This invention relates to identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides. More particularly, a polypeptide of the present invention is xe2x80x9cheparanasexe2x80x9dobtainable from the human SV40-transformed fibroblast cell line ATCC CCL 75.1. The heparanase is an endoglucuronidase capable of specifically degrading heparan sulfate into 6 to 20 kDa fragments.

This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides. More particularly, a polypeptide of the present invention is xe2x80x9cheparanasexe2x80x9d obtainable from the human SV40-transformed fibroblast cell line ATCC CCL 75.1. The heparanase is an endoglucuronidase capable of specifically degrading heparan sulfate into 6 to 20 kDa fragments. The invention also relates to vectors and host cells, comprising a polynucleotide of the invention. Furthermore, the present invention relates to antibodies directed to polypeptides according to the present invention, to pharmaceutical compositions comprising such antibodies or polypeptides, and to assay systems suitable for identifiying agonists or antagonists of such polypeptides.
Heparanase was first identified in murine metastatic melanoma cells by Nakajima et al. (Nakajima et al., Science 220: 611-613, 1983). They concluded the heparan sulfate degrading enzyme responsible is an endoglucuronidase, cleaving linkage between GIcA and GIcNAc, and named it heparanase (Nakajima et al., J. Biol. Chem. 259: 2283-2290, 1984). The heparanase is a hydrolase distinguished from Flavobacterium heparitinase and heparinase (Ototani, N. et al., Carbohydrate Res. 88: 291-303, 1981) which are eliminases capable of specifically degrading heparan sulfate and heparin, respectively into di- and tetra-saccharides (Nakajima et al., J. Biol. Chem. 259: 2283-2290, 1984).
Heparanase-like activity has been found in several normal and tumor cells and tissues as reviewed by Nakajima et al. (J. Cell. Biochem. 36: 157-167, 1988). According to the reports from various laboratories the existance of at least three different types of heparin and/or heparan sulfate-degrading endoglucuronidase has been predicted. The melanoma heparanase degrades heparan sulfate but is not active on heparin. The human platelet heparanase depolymerizes both heparin and heparan sulfate and cleaves xcex2-glucuronidic linkages in the antithrombin-binding domain of heparin (Thunberg et al., J. Biol. Chem. 257:10278-10282, 1982). Another endoglucuronidase from mastocytoma cells catalyzes the depolymerization of macromolecular heparin proteoglycans into fragments similar in size to commercial heparin (xc3x96gren and Lindahl, J. Biol. Chem. 250:2690-2697,1975). The mastocytoma enzyme has little or no activity against heparan sulfate and does not cleave the antithrombin-binding regions of heparin.
The enzymatic characteristics of heparanase have been studied in several laboratories. Nakajima et al. found that heparanase does not degrade highly sulfated heparin of porcine mucosa and bovine lung, chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate, and hyaluronic acid (Nakajima et al., J. Biol. Chem. 259: 2283-2290,1984). They also reported that heparanase is inhibited by heparin described above but not by an exoglucuronidase inhibitor, 1,4-saccharolactone (Nakajima et al., J. Biol. Chem. 259: 2283-2290,1984). Highly sulfated heparan sulfate produced by vascular endothelial cells is relatively resistant to heparanase as compared with bovine lung and kidney heparan sulfate and is cleaved by heparanase into large molecular size fragments (Nakajima et al., J. Biol. Chem. 259: 2283-2290, 1984). Thus they suggested the domain structures of heparan sulfate recognized by heparanase. Bai, X. et al (J. Biol. Chem. 272: 23172-23179, 1997) have recently shown with a use of a mutant cell line of CHO cell that 2-O-sulfate uronic acid is important for heparanase recognition of heparan sulfate and its enzymatic activity. Bame, K. J. et al (J. Biol. Chem. 272: 2245-2251, 1997) have predicted the existance of two types of heparanase, one cleaving near the reducing end and the other cleaving near the non-reducing end of highly sulfated region of heparan sulfate. Schmidtchen, A. et al. (Eur. J. Biochem. 223: 211-221, 1994) have also proposed the model of heparanase cleavage site from experiments that heparanase treatment generated low-sulfated, GIcNAc-containg heparan sulfate fragments of approximately 7 kDa in molecular size.
Various methods for detecting heparanase activity are reported including (i) polyacrylamide gel electrophoresis (Nakajima, M. et al., Science 220: 611-613, 1983), (ii) gel filtration chromatography (Nakajima, M. et al., J. Biol. Chem. 259: 2283-2290, 1984), (iii) high speed gel permeation chromatography (Irimura, T. et al., Anal. Biochem, 130: 461-468, 1983) (iv) solid-phase substrates of heparanase (Nakajima, M. et al., Anal. Biochem. 157: 162-171, 1986; U.S. Pat. No. 4,859,581), (v) radio-labeled and florescein-labeled heparan sulfate for detection of heparanase activity (U.S. Pat. No. 4,859,581, WO 9504158A), (vi) use of chicken histidine rich glycoprotein (cHRG), taking advantage that heparanase treated heparan sulfate fragment has low affinity to cHRG.
Various methods for purifying heparanase have been disclosed in WO 9102977A and WO 9504158A: the former is a method for preparation of the native heparanase by using chromatographic procedure, and the later is a method for purifying heparanase having activity of endo-N-acetylglucosaminidase.
Biochemical, biological, and pathological studies of heparan sulfate proteoglycans have led to examine the role of heparanase in various diseases. Heparan sulfate is a major component of basement membranes which are continuous sheets of extracellular matrices separating parenchymal cells from underlying interstitial connective tissues. Basement membranes have characteristic permeability and play a role in maintaining normal tissue architecture. Heparan sulfate proteoglycans promote basal lamina matrix assembly by enhancing the interactions of collagenous and noncollagenous protein components while protecting them against proteolytic attack. Heparan sulfate is also a real barrier against cationic and large molecules in the basement membrane. Thus, the destruction of heparan sulfate proteoglycan barrier is an important step during the penetration of basement membranes by both normal and tumor cells (Nakajima, M. et al., J. Cell. Biochem. 36:157-167, 1988).
Most cancer mortality is the result of metastasis to regional and distant metastases. Metastasis formation occurs via a sequential and complex series of unique interactions between tumor cells and normal host cells and tissues. During the metastasis formation migrating tumor cells confront natural barriers such as connective tissues and basement membranes. The ability of malignant cells to penetrate these barriers depends on the presence of tumor and/or host enzymes capable of degrading stromal and basement membrane components. In fact several tumor cell-associated proteinases and glycosidases have been implicated in the tumor cell invasion and metastasis and their activities correlate with metastatic potential in several types of malignant cells. The enzymatic degradation of heparan sulfate proteoglycans in vascular subendothelial basement membranes followed by release of heparan sulfate fragments are achieved by metastatic tumor cells, angiogenic endothelial cells, and inflammatory cells. A good correlation between heparanase activity and metastatic potential has been found in several types of malignant tumors such as melanoma, T cell lymphoma, fibrosarcoma, and rhabdomyosarcoma (Nakajima et al., Science220: 611, 1983; Vlodavsky et al., Cancer Res. 43: 2704, 1983; Ricoveri and Cappelletti, Cancer Res. 46: 3855, 1986; Becker et al., J. Natl. Cancer Inst. 77: 417, 1986).
