CA2233296A1 - Compound having sequence homology with lipoprotein associated phospholipase a2 (lp-pla2)/paf acetyl hydrolase - Google Patents

Abstract

Polynucleotides which encode the polypeptide of SEQ ID NO 1, which has +/- 40 % homology with Lipoproteine Associated Phospholipase A2 (Lp-PLA2)/platelet Activating Factor acetyl hydrolase (PAF acetyl hydrolase).

Description

COMPOUND HAVING SEQUENCE HOMOLOGY WITH L~ N ASSOClAlED ~ rtl0-LIPASE A2 (1~PLA2yP~F ~CEl~L HYDROL~SE
The present invention relates to the use of inhibi~ors of a polypeptide in the therapy. The present invention also relates to the polypeptide, to polynucleotides encoding the polypeptide, to recombinant host cells transformed with DNA encoding the polypeptide and to the use of the polypeptide in identifying compounds which are potentially useful in therapy.
Lipoprotein Associated Phospholipase A2 (Lp-PLA2) is also known in the art as Platelet Activating Factor Acetyl Hydrolase (PAF acetyl hydrolase). During the conversion of LDL to its oxidised form, Lp-PLA2 is responsible for hydrolysing the sn-2 ester of oxidatively modified phosphatidylcholine to give lyso-phosphatidylcholine and an oxidatively modified fatty acid. Both of these products of Lp-PLA2 action are potent chemoattractants for circulating monocytes. As such, this enzyme is thought to be responsible for the accumulation of cells loaded with cholesterol ester in the arteries, causing the characteristic 'fatty streak' associated with the early stages of atherosclerosis. Inhibition of the Lp-PLA2 enzyme would therefore be expected to stop the build up of this fatty streak (by inhibition of the formation of lysophosphatidylcholine), and so be useful in the treatment of atherosclerosis. Lp-PLA2 inhibitors may also have a general application in any disorder that includes lipid peroxidation in conjunction with the enzyme activity, for example in addition to conditions such as atherosclerosis and diabetes other conditions such as rheumatoid arthritis, stroke, myocardial infarction, reperfusion injury, acute and chronic infl~mm~tion and various neulupsychiatric illnçcces (e.g., schizophrenia, ref. Psychopharmacology Bulletin, 31: 159-165, 1995).
The amino acid and DNA sequence of the enzyme lipoprotein associated Lp-PLA2 is disclosed in W095/00649.
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, the polypeptide of the present invention is a novel lipase. The invention also relates to inhibiting the action of such polypeptides.
In accordance with one aspect of the present invention, there is provided a novel polypeptide which is a lipase having the amino acid sequence given in SEQ ID
NO 1, or a fragments, analogs or derivative thereof. The polypeptide of the present invention is of human origin.
Hereinafter the term polypeptide(s) will be used to refer to the lipase and its fr~gment.c, analogs and derivatives In accordance with another aspect of the present invention, there are provided polynucleotides (DNA or RNA) which encode such polypeptides.

CA 02233296 l998-03-27 In accordance with a preferred aspect of the present invention, there is provided a polynucleotide which encodes for the polypeptide having the amino acid sequence of SEQ ID NO 1.
In particular, the invention provides a polynucleotide having the DNA
sequence given in SEQ ID NO 2.
cDNA molecules showing extentlPd identity sections with the cDNA of SEQ
ID NO 2 have been identified in cDNA libraries from the following tissues: foetal heart, pineal gland, activated T cells, microvascular endothelial cells and secondary breast tumour The polynucleotide of SEQ ID NO 2 was discovered in a cDNA
lo library derived from prostate (benign possible hyperplasia). It is structurally related to the lipase family. It contains an open reading frame encoding a protein of about 393 amino acid residues. The protein exhibits the highest degree of homology to Lp-PLA2 (WO95/00649, WO 95/09912~ with 40% identity and 60% ~imil~rity over a 390 amino acid stretch. Although the overall identity is only 40% the residues i~lentifit-.d for the catalytic triad in Lp-PLA2 (WO 95/09912) are conserved between the two polypeptides implying that they are likely to have a similar biochemicalfunction. The positions of the Ser, Asp,and His, are underlined below. SEQ ID NO1, is the lower seqence and Lp-PLA2 the upper sequence. Vertical lines in~ at~P
i~lenti~ residues.
238 DIDHGKPVKNAIRLKFDMEQLKDSIDREKIAVIGH~FGGATVIQTLSEDQ 287 ::. 1..1 1 :. :1: 11:.11..::11:11111111.1 .1..:

