to yield both the 8 kDa and the 56 kDa subunits of the human heparanase enzyme. Fragments of human heparanase polypeptides are also provided. Unless otherwise indicated, any reference herein to a “human heparanase enzyme” will be understood to refer to a non-covalently associated complex of the 56 kDa and the 8 kDa human h
Fairbanks Michael B.
Heinrikson Robert Leroy
Mildner Ana M.
Epps Janet
Kerber Lori E.
McGarry Sean
Pharmacia and Upjohn Company
Rehberg Edward F.
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