Similar observations have been reported from several other laboratories using different types of tumors as reviewed by Nakajima et al. (J. Cell. Biochem. 36: 157-167, 1988) and Vlodavsky et al. (Cancer Metastasis Rev., 9: 203-226, 1990), suggesting that heparanase plays a critical role in cell penetration through vascular basement membranes during the blood-borne metastasis, angiogenesis, and inflammatory cell migration. Therefore, heparanase inhibition leads to suppression of tumor cell extravasation and angiogenesis resulting in the inhibition of tumor metastasis. U.S. Pat. No. 5,262,403A describes glycosaminoglycan derivatives and their use as inhibitors of tumor invasiveness of metastatic profusion. U.S. Pat. No. 4,882,318 describes heparin derivatives as inhibitors of tumor metastasis and angiogenesis.
Various molecules such as fibroblast growth factor (FGF), antithorombin III, platelet factor IV, vascular endothelial growth factor (VEGF), interferon-gamma (IFN-g), hepatocyto growth factor (HGF), kinases, phosphatases lipoprotein lipase, IP-10, herpes simplex virus type I are known to bind to heparan sulfate. Among all, extensive studies on interaction of bFGF and heparan sulfate have been reported from various laboratories(for review: Schlessinger, J. et al. Cell. 83: 357-360, 1995). Modulations of the interactions and bioavailability of these molecules by heparanase have been reported by several groups. Whitelock. J. M. et al. (J. Biol. Chem. 271: 10079-10086, 1996) have shown heparanase as the most efficient agent among the enzymes they have tested (plasmin, collagenase, thrombin, and stromelysin) to release bound growth factors from perlecan, one of the heparan sulfate proteoglycans. They have speculated the release of bFGF from heparan sulfate oligosaccaride chains would lead to regeneration of tissues at sites of injury in the wound healing process.
Hoogewerf. A. J. et al. have identified heparanase from human platelets and examined its enzymatic characteristics (J. Biol.Chem. 270: 3268-3277, 1995). The N-terminal amino acid sequence of heparanase from human platelets, which they identified, revealed it as Connective Tissue Activating Peptide-III (CTAP-III), one of a CXC Chemokine family, and its mode of action was an endo-N-acetylglucosaminidase, degrading heparan sulfate into dissachrides. In their discussion they referred to the dual function of CTAP-III as both a heparanase and a neutrophil chemoattractant, suggesting its functions in various pathologies. For example, heparanase activity of the chemokine could down-regulate inflammation by degrading focal sites of chemokine anchoring on the surface of the inflammed endothelium. In vascular pathologies, degradation of vascular heparan sulfate by CTAP-III would remove antithrombin III binding sites and promote thrombogenesis.
Lider et al. have shown a disaccharide that inhibits tumor necrosis factor alpha is formed from the extracellular matrix by the enzyme heparanase (Proc. Natl. Acad. Sci. U.S.A. 92: 5037-5041, 1995). When T-cells are activated by the antigens presented on antigen-presenting cells, they produce effector molecules, such as an inflammatory cytokines, tumor necrosis factor-alpha (TNF-a) and an enzyme, heparanase. As a consequence, heparanase disrupts heparan sulfate molecules in extracellular matricies and/or cell surfaces and produces dissaccharides of heparan sulfate, which in turn down-regulates the inflammatory activity of activated T-cells. The negative feedback control of T-cell mediated inflammation by heparanase implies that administering such enzyme molecules therapeutically might succeed in immune system modulation.
Gilat, D. et al. have shown that the heparanase serves as a T-cell adhesion molecule at physiological pH (J. Exp. Med. 181: 1929-1934, 1995). At a physiological pH, the relatively quiescent enzyme, heparanase appears to act as a lectin-like proadhesive molecule that can organize the recruitment of resting T cells in extravascular loci. Therefore, the heparanase-mediated ECM-anchored CD4+ T cells could readily respond to costimulatory signals elicited by specifically activated adjacent immune cells.
Beta-amyloid is a major component of the senile plaques characteristic of Alzheimer""s disease (Snow, A. D. et al., Neuron 12: 219-234, 1996). Histochemical and immunocytochemical studies have shown that heparan sulfate proteoglycans and glycosaminoglycans colocalize with beta-amyloid protein in senile plaques of Alzheimer""s disease. McCubbin et al (Biochem J. 256: 775-783, 1988) have shown that heparan sulfate removed all the random coil structure of serum amyloid polypeptide and converted into beta-sheet beta-turn conformation leading to aggregation of amyloid-beta fibrils. Glypican, one of heparan sulfate proteoglycans was shown to binds to the amyloid precursor protein of Alzheimer""s disease and inhibits amyloid precursor protein-induced neurite outgrowth (Williamson et al., J. Biol. Chem. 271: 31215-31221, 1996). These findings suggest that one mechanism to prevent the complex formation of beta-amyloid with heparan sulfate proteoglycan that lead to deposition of amyloid would be to degrade the proteoglycan. Small molecules, which mimic elements of the heparan sulfate structure, can interfere with both in vivo induction and persistence of amyloid protein, and furthermore can interfere with the induction of amiloid beta-sheet formation and the amyloid-beta fibrinogenesis in vitro by heparan sulfate (Kisilevsky et al., Nature Med. 1: 143-148, 1995). Thus, heparanase capable of degrading heparan sulfate can be used as a therapeutic tool to prevent beta-amyloid deposition in senile plaques.
In view of the various features of enzymes showing heparanase-like activities, which are well suited for use in the pharmaceutical field, there is an ongoing need to provide further polypeptides showing such activities. Preferably, such polypeptides show an advantageous behaviour compared to that of known enzymes. Moreover, there is a need for providing a polynucleotide encoding such a polypeptide, in order to be able to prepare sufficient amounts of such a polypeptide by means of recombinant expression technologies.
The present invention relates to polypeptide showing the biological activity of human heparanase obtainable from the SV-40 transformed human fibroblast cell line ATCC CCL 75.1, or a functional derivative, a functional fragment or a functional analogue thereof.
The present invention further relates to a polynucleotide comprising a nucleotide sequence encoding a such a polypeptide.
In another aspect the invention relates to a process for preparing such a polypeptide or such a polynucleotide.
Moreover, the present invention relates to a hybrid vector comprising such a polynucleotide, and to a host cell transformed with such a hybrid vector.
It is a further object of the present invention to provide an antibody specifically recognizing and binding to such a polypeptide and to a method of diagnosis utilizing such an antibody.
Further objects of the present invention relate to pharmaceutical compositions comprising such a polypeptide or antibody, and to a method of treatment comprising administration of such a polypeptide or antibody.
In a yet further aspect the present invention provides an assay system and a method for identifying a substance capable of modulating the biological activity or expression of such a polypeptide, and substances obtainable by such a method.
Another aspect of the present application relates to an oligonucleotide or a derivative thereof capable of specifically hybridizing with a polynucleotide according to the present invention.
In a further aspect, the present invention is directed to the use of such a polynucleotide in gene therapy.
In accordance with one aspect of the present invention, there is provided a polypeptide showing the biological activity of human heparanase obtainable from SV-40 transformed human fibroblast cell line ATCC CCL 75.1, or a functional derivative, a functional fragment or a functional analogue thereof. In particular, such a polypeptide is an endoglucuronidase capable of specifically cleaving heparan sulfate into fragments having a size range of from about 6 kDa to about 20 kDa. Said human cell line is the cell line WI-38 VA 13 subline 2RA, available from ATCC (American Type Culture Collection) under accession number ATCC CCL 75.1. It is an SV40 virus-transformed derivative of the WI-38 fibroblast cell line (ATCC CCL 75), which is a diploid cell line from normal embryonic (3-month gestation) lung tissue of a Caucasian female.
In a preferred embodiment thereof a polypeptide according to the present invention is the mature form of the polypeptide having the amino acid sequence set forth in SEQ ID NO 2.In particular, the mature polypeptide has the amino acid sequence of amino acids 158 to 543 set forth in SEQ ID NO 2.