288 RFRCGIAL~AWMFPLGDEVYSRIPQPLFFINSEYFQYPANIIKMKKCYSP 337 .111::111111111: :.1.: ..1:1111.1 11 ..: 111.:..

338 DKERKMITIRGSV~QNFADFTFATGKIIGHML.. KLKGDIDSNAAIDLSN 385 ..:.::11: 1111.. .11.1.I1.:11.:: . :1.:1. .: ::
302 HEQSRIITVLGSV~RSQTDFAFVTGNLIGKFFSTETRGSLDPYEGQEVMV 351 Tissue sources of this enzyme suggest a role in the biology of the vasculature as well as certain c~n~prc As such, an inhibitor of this polypeptide could find utility in disease states such as atherosclerosis, hypertension, endothelial dysfunction, myocardial infarction, reperfusion injury, and certain cancers. In addition. thehomology to Lp-PLA2 suggests that this novel enzyme could play similar roles and as such inhibitors may find utility in atherosclerosis, myocardial infarction, reperfusion 40 injury, acute and chronic in~l~mm~tion, rheumatoid arthritis, stroke, diabetes and neulop~ychiatric illne.cses The polynucleotide of the present invention may be in the form of RNA or in the form of DNA, 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 polypeptide may be identical to the coding sequence shown in SEQ ID
NO 1 or may be a different coding sequence which, as a result of the rednn-l~n< y or degeneracy of the genetic code, encodes the same polypeptide as the DNA of SEQ ID
s NO 1.
The present invention includes variants of the hereinabove described polynucleotides which encode fragmentc, analogs and derivatives of the polypeptide having the amino acid sequence of SEQ ID NO 1. The variant of the polynucleotidemay be a naturally occurring allelic variant of the polynucleotide or a non-naturally occurring variant of the polynucleotide.
Thus, the present invention includes polynucleotides encoding the same polypeptide as shown in SEQ ID NO 1 as well as variants of such polynucleotides which variants encode for a fragment, derivative or analog of the polypeptide. Such nucleotide variants include deletion variants, substitution variants and addition or insertion variants.
The polynucleotide may have a coding sequence which is a naturally occurring allelic variant of the coding sequence of SEQ ID NO 2. As known in theart, 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 subst~n~i~lly alter the function of the encoded polypeptide.
The polynucleotide which encodes for the polypeptide of SEQ ID NO 1 may include: only the coding sequence for the polypeptide; the coding sequence for the polypeptide and additional coding sequence such as a leader or secretory sequence or a proprotein sequence; the coding sequence for the polypeptide (and optionally 2s additional coding sequence) and non-coding sequence, such as introns or non-coding sequence 5' and/or 3' of the coding sequence for the mature polypeptide.
Thus, the term "polynucleotide encoding a polypeptide" encomp~cses a polynucleotide which includes only coding sequence for the polypeptide as well as a polynucleotide which jnchl(les additional coding and/or non-coding sequence.
The present invention therefore includes polynucleotides, wherein the coding sequence for the 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 leader sequence which functions as a secretory sequence for controlling transport of a polypeptide from the cell. The polypeptide having a 3s leader sequence is a preprotein and may have the leader sequence cleaved by the host cell to form the mature form of the polypeptide. The polynucleotides may also ~ encode for a proprotein which is the mature protein plus additional ~' amino acid residues. A mature protein having a prosequence is a proprotein and is an inactive WO 97~1~984 PCT/GB95/02320 form of the protein. Once the prosequence is cleaved an active mature protein remains.
Tnus, 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 s both a prosequence and a presequence (leader sequen-~e).
The polynucleotides of the present invention may also have the coding sequence fused in frame to a marker sequence which allows for pllrifi~ti~n of the polypeptide of the present invention. The marker sequence may be a hexa-hi~ti~1ine tag supplied by a pQE-9 vector to provide for purification of the mature polypeptide 10 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 m~mm~ n host, e.g. COS-7 cells, is used. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984)).
The present invention further relates to polynucleotides which hybridize to 5 the hereinabove-described sequences if there is at least 50% and preferably 70%
identity between the sequences. The present invention particularly relates to polynucleotides which hybridize under stringent conditions to the hereinabove-described polynucleotides . As herein used, the term "stringent conditions" means hybridization will occur only if there is at least 95% and preferably at least 97%
20 identity between the sequences. The polynucleotides which hybridize to the hereinabove described polynucleotides in a preferred embodiment encode polypeptides which retain substantially the same biological function or activity as the polypeptide of SEQ ID NO 1.
The terrns "fragment," "derivative" and "analog" when referring to the 2s polypeptide of SEQ ID NO l, means a polypeptide which retains ecse~ti~lly the same biological function or activity as such polypeptide. Thus, an analog includes a proprotein which can be activated by cleavage of the proprotein portion to produce an active mature polypeptide.
The polypeptide of the present invention may be a recombinant polypeptide, 30 a natural polypeptide or a synthetic polypeptide, preferably a recombinant polypeptide.
The fragment, derivative or analog of the polypeptide of SEQ ID NO 1 may be ~i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid35 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 ~eemed to be within the scope s 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 "isolated" means that the material is removed from its ori~:in~l environment (e.g., the natural environment if it is naturally occurring). For eY~mp~
o a naturally-occurring polynucleotide or polypeptide present in a living animal is not tPd, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment. The polypeptide is preferably in purified form. By purifiedform is meant at least 80%, more preferably 90%, still more preferably 95% and most preferably 99% pure with respect to other protein cont~min~ntc The DNA of the present invention also makes possible the development by homologous recombination or "knockout" stategies (Kapecchi, Science, 244,:1288-1292 (1989) of ~nim~ll.c that fail to express, or express a variant form of this enzyme The present invention also relates to vectors which include polynucleotides of the present invention, host cells which are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinanttechniques.
2s In accordance with yet a further aspect of the present invention, there is therefore provided a process for producing the polypeptide of the invention by recombinant techniques by expresssing a polynucleotide encoding said polypeptide in a host and recovering the expressed product. Alternatively, the polypeptides of the invention can be synthetically produced by conventional peptide synthPsi7Prs.
Host cells are genetically engineered (tr~n~duced or transformed or transfected) with the vectors of this invention which may be, for example, a cloning vector or an expression vector. The vector may be, for example, in the form of aplasmid, a cosmid, a phage, etc. The engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the genes. The culture conditions, such astemperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.

