Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
1998-10-28
2001-06-05
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S252300, C435S320100, C536S023100, C536S023200
Reexamination Certificate
active
06242238
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to isolated or recombinant mammalian endoglucuronidase enzymes, polypeptides and peptides, in particular human platelet heparanase, genetic sequences encoding the same and uses therefor, for example in the determination and characterisation of chemical compounds, proteins, polypeptides, small molecules and macromolecules capable of inhibiting metastasis, angiogenesis, angioplasty-induced restenosis, atherosclerosis, inflammation, promote wound healing and otherwise modulate physiological processes involving heparanase cleavage of heparan sulphate. The invention further relates to a method of altering, modifying or otherwise modulating the level of expression of mammalian heparanase in a cell. A further aspect of the invention relates to immunoreactive molecules capable of binding to and/or inhibiting mammalian heparanase, in particular monoclonal antibodies. A still further aspect of the invention contemplates the use of heparanase as an agent to inhibit the processes of neovascularisation.
GENERAL
Bibliographic details of the publications numerically referred to in this specification are collected at the end of the description.
This specification contains nucleotide and amino acid sequence information prepared using the programme PatentIn Version 2.0, presented herein after the bibliography. Each nucleotide or amino acid sequence is identified in the sequence listing by the numeric indicator <210> followed by the sequence identifier (e.g. <210>1, <210>2, etc). The length, type of sequence (DNA, protein (PRT), etc) and source organism for each nucleotide or amino acid sequence are indicated by information provided in the numeric indicator fields <211>, <212> and <213>, respectively. Nucleotide and amino acid sequences referred to in the specification are defined by the information provided in numeric indicator field <400> followed by the sequence identifier (eg. <400>1, <400>2, etc).
The designation of nucleotide residues referred to herein are those recommended by the IUPAC-IUB Biochemical Nomenclature Commission, wherein A represents Adenine, C represents Cytosine, G represents Guanine, T represents thymine, Y represents a pyrimidine residue, R represents a purine residue, M represents Adenine or Cytosine, K represents Guanine or Thymine, S represents Guanine or Cytosine, W represents Adenine or Thymine, H represents a nucleotide other than Guanine, B represents a nucleotide other than Adenine, V represents a nucleotide other than Thymine, D represents a nucleotide other than Cytosine and N represents any nucleotide residue.
The designations for amino acid residues referred to herein are set forth in Table I.
As used herein the term “derived from” shall be taken to indicate that a specified integer may be obtained from a particular source albeit not necessarily directly from that source.
Throughout this specification, unless the context requires otherwise, the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated step or element or integer or group of steps or elements or integers but not the exclusion of any other step or element or integer or group of elements or integers.
BACKGROUND OF THE INVENTION
Tissue invasion by blood-borne malignant tumour cells and leukocytes involves their adhesion to the luminal surface of the vascular endothelium, passage through the vascular endothelial cell layer and the subsequent degradation of the underlying basal lamina and extracellular matrix (ECM) with a battery of secreted and/or cell surface protease and glycosidase activities (Nakajima et al., 1983; Schmitt et al., 1992; Vlodavsky et al., 1992).
Studies have shown that while the initial entrapment of metastatic tumour cells by the capillary endothelium is platelet-independent, platelet aggregation which occurs shortly thereafter can lead to platelet activation and degranulation, resulting in gap formation and retraction of endothelial cells, exposing the underlying basement membrane to adhesion by the tumour cells (Tanaka et al., 1986; Crissman et al., 1985; Yahalom et al., 1985).
The basal lamina and underlying connective tissue stroma consist predominantly of a complex network of fibronectin, laminin, collagen type IV and vitronectin, each of which interact with heparan sulphate (HS) side chains of heparan sulphate proteoglycans (HSPG) embedded with the matrix (Yurchenco and Schittny, 1990).
HS chains generally consist of clusters of sulphated disaccharide units (predominantly N-sulphated glucosamine linked 1-4 to &agr;-L-iduronic acid residues) separated by lowly or non-sulphated regions (predominantly disaccharide units of N-acetylated glucosamine linked 1-4 to &bgr;-D-glucuronic acid) (Turnbull and Gallagher, 1990; 1991).
In work leading up to the present invention, the inventors sought to isolate and characterise enzymes, proteins, polypeptides and peptides which are capable of cleaving the HS side chains of HSPG embedded in the matrix and genetic sequences encoding same. The genetic sequences thus derived provide a means for assisting the disassembly of the ECM and facilitating cell migration, when expressed at the matrix site or transported thereto.
The genetic sequences of the present invention further provide the means for developing a wide range of therapeutic and prophylactic pharmaceutical compounds to inhibit metastasis, neovascularisation, angiogenesis, angioplasty-induced restenosis, atherosclerotic plaque formation and inflammation and/or to promote wound healing, amongst others.
SUMMARY OF THE INVENTION
One aspect of the invention provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding a polypeptide capable of hydrolysing glycosidic bonds in HS.
A second aspect of the invention provides an isolated nucleic acid molecule comprising a sequence of nucleotides which encodes or is complementary to a sequence which encodes a mammalian endoglucuronidase polypeptide, in particular heparanase or fragment or derivative thereof. More particularly, the mammalian endoglucuronidase polypeptide comprises an amino acid sequence as set forth in any one or more <400>1-11 or <400>13 or <400>15 or <400>17 or <400>19 or <400>23 or is at least 40% identical thereto.
A further aspect of the invention provides an isolated nucleic acid molecule which is at least 40% identical to the nucleotide sequence set forth in any one of <400>12 or <400>14 or <400>16 or <400>18 or a homologue, analogue or derivative thereof, or a complementary sequence thereto.
A still further aspect of the present invention provides a genetic construct which expresses a recombinant endoglucuronidase activity, in particular heparanase activity or an active site thereof.
Another aspect of the invention provides a recombinant mammalian endoglucuronidase polypeptide, in particular heparanase or fragment or derivative thereof.
Still yet another aspect of the invention contemplates a method of identifying a modulator of heparanase activity, said method comprising assaying recombinant heparanase activity in the presence of a potential modulator and comparing said activity to the activity of recombinant heparanase in the absence of said potential modulator.
A further aspect of the invention contemplates an inhibitor of a mammalian endoglucuronidase polypeptide, in particular a mammalian heparanase. The inhibitor molecules encompassed by the invention are particularly useful as inhibitors of metastasis, angiogenesis, wound healing, angioplasty-induced restenosis, arteriosclerosis, atherosclerosis, inflammation or other physiological or medical condition wherein heparanase activity is elevated.
In still yet another aspect of the invention there is contemplated the use of recombinant heparanase or an active fragment or derivative thereof to inhibit neovascularisation and its associated processes involved in the regulation of tissue
Freeman Craig Geoffrey
Hamdorf Brenton James
Hulett Mark Darren
Parish Christopher Richard
Achutamurthy Ponnathapu
Burns Doane , Swecker, Mathis LLP
Rao Manjunath N.
The Australian National University
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