The polypeptide having the amino acid sequence set forth in SEQ ID NO 2 (i.e. amino acids 1 to 543) is the predicted precursor form of the protein where approximately the first 24 amino acids represent the leader sequence and the first 157 amino acids are the prosequence. Such a precursor polypeptide forms another aspect of the present invention.
The present invention further provides a process for the preparation of a polypeptide of the invention, said process comprising chemical synthesis, recombinant DNA technology or a combination of these methods.
In accordance with another aspect of the present invention, there is provided an isolated nucleic acid molecule (polynucleotide) comprising a nucleotide sequence encoding a polypeptide according to the present invention, i.e. a polypeptide showing the biological activity of human heparanase obtainable from SV-40 transformed human fibroblast cell line ATCC CCL 75.1, or coding for the amino acid sequence of a functional derivative, a functional fragment or a functional analogue thereof. Preferably such a polynucleotide comprises a nucleotide sequence encoding such a polypeptide having the amino acid sequence of amino acids 158 to 543 set forth in SEQ ID NO 2, said polypeptide being the mature form of the heparanase of the present invention. In another embodiment such polynucleotide comprises a nucleotide sequence encoding a polypeptide having the amino acid sequence of amino acids 1 to 543 set forth in SEQ ID NO 2, said polypeptide being the precursor form of the heparanase of the present invention. Another preferred embodiment of the present invention relates to a polynucleotide having the nucleotide sequence set forth in SEQ ID NO 1.
A polynucleotide encoding a polypeptide of the present invention may be obtained from a cDNA library derived from human carcinoma cells, placenta, peripheral blood leucocytes, or lung. In particular, the polynucleotide described herein is isolated from a cDNA library derived from human SV-40 transformed fibroblast cell line ATCC CCL 75.1. The cDNA insert is 3726 base pairs (bp) in length and contains an open reading frame encoding a protein 543 amino acids in length of which approximately the first 24 amino acids represent the leader sequence and first 157 amino acids represent the prosequence. Thus, the mature form of the polypeptide of the present invention consists of 386 amino acids after the 157 amino acid prosequence (which includes the approximately 24 amino acid leader sequence) is cleaved. The polypeptide may be found in lysosomes of, or extracellularly near, carcinoma cells.
The polynucleotide of the present invention may be in the form of RNA or in the form of DNA, which RNA includes mRNA and pre-mRNA, and which DNA includes cDNA, genomic DNA and synthetic DNA. The DNA may be double-stranded or single-stranded, and if single stranded may be the coding strand or non-coding (anti-sense) strand. The coding sequence which encodes the mature polypeptide or the precursor form may be identical to the coding sequence contained in SEQ ID NO 1, or may be different from that coding sequence, as a result of the redundancy or degeneracy of the genetic code, but encodes the same, mature polypeptide or the precursor form thereof as the cDNA of SEQ ID NO 1.
In preferred embodiments, the polynucleotide according to the present invention may include: only the coding sequence for mature polypeptide; the coding sequence for the leader or secretory sequence or a proprotein sequence; the coding sequence for the mature polypeptide (and optionally additional coding sequence) and non-coding sequence, such as introns or non-coding sequence 5xe2x80x2 and/or 3xe2x80x2 of the coding sequence for the mature polypeptide.
Thus, the term xe2x80x9cpolynucleotide comprising a nucleotide sequence encoding a polypeptidexe2x80x9d encompasses a polynucleotide which includes only coding sequence for the polypeptide as well as a polynucleotide which includes one or more additional coding and/or non-coding sequences.
The present invention further relates to variants of the hereinabove described polynucleotides which encode for fragments, analogs and derivatives of the polypeptide having the deduced amino acid sequence of SEQ ID NO 2. The variant of the polynucleotide may be a naturally occurring allelic variant of the polynucleotide or a non-naturally occurring variant of the polynucleotide. The present invention also relates to polynucleotide probes constructed form the polynucleotide sequence of SEQ ID NO 1 or a segment of the sequence of SEQ ID No 1 amplified by the PCR method, which could be utilized to screen an above mentioned cDNA library to deduce the polypeptide of the present invention.
Thus, the present invention includes polynucleotides encoding the same mature polypeptide of the present invention as well as variants of such polynucleotides which variants encode for a fragment, derivative or analog of such a polypeptide. Such nucleotide variants include deletion variants, substitution variants and addition or insertion variants.
As hereinabove indicated, the polynucleotide may have a coding sequence which is a naturally occurring allelic variant of the coding sequence of SEQ ID NO 1. As known in the art, an allelic variant is an alternate form of a polynucleotide sequence which may have a substitution, deletion or addition of one or more nucleotides, which does not substantially alter the function of the encoded polypeptide.
The present invention also includes polynucleotides, wherein the coding sequence for the mature polypeptide may be fused in the same reading frame to a polynucleotide sequence which aids in expression and secretion of a polypeptide from a host cell, for example, a controlling transport of a polypeptide from the cell. The polypeptide having a leader sequence is a preprotein and may have the leader sequence cleaved by the host cell to form a mature form of the polypeptide. The polynucleotides may also encode for a pro-protein which is the mature protein plus additonal N-terminal and C-terminal amino acid residues. A mature protein having a prosequence is a pro-protein and may in some cases be an inactive form of the protein. Once the prosequence is cleaved an active mature protein remains.
Thus, for example, the polynucleotide of the present invention may encode for a mature protein, or for a protein having a prosequence or for a protein having both a presequence (leader sequence) and a prosequence.
The polynucleotides of the present invention may also have the coding sequence fused in frame to a marker-sequence which allows for purification of the polypeptide of the present invention. The marker sequence may be a hexa-histidine tag supplied by a pProEX-HTb (Gibco BRL) vector to provide for purification of the mature polypeptide fused to the marker in the case of a bacterial host, or, for example, the marker sequence may be a hemagglutinin (HA) tag when a mammalian host, e.g. COS7 cells, is used. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, I., et al. Cell, 37:767 (1984)).
A polynucleotide according to the present invention can be prepared by a process comprising chemical synthesis, recombinant DNA technology, polymerase chain reaction or a combination of these methods. Such a process forms a further aspect of the present invention.
The terms xe2x80x9cfunctional fragment,xe2x80x9d xe2x80x9cjunctional derivativexe2x80x9d and xe2x80x9cfunctional analoguexe2x80x9d when referring to a polypeptide according to the present invention, means a polypeptide which retains essentially the same biological function or activity as such polypeptide. Thus, an analogue may include a proprotein portion to produce an active mature polypeptide.
The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide, preferably a recombinant polypeptide.
The functional fragment, derivative or analog of a polypeptide of the present invention may be (i) one in which one or more of the amino acid resideus are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the mature polypeptide, such as a leader or secretory sequence or a sequence which is employed for purification of the mature polypeptide or a proprotein sequence. Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.
The polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity. The term xe2x80x9cisolatedxe2x80x9d means that the material is removed from its original environment (e.g., the natural environment if it is naturally occuring). For example, a naturally-occuring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide (e.g. a DNA or a RNA molecule) or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. A polynucleotide of the present invention could be a part of a vector and/or such a polynucleotide or polypeptide could be a part of a composition, and still be isolated in that such a vector or composition is not a part of its natural environment.
The present invention also relates to a hybrid vector comprising a polynucleotides of the present invention, said hybrid vector being operably linked to suitable control sequences. Preferably, such a hybrid vector is an expression vector. Furthermore, the present invention relates to a host cell transformed with a hybrid vector of the present invention, i.e. which host cell is genetically engineered with such a hybrid vector. In another aspect, the present invention relates to a recombinant process for the preparation of a polypeptide of the present invention, said process comprising (i) cultivation of a host cell transformed with a hybrid vector of the present invention under conditions suitable for performing expression of the polypeptide, and (ii) isolation of the thus-expressed polypeptide.
Host cells are genetically engineered (transduced or transformed or transfected) with the vectors of this invention which may be, for example, a cloning vectors or an expression vector. The vector may be, for example, in the form of a plasmid, a viral particle, a phage, etc.
The engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promotes, selecting transformants or amplifying the Heparanase gene. The culture conditions, such as temperature, pH and the like, are those used with the host cell selected for expression, and will be apparent to the ordinarily skilled art.
The polynucleotide of the present invention may be employed for producing a polypeptide by recombinant techniques. Thus, for example, the polynucleotide sequence may be included in any one of a variety of expression vehicles, in particular vectors or plasmids for expressing a polypeptide. Such vectors include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA; yeast plasmid; vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, baculovirus, and pseudorabies. However, any other plasmid or vector may be used as long as it is replicable and viable in the host.
As hereinabove indicated, the appropriate DNA sequence may be inserted into the vector by a variety of procedures. In general, the DNA sequence is inserted into appropriate restriction endonuclease sites by procedures known in the art.
The DNA sequence in the expression vector is operatively linked to (an) appropriate expression control sequence(s) (promoter) to direct mRNA synthesis. As representative examples of such promoters, there may be mentioned: LTR or SV40 promoter, the E.coli. lac or trp, the expression of genes in prokaryotic or eukaryotic cells or their viruses. The expression vector also contains a ribosome binding site for translation initiation and a transcription terminator. The vector may also include appropriate sequences for amplifying expression.
In addition, the expression vectors preferably contain a gene to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E.coli.
The vector containing the appropriate DNA sequence as hereinabove described, as well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the host to express the protein. As representative examples of an appropriate host, there may be mentioned: bacterial cells, such as E.coli, Salmonella typhimurium; Streptomyces; fungal cells, such a s yeast; insect cells such as Drosophila and Sf9; animal cells such as CHO and COS; plant cells, etc. The selection of an appropriate host is deemed to be within the scope of those skilled in the art fom the teachings herein.
More particularly, the present invention also includes recombinant constructs comprising one or more of the sequences as broadly described above. The constructs comprise a vector, such as a plasmid or viral vector, into which a sequence of the invention has been inserted, in a forward or reverese orientation. The construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence. Large numbers of suitable vectors and promoters are known to those of skill in the art, and are commercially available. The following vectors are provided by way of example. Bacterial:pQE70, pQE60, pQE-9 (Qiagen)pBs, phagescript, psiX174, pBluescript SK, pBsKS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene); pTrc99A, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia). Eukaryotic: pWLneo, pSV2cat, pOG44, pXT1, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any other plasmid or vector may be used as long as it is replicable and viable in the host.
Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers. Two appropriate vectors are pKK232-8 and pCM7. Particularly named bacterial promoters include lac, lacZ, T3, T7, gpt, lambda PR, PI and trp. Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art. In a further embodiment, the present invention relates to host cells containing the above-described construct. The host cell can be a higher eukaryotic cell such as mammalian cell, or a lower eukaryotic cell, such as a yeast cell, e.g. S. cerevisiae, or the host cell can be a prokaryotic cell, such as a bacterial cell, e.g. E. coli. Introduction of the construct into the host cell can be effected by clacium phospahe transfection, DEAE-Dextran mediated trasfection, or electorporation (Davis, L., Dibner, M., Battey I., Basic Methods in Molecular Biology, 1986)).
The constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence. Alternatively, the polypeptides of the invention can be synthetically produced by conventional peptide synthesizers.
Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA costructs of the present invention. Appropriate cloning an expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook. Et al., Molecular Cloning: A Laboratory Manual, Second Edition, (Cold Spring Harbor, N.Y., 1989), the disclosure of which is hereby incorporated by reference.
Transcription of a DNA encoding the polypeptides of the present invention by higher eukaryotes is increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 19 to 300 bp, that act on a promoter to increase its transcription. Examples include the SV40 enhancer on the late side of the replication origin (bp 100 to 270), a cytomegalovirus early promoter enhancer, a polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
Generally, recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of E. coli and S cerevisiae TRP1 gene, and a promoter derived form a highly-expressed gene to direct transcription of a downstream structural sequence. Such promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), alpha factor, acid phosphatase or heat shock proteins, among others. The heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of a translated protein into the periplasmic space or extracellular medium. Optionally, the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparing desired characteristics, e.g., stabilization or simplified purificatio of expressed recominant product.
Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading phase with a functional promoter. The vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vetor and to, if desirable, provide amplification within the host. Suitable prokaryotic hosts for transformation include E.coli, Bacillus subilis, Salmonellatyphimurium and various species within the genera Pseudomonas, Streptomyce, and Staphylococcus, although others may also be employed as a matter of choice.
As a representative but nonlimiting example, useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBK322 (ATCC37017). Such commercial vectors include, for, example, PKK223-3 (Pharmacia) and GEM1 (Promega Biotec, Madison, Wis., USA). These pBR3222 xe2x80x9cbackbonexe2x80x9d sections are combined with an appropriate promoter and structural sequence to be expressed. Various mammalian cell culture systems can also be employed to express recombint protein. Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell, 23:175 (1981), and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines. Mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5xe2x80x2 flaking nontranscribed sequences. DNA sequences derived from the SV40 viral genome for example, SV40 origin, early promoter, enhancer, splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.
A thus-expressed polypeptide of the present invention is recovered and purified from recombinant cell cultures by methods used heretofore, including detergent homoginates, Heparin-Sepharose chromatography, cation exchange chromatography, Con A-Sepharose chromatography, gel-filtration chromatography, and hydrophobic interaction chromatography.
A polypeptide of the present invention may be purified products naturally expressed from a high expressing cell line, or a product of chemical synthetic procedures, or produced by recombinant techniques from a prokaryotic or eukaryotic host (hor example, by bacterial, yeast, higher plant, insect and mammalian cells in culture). Depending upon the host employed in a recombinant production procedure, a polypeptide of the present invention may be glycosylated with mammalian or other eukaryotic carbohydrates or may be non-glycosylated. A polypeptide of the invention may also include an initial methionine amino acid residue.
The polynucleotides of the present invention are also valuable for chromosome identification. The polynucleotide is specifically targeted to and can hybridize with a particular location on an individual human chromosome. Moreover, there is a current need for identifying paricular sites on the chromosome. Few chromosome marking reagents based on actual sequence data (repeat polymorphism""s) are presently available for marking chromosomal location. The mapping of DNAs to chromosomes according to the present invention is an important first step in correlating those sequences with genes associated with diseases.
Briefly, sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25b bp) from the cDNA Computer analysis of the cDNA is used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the primer will yield an amplified fragment.
PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular DNA to a particular chromosome. Using the present invention with the same oligonucleotide primers, sublocalization can be achieved with panels of fragments from specific chromosomes or pools of large genomic clones in an analogous manner. Other mapping strategies that can similarly be used to map to its chromosome include in situ hybridization, prescreening with labeled flow-sorted chromosomes and preselection by hybridization to construct chromosome specific-cDNA libraries.
Fluorescence in situ hybridization (FISH) of a cDNA clone to a metaphase chromosomal spread can be used to provide a precise chromosomal location in one step. This technique can be used with cDNA as short as 500 or 600 bases; however, clones larger than 2,000 bp have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection. FISH requires use of the clone from which the EST was derived, and the longer the better. For example, 2,000 bp is good, 4,000 is better, and more than 4,000 is probably not necessary to get good results a reasonable percentage of the time. For a review of this technique, see Verma et al., Human Chromosomes* a Manual of Basic Techniques. Pergamon Press, New York (1988).
Once a sequence has been mapped to a precise chromosomal location, the physical positio of the sequence on the chromosome can be correlated with genetic map data. (Such data are found, for example, in V. McKusck, Mendelian Inheritance in Man (available on line through Johns Hopkins University welch Medical Library). The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheritance of physicall adjacent genes).
Next, it is necessary to determine the differences in the cDNA or genomic sequence between affected and unaffected individuals. If a mutation is observed in some or all of the affected individuals but not in any normal individuals, then the mutation is likely to be the causative agent of the disease.
With current resolution of physical mapping and genetic mapping techniques, a cDNA precisely localized to a chromosomal region associated with the disease could be one of between 50 and 500 potential causative genes. (This assumes 1 megabase mapping resolution and one gene per 20 kb)
Comparison of affected and unaffected individuals generally involves first looking for structural alterations in the chromosomes, such as deletions or translocations that are visible form chromosome spreads or detectable using PCR based on that cDNA sequence. Ultimately, complete sequencing of gened from several individuals is required to confirm the presence of a mutation and to distinguish mutations from polymorphisms.
In another aspect the present invention relates to an oligonucleotide or a derivative thereof, or a salt thereof where salt-forming groups are present, which is specifically hybridizable with the nucleotide sequence set forth in SEQ ID NO 1, said oligonucleotide or derivative thereof comprising nucleoside units or analogues of nucleoside units sufficient in number and identity to allow such hybridization.
Such an oligonucleotide may have a length of, e.g., from about 5 to about 100 or to even several hundred nucleoside units or analogs thereof, depending on the intended use.
An oligonucleotide of the invention may be used as a cloning or sequencing primer or probe. Another use relates to stimulating and inhibiting a polypeptide of the present invention in vivo by the use of sense or anti-sense technology. These technology can be used to control gene expression through triple-helix formation on double-stranded DNA or antisense-mechanisms on RNA, both of which methods are based on binding of such an oligonucleotide to DNA or RNA. For example, the 5xe2x80x2 coding portion of the polynucleotide sequence, which encodes for the mature polypeptide of the present invention, is used to design an antisense RNA oligonucleotide of from about 10 to about 40 base pairs in length. A DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription (for triple-helix technology see Lee et al, Nucl. Acids Res., 6:3073 (1979); Cooney et al, Science, 241:456 (1988); and Dervan et al, Science, 251:1360 (1991)), thereby preventing transcription and the production of Heparanase. The antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of an mRNA molecule into a polypeptide of the present invention (for antisensexe2x80x94technology see Okano, J. Neuroch., 56:569 (1991); Oligodeoxynucleotides as Antisense inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988)).
Alternatively, the oligonucleotides described above can be delivered to cells by procedures in the art such that the anti-sense RNA or DNA may be expressed in vivo to inhibit production of a polypeptide of the present invention in the manner described above.
Antisense constructs to a polypeptide of the invention, therefore inhibit the action of such a polypeptide and may be used for treating certain disorders, for example, cancer and cancer metastasis, since elevated levels of such a polypeptide is found in highly metastatic cell lines.
The polypeptides, their functional fragments, derivatives or analogs thereof, or a cell expressing them can be used as an immunogen to produce antibodies thereto.
Therfore, the present invention relates to an antibody which specifically recognizes and binds to a polypeptide of the invention.
Such an antibody can be, for example, a polyclonal or a monoclonal antibody. The present invention also includes chimeric, single chain and humanized antibodies, as well as Fab fragments, or the product of and Fab expressing library. Various procedures known in the art may be used for the production of such antibodies and fragments.
Antibodies generated against a polypeptide of the present invention can be obtained by direct injection of the polypeptide into an animal or by administering the polypeptide to an animal, preferably human. The antibody so obtained will then bind the polypeptide itself. In this manner, even a sequence encoding only a fragment of the polypeptide can be used to generate antibodies binding the whole native polypeptide. Such an antibody can then be used to isolate the polypeptide from tissue expressing that polypeptide. For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler and Milstein, 1975, Nature, 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1985, Imunology Today 4:72), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole, et al., 1985, in Monoclonal Antibodies and Cancer Theraph, Alan R. Liss, Inc., pp.77-96).
Techniques described for the production of single chain antibodies (e.g. U.S. Pat. No. 4,946,778) can be adapted to produce single chain antibodies to immunogenic polypeptide products of this invention.
An antibodie specific to a polypeptide of the present invention may further be used to inhibit the biological action of the polypeptide by binding to the polypeptide. In this manner, the antibodies may be used in therapy, for example, to treat cancer since mRNA of such a polypeptide and the polypeptide itself is increased in SV-40 transformed fibroblasts.
Further, such antibodies can detect the presence or absence of a polypeptide of the present invention and the level of concentration of such polypeptide and, therefore, are useful as diagnostic markers for the diagnosis of disorders such as cancer, cancer metastasis, and angiogenesis.
Hence, the present invention relates to a method of diagnosis of conditions resulting from shortage or lack of a polypeptide of the present invention, in particular of a polypeptide showing the biological activity of human heparanase obtainable from the SV-40 transformed human fibroblast cell line ATCC CCL 75.1, or a functional derivative, a functional fragment or a functional analogue thereof, inclusive the respective preferred embodiments thereof, or resulting from excessive acitvity or overexpression of such a polypeptide, said method comprising contacting cells or tissues or body fluids from an animal inclusive man suspected of having such a condition with an antibody of the present invention.
In a further aspect, the present invention relates to a method for identifying a substance capable of modulating the biological activity or expression of a polypeptide of the present invention, said method comprising contacting such a polypeptide or a functional derivative, a functional fragment or a functional analogue thereof, or a cell capable of expressing such a polypeptide, functional derivative, functional fragment or functional analogue, with at least one compound or agent whose ability to modulate the biological activity or expression of said polypeptide is sought to be investigated, and determining the change of the biological activity or the expression of said polypeptide, derivative, fragment or analogue caused by the substance.
Another embodiment of the present invention relates to an assay system for testing a substance for its capability of binding to or having functional effects on a polypeptide of the present invention, said assay system comprising a polypeptide as of the present invention, or a functional derivative, a functional fragment or a functional analogue thereof, or a cell expressing such a polypeptide, functional derivative, functional fragment or functional analogue.
In this context, the present invention is also directed to substance obtainable by an identification-method as described above, said substance being an agonist or an antagonist of a polypeptide of the present invention.
Thus, the present invention is also directed to antagonists and inhibitors of a polypeptide of the present invention. The antagonists and inhibitors are those substances which inhibit or eliminate the function of such a polypeptide. The present invention further relates to agonists and stimulators of a polypeptide of the present invention. The agonists and stimulators are those substances which enhance the function or activity or the expression of such a polypeptide.
For example, an antagonist may bind to a polypeptide of the present invention and inhibit or eliminates its function. The antagonist, for example, could be an antibody against the polypeptide which eliminated the activity of Heparanase by binding to Heparanase, or in some cases the antagonist may be an oligonucleotide. An example of an inhibitor is a small molecule inhibitor which inactivates the polypeptide by binding to and occupying the catalytic site, thereby making the catalytic site inaccessible to a substrate, such that the biological activity of Heparanase is prevented. Examples of small molecule inhibitors include but are not limited to small carbohydrate or carbohydrate-like molecules.
In these ways, the antagonists and inhibitors may be used to treat cancer, angiogenesis by preventing heparanase from functioning to break down extracellular matrix and release heparan sulfate from extracellular matrix and cell surface.
The antagonists and inhibitors may be employed in a composition with a pharmaceutically acceptable carrier, including but not limited to saline, buffered saline, dextrose, wate, glycerol, ethanol and combinations thereof. Administration of inhibitors of the polypeptides of the present invention are preferably systemic. Suramin, a polysulfonated naphthylurea, was shown to strongly inhibit murin melanoma heparanase and its invation (Nakajima, M. et al. J. Biol. Chem. 266, p9661-9666, 1991). 2,3-O-desulfated heparin was shown to inhibit heparanase activity, tumor growth of a subcutaneous human pancreatic (Ca-Pan-2) adenocarcinoma in nude mice and prolonged the survival times of C57BL/6N mice in a B16-F10 melanoma experimental lung metastasis assay (Lapierre, F. et al. Glycobiology vol. 6, 335-366, 1996).
In particular, the present invention also relates to an assay for identifying the above-mentioned substances, e.g. small molecule inhibitors, which are specific to the polypeptides and prevent them from functioning or prevent their expression. Either natural carbohydrate substrates or synthetic carbohydrates would be used to assess endo-glycosidase activity of the polypeptide, and the ability of inhibitors to prevent this activity could be the basis for a screen to identify compounds that have therapeutic activity in disorders of excessive activity or overexpression of a polypeptide according to the present invention.
In particular, the present invention also relates to an assay for identifying the above-mentioned substances, e.g. small molecule stimulators, which are specific to the polypeptides and enhance its function or expression. Either natural carbohydrate substrates or synthetic carbohydrates would be used to assess endo-glycosidase activity of the d the ability of stimulators to enhance this activity could be the basis for a screen to identify compounds that have therapeutic activity in disorders which are resultant of shortage or lack of a polypeptide according to the present invention.
A further aspect relates to a polypeptide according to the present invention for use in medicine. In particular, the invention relates to the use of a polypeptide according to the present invention in the preparation of a pharmaceutical composition for the treatment of a disease resulting from shortage or lack of said polypeptide. Instead of a polypeptide of the present invention, an agonist of the polypeptide or an expression inducer/enhancer of such a polypeptide may be used for the medicinal purposes. Such diseases are, for example, trauma, autoimmune diseases, skin diseases, cardiovascular diseases and nervous system diseases including Alzheimer""s disease.
Another aspect relates to an antibody according to the present invention for use medicine. In particular, the invention relates to the use of an antibody according to the present invention in the preparation of a pharmaceutical composition for the treatment of a disease resulting from excessive acitvity or overexpression of a polypeptide of a polypeptide according to the present invention. Instead of a antibody of the present invention, an antagonist of the polypeptide or an expression inhibitor of such a polypeptide may be used for the medicinal purposes. Such diseases are. for example, cancer, cancer metastasis, angiogenesis and inflammation including arthritis.
The invention moreover is directed to a pharmaceutical composition suitable for administration to a warm-blooded animal inclusive man suffering from a disease resulting from shortage or lack of a polypeptide of the present invention, said composition comprising such a polypeptide together with at least one pharmaceutically acceptable carrier and/or excipient. In this context, the invention is directed to a method of treatment of a disease resulting from shortage or lack of a polypeptide of the invention said method comprising administration of a suitable amount of such a polypeptide. As mentioned above, instead of such a polypeptide said composition or method of treatment may comprise or utilize an agonist of the polypeptide or an expression inducer/enhancer of such a polypeptide.
Furthermore, the invention is directed to a pharmaceutical composition suitable for administration to a warm-blooded animal inclusive man suffering from a disease resulting from excessive acitvity or overexpression of a polypeptide of the present invention, said composition comprising an antibody of the present invention together with at least one pharmaceutically acceptable carrier and/or excipient. In this context, the invention relates to a method of treatment of a disease resulting from excessive acitvity or overexpression of a polypeptide of the present invention, said method comprising administration of a suitable amount of an antibody of the present invention. As mentioned above, instead of such an antibody said composition or method of treatment may comprise or utilize an antagonist of the polypeptide or an expression inhibitor of such a polypeptide.
Further ingredients, i.e. a carrier or excipient, of a pharmaceutical composition of the present invention may be those known in the art, in particular those as decribed herein above.
In another aspect the present invention relates to a polynucleotide of the invention for use in gene therapy.
The present invention will be further described with reference to the following examples; however, it is to be understood that the present invention is not limited to such examples. All parts or amounts, unless otherwise specified, are by weight.
In order to facilitate understanding of the following examples, certain frequently occuring methods and/or terms will be described.
xe2x80x9cPlasmidsxe2x80x9d are designated by a lower case preceded and/or followed by capital letters and/or numbers. The starting plasmids herein are either commercially available, publicly availabe on an unrestricted basis, or can be constructed from available plasmids in accord with published procedures. In addition, equivalent plasmids to those described are known in the art and will be apparent to the ordinarily silled artisan.
xe2x80x9cDigestionxe2x80x9d of DNA refers to catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences in the DNA. The various restriction enzymes used herein are commercially available and their reaction conditions, cofactors and other requirement were used as would be known to the ordinarily skilled artisan. Fro analytical purpose, typically 1 microg of plasmid or DNA fragment is used with about 2 units of enzyme in about 20 microl of buffer solution. For the pupose of isolating DNA fragments for plasmid construction, typically 5 to 50 microg of DNA are digested with 20 to 250 units of enzyme in a larger volume. Appropriate buffers and substrate amounts for particular restriction enzymes are specified by the manufacturer. Incubation times of about 1 hour at 37xc2x0 C. are ordinarily used, but may vary in accordance with the supplier""s instructions. After digestion the reaction is electrophoresed directly on a polyacrylamide gel to isolate the desired fragment.
Size separation of the cleaved fragments is performed using 8 percent polyacrylamide gel described by Goeddel, D. et al., Nucleic Acis Res., 8*4057 (1980 ).
xe2x80x9cOligonucleotidesxe2x80x9d refers to either a single stranded polydeoxynucleotide or two complementary strande polydeoxynucleotide strands which may be chemically synthesized.
Such synthetic oligonucleotides have no 5xe2x80x2 phosphate and thus will not ligate to another oligonucleotide without adding a phosphate with an ATP in the presence of a kinas. A synthetic oligonucleotide will ligate to a fragment that has not been dephosphorylated.
xe2x80x9cLigationxe2x80x9d refers to the process of forming phosphodiester bonds between two double strande nucleic acids. Unless otherwise provided, ligation may be accomplished using known buffers and conditions with 10 units of T4 DNA ligase per 0.5 microg of approximately equimolar amounts of the DNA fragments to be ligated. Unless otherwise stated, transformation is performed as described in the methods of Graham, F. and Van Der Eb, A., Virology, 52:456-457 (1973).


A system is disclosed for converting energy from the electromagnetic quantum vacuum available at any point in the universe to usable energy in the form of heat, electricity, mechanical energy or other forms of power. By suppressing electromagnetic quantum vacuum energy at appropriate frequencies a change may be effected in the electron energy levels which will result in the emission or release of energy. Mode suppression of electromagnetic quantum vacuum radiation is known to take place in Casimir cavities. A Casimir cavity refers to any region in which electromagnetic modes are suppressed or restricted. When atoms enter into suitable micro Casimir cavities a decrease in the orbital energies of electrons in atoms will thus occur. Such energy will be captured in the claimed devices. Upon emergence form such micro Casimir cavities the atoms will be re-energized by the ambient electromagnetic quantum vacuum. In this way energy is extracted locally and replenished globally from and by the electromagnetic quantum vacuum. This process may be repeated an unlimited number of times. This process is also consistent with the conservation of energy in that all usable energy does come at the expense of the energy content of the electromagnetic quantum vacuum. Similar effects may be produced by acting upon molecular bonds. Devices are described in which gas is recycled through a multiplicity of Casimir cavities. The disclosed devices are scalable in size and energy output for applications ranging from replacements for small batteries to power plant sized generators of electricity.

Max Planck proposed the concept of zero-point energy in 1912. The idea was then studied by Albert Einstein and Otto Stern in 1913. In 1916 Walther Nernst proposed that the Universe was filled with zero-point energy. The modern field of stochastic electrodynamics is based upon these ideas.
At that same time the structure and stability of the atom were puzzles. The Rutherford model of the atom was based on analogy to the motions of planets (electrons) around the Sun (the nucleus). However this was not feasible. The orbiting electron(s) would emit Larmor radiation, quickly losing energy and thus spiraling into the nucleus on time scales less than one-trillionth of a second, thereby rendering stable matter impossible. It is now known within the context of stochastic electrodynamics (SED) theory that a possible solution involves the absorption of zero-point energy. It was shown in 1975 by Boyer that the simplest possible atom and atomic state, the hydrogen atom in its ground state, would be in a state of equilibrium between Larmor radiation and absorption of zero-point energy at the correct radius for a classical Rutherford hydrogen atom.
Since this solution was not known in 1913, Niels Bohr followed a different path by simply postulating that only discrete energy levels were available to the electron in an atom. This line of reasoning let to the development of quantum theory in the 1920s. The concept of classical zero-point energy was forgotten for a decade. However the same concept found itself reborn in a quantum context in 1927 with the formulation of the Heisenberg uncertainty principle. According to the principle, the minimum energy of a harmonic oscillator has the value hf/2, where h is Planck's constant and f is the frequency. It is thus impossible to remove this last amount of random energy from an oscillating system.
Since the electromagnetic field also must be quantized in quantum theory, a parallel is drawn between the properties of a quantum oscillator and the waves of the electromagnetic field. It is concluded that the minimum energy of any possible mode of the electromagnetic field, consisting of frequency, propagation direction and polarization state, is hf/2. Multiplying this energy by all possible modes of the field gives rise to the electromagnetic quantum vacuum, which has identical properties—energy density and spectrum—to the classical zero-point energy studied by Planck, Einstein, Stern and Nernst a decade previously.
The line of inquiry involving classical physics plus the addition of a classical zero-point field was reopened in the 1960s by Trevor Marshall and Timothy Boyer and has been named stochastic electrodynamics (SED). SED asks the question: "Which quantum properties, processes or laws can be explained in terms of classical physics with the only addition being a zero-point electromagnetic field." Two of the early successes were a classical derivation of the blackbody spectrum (i.e. one not involving quantum physics) and the discovery that a classically orbiting electron in a hydrogen atom emitting Larmor radiation but absorbing zero-point radiation would have an equilibrium orbit at the classical Bohr radius. An initial approach to this problem by Timothy Boyer (1975) was perfected by H. E. Puthoff (1987). Their analyses treated the orbiting electron as a harmonic oscillator.
This result underwent a major new development with the recent work of Daniel Cole and Y. Zou which simulated the orbit of a classical electron in a true Coulomb field of a hydrogen nucleus and found that such a realistic electron would find itself in a range of distances from the nucleus, in agreement with quantum mechanics, owing to the random nature of the emission and absorption processes. The mean position is at the correct Bohr radius, but the actual distribution of positions very precisely duplicates the electron probability distribution of the corresponding Schrödinger equation in which the electron is regarded as being represented by a wave function. (In the SED representation the electron is "smeared out" not because it is a wave function, but because as a point-like particle it is subject to the continuous perturbations of the electromagnetic quantum vacuum fluctuations.)
A clear consequence of this theory is that a reduction of the electromagnetic quantum vacuum at the frequency corresponding to the orbit of the electron will result in a decay of the orbit since there will thereby be an imbalance in the Larmor radiation vs. absorption.
The electromagnetic quantum vacuum energy spectrum is proportional to the cube of the frequency. If the vacuum energy is suppressed at the frequency of the "normal" orbit of the electron, this will cause the electron to spiral inward to a higher frequency orbit. In this fashion it will then encounter a new equilibrium situation with the electromagnetic quantum vacuum energy spectrum owing to that spectrum's increase with the cube of the frequency.
If the SED interpretation is correct for the hydrogen atom as the analyses of Boyer, Puthoff, Cole and Zou indicate, it must apply as well to all other atoms and their multi-electron configurations. In that case, a transition of an electron from an excited state to a lower energy state involves a rapid decay from one stable orbit to another, not an instantaneous quantum jump. The details of the bases for stability of electron orbits has yet to be determined by SED theory, but the logical extrapolation from the single-electron hydrogen case is clear: electron orbits in all atoms must be determined by an emission vs. absorption balance and thus are subject to modification involving mode suppression of the electromagnetic zero-point field at appropriate frequencies.
It is claimed that modification of electron orbits is in essence the same process as natural transition between energy levels of electrons in atoms and therefore that the energy released in such a process can be captured in the same way as ordinary transition energy.
By moving an atom into and out of a microstructure that suppresses appropriate modes of the electromagnetic quantum vacuum, an extraction of energy from the electromagnetic quantum vacuum may be accomplished. This can be done with micro Casimir cavities.
The electromagnetic quantum vacuum as a real source of energy is indicated by the Lamb shift between s and p levels in hydrogen, van der Waals forces, the Aharanov-Bohm effect, and noise in electronic circuits.
However the most important effect of the electromagnetic quantum vacuum is the existence of the Casimir Force, a force between parallel conducting plates which may be interpreted as a radiation pressure effect of electromagnetic quantum vacuum energy. Electromagnetic waves in a cavity whose walls are conducting are constrained to certain wavelengths for reasons having to do with transverse component boundary conditions on the wall surfaces. As a result, in a Casimir cavity between parallel plates there will be, in effect, an exclusion of radiation modes whose wavelengths are longer than the separation of the plates. An overpressure of electromagnetic quantum vacuum radiation on the outside then pushes the plates together. An extensive literature exists on the Casimir force and the reality of the force has moved from laboratory experimentation to micro-electro-mechanical structures (MEMS) technology both as a problem (so-called "stiction") and as a possible control mechanism.
The exclusion of modes does not begin all at once at the wavelength equivalent to the plate separation, d. Mode suppression will be strongest for wavelengths of d or greater, but will begin to occur as well for wavelengths falling in between the "stairway" d/n, with the effect diminishing as n increases. We propose to use the partial suppression of modes for wavelengths shorter than d occurring in this fashion in order to be able to employ Casimir cavities of the maximum possible physical size.
Researchers have shown that thermodynamic laws are not violated when energy is "extracted" from zero-point energy, as energy is still conserved and the second law is not violated. Cole and Puthoff have carried out and published thermodynamic analyses showing that there is no violation. Indeed, a thought experiment by Forward (1984) showed a simple, but not practical, energy extraction experiment.
In the stochastic electrodynamics (SED) interpretation of the hydrogen atom, the ground state is interpreted as effectively equivalent to a classically orbiting electron whose velocity is c/137. The orbit is stable at the Bohr radius owing to a balance between classical electromagnetic emission and absorption from the electromagnetic zero-point field. This view, first obtained by Boyer (1975) and subsequently refined by Puthoff (1987) has been further strengthened by the detailed simulations of Cole and Zou (2003, 2004) which demonstrate that the stochastic motions of the electron in this interpretation reproduce the probability density distribution of the Schrödinger wave function. Note that one apparent difference between this interpretation and that of quantum mechanics is that in quantum mechanics the 1s state of the electron is regarded as having zero angular momentum, whereas in the SED interpretation the electron has an instantaneous angular momentum of mcr/137=h/2π. However SED simulations by Nickisch have shown that the time-averaged angular momentum is zero just as in the quantum case owing to the zero-point perturbations on the orbital plane. Thus averaged over enough "orbits" this "classical electron" will fill a spherical symmetric volume around the nucleus having the same radial probability density as the Schrödinger wave function and zero net angular momentum, completely consistent with quantum behavior.
The Bohr radius of the atom in the SED view is 0.529 A (Angstroms). This implies that the wavelength of zero-point radiation responsible for sustaining the orbit is 2*π*0.529*137=455 A (0.0455 microns). It is claimed that suppression of zero-point radiation at this wavelength and shorter in a Casimir cavity will result in the decay of the electron to a lower energy state determined by a new balance between classical emission of an accelerated charge and zero-point radiation at λ<455 A, where λ depends on the Casimir plate separation, d. Note that the tail end of the quantum probability density of the electron (as well as the SED simulation of Cole and Zou) extends beyond five Bohr radii, so that some change in the energy balance could be accomplished even at considerably longer wavelengths of perhaps 0.1 microns-0.2 microns
Since the frequency of this orbit is 6.6×1015 s−1, no matter how quickly the atom is injected into a Casimir cavity the process will be a slow one as experienced by the orbiting electron. We therefore assume that the decay to a new sub-Bohr ground state will involve gradual release of energy in the form of heat, rather than a sudden optical radiation signature.
Since the binding energy of the electron is 13.6 eV, we assume that the amount of energy released in this process would be on the order of 1 to 10 eV for injection of the hydrogen atom into a Casimir cavity of d=250 A or thereabouts (and perhaps even a larger cavity as noted above). Upon exiting the cavity the electron would absorb energy from the zero-point field and be re-excited to its normal state. The energy (heat) extracted in the process comes at the expense of the zero-point field, which in the SED interpretation flows at the speed of light throughout the Universe. We are in effect extracting energy locally and replenishing it globally. Imagine extracting thimbles-full of water from the ocean. Yes, the ocean is being depleted thereby, but no practical consequences ensue.
Since naturally occurring hydrogen at standard temperature and pressure (STP) is a two-atom molecule, a dissociation process would need to precede an injection of hydrogen atoms into a Casimir cavity. We avoid this complication and take advantage of multi-electron modification by working with monatomic (noble) gases which also have the advantage of being safe and inexpensive.
We work with naturally occurring monatomic gases for three reasons:                (1) No dissociation process is required.        (2) Heavier element atoms are approximately two to four times larger than hydrogen and thus can utilize and be affected by a larger Casimir cavity which is easier to fabricate.        (3) Heavier elements have numerous outer shell electrons, several of which may be simultaneously affected by the reduction of zero-point radiation in a Casimir cavity.
The following five noble gases are potentially suitable:                He (Z=2, r=1.2 A)        Ne (Z=10, r=1.3 A),        Ar (Z=18, r=1.6 A)        Kr (Z=36, r=1.8 A)        Xe (Z=54, r=2.05 A).
All of these elements contain ns electrons. He has two 1s electrons. Ne has two each of 1s and 2s electrons. Ar has two each of 1s, 2s, and 3s electrons. Kr has two of each of 1s, 2s, 3s, and 4s electrons. Xe has two of each of 1s, 2s, 3s, 4s and 5s electrons.
Assuming an outermost electron which is completely shielded by the other electrons (a crude assumption), its orbital velocity would scale as r−1/2 (the familiar Keplerian period squared proportional to semi-major axis cubed relationship) and thus λ proportional to r/v) will scale as r3/2. If that is the case, then the larger radii translate as r3/2 into larger Casimir cavities having an effect on the energetics of the outer electron shells. We would therefore expect that a Casimir cavity having d=0.1 microns (or perhaps even as large as one micron would have an effect on reducing the energy levels of the outermost pair of s electrons . . . and possibly also p electrons and intermediate shell s electrons as well.
It is reasonable to expect that a 0.1 microns Casimir cavity would result in a release of 1 to 10 eV for each injection of a He, Ne, Ar, Kr or Xe atom into such a cavity.
According to a Jordan Maclay, who has done theoretical Casimir cavity calculations, a long cylindrical cavity results in an inward force on the cavity. In the "exclusion of modes" interpretation of the Casimir force, this implies that a cylindrical cavity of diameter 0.1 micron would yield the desired decay of outer shell electrons and subsequent release of energy.
It is now recognized that an electromagnetic quantum vacuum field is formally necessary for atomic stability in conventional quantum theory (Milonni 1994). In the field of physics known as stochastic electrodynamics, this concept has been shown by theory and simulations to underlie the ground state of the electron in the hydrogen atom. The classical Bohr orbit is determined by a balance of Larmor emission and absorption of energy from the zero-point fluctuations of the electromagnetic quantum vacuum in SED theory. It follows that upon suppression of appropriate zero-point fluctuations the balance will be upset causing the electron to decay to a lower energy level not ordinarily found in nature with a release of energy during this transition. A Casimir cavity of the proper dimensions can accomplish this suppression of zero-point fluctuations. A Casimir cavity refers to any region in which electromagnetic modes are suppressed or restricted. Upon entering such a properly designed Casimir cavity the electron energy level will shift and energy will be released. Upon exiting the Casimir cavity the electron will return to its customary state by absorbing energy from the ambient zero-point fluctuations. This permits an energy extraction cycle to be achieved at the expense of the zero-point fluctuations. Although it has not yet been proven theoretically, a similar balance of Larmor emission and absorption of energy from the zero-point fluctuations must underlie the electron states of all atoms, not just hydrogen, permitting any atom to be used as a catalyst for extraction of zero-point energy (the energy associated with the zero-point fluctuations). An analogous process is also believed to underlie molecular bonds, yielding a similar energy extraction cycle.
The following is a list of patents that deal with related phenomena:
U.S. Pat. No. 5,018,180, Energy conversion using high charge density, Kenneth R. Shoulders. This concerns the production of charge clusters in spark discharges. It is conjectured that the electrostatic repulsion of charges is overcome in charge clusters by a Casimir-like force. This invention does not deal with energy release from atoms in Casimir cavities and is therefore not relevant to the present invention.
U.S. Pat. No. 5,590,031, System for converting electromagnetic radiation energy to electrical energy, Franklin B. Mead and Jack Nachamkin. This invention does not deal with energy release from atoms in Casimir cavities and is therefore not relevant to the present invention.
U.S. Pat. No. 6,477,028, Method and apparatus for energy extraction, Fabrizio Pinto. Proposes to vary one or more of a variety of physical factors that affect the Casimir force, or by altering any of a variety of environmental factors that affect such physical factors and thereby render a Casimir force system as non-conservative. This invention does not deal with energy release from atoms in Casimir cavities and is therefore not relevant to the present invention.
U.S. Pat. No. 6,593,566, Method and apparatus for energy extraction, Fabrizio Pinto. A method and apparatus for accelerating and a decelerating particles based on particle surface interactions. This invention does not deal with energy release from atoms in Casimir cavities and is therefore not relevant to the present invention.
U.S. Pat. No. 6,665,167, Method for energy extraction-I, Fabrizio Pinto. Similar to U.S. Pat. No. 6,477,028. This invention does not deal with energy release from atoms in Casimir cavities and is therefore not relevant to the present invention.