CA 02233296 l998-03-27 Suitable expression vectors include chromosomal, nonchromosomal and synthedc DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA;
baculovirus; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, and s pseudorabies. However, any other vector may be used as long as it is replicable and viable in the host.
The appropriate DNA sequence may be inserted into the vector by a variety of procedures. In general, the DNA sequence is inserted into an applo~ te restrictiQn endonuclease site(s) by procedures known in the art. Such procedures and 10 others are deemed to be within the scope of those skilled in the art.
The DNA sequence in the expression vector is operatively linked to an applopriate 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. Iac or trp, the phage lambda PL promoter and other promoters 15 known to control expression of genes in prokaryotic or eukaryotic cells or their viruses. The expression vector also contains a ribosome binding site for tr~ncl~tion initiation and a transcription terminator. The vector may also include applo~liate sequences for amplifying expression.
In addition, the expression vectors preferably contain one or more selectable 20 marker genes 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 gene can be placed under the control of a promoter, ribosome binding site (for bacterial expression) and, optionally, an operator (collectively referred to 25 herein as "control" elements), so that the DNA sequence encoding the desired protein is tr~nccrihed into RNA in the host cell transformed by a vector cor t~ining this e,~lession construction. The coding sequence may or may not contain a signal peptide or leader sequence. The protein sequences of the present invention can be expressed using, for example, the E. col~ tac promoter or the protein A gene (spa) 30 promoter and signal sequence. Leader sequences can be removed by the bacterial host in post-tr~nclslti~nal processing. 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. Particular named bacterial promoters include lacI, lacZ, T3, T7, gpt, lambda PR, PL and trp.
35 Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, earlyand late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the approp~iate vector and promoter is well within the level of ordinary skill in the art.

In addition to control sequences, it may be desirable to add regulatory sequences which allow for regulation of the expression of the prooein sequences relative to the growth of the host cell. Regulatory sequences are known to those of skill in the art, and examples include those which cause the expression of a gene to be 5 turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Other types of regulatory elements may also be present in the vector, for example, enhancer sequences.
An expression vector is constructed so that the particular coding sequence is located in the vector with the appropriate regulatory sequences, the positioning and 10 orientation of the coding sequence with respect to the control sequences being such that the coding sequence is transcribed under the "control" of the control sequences (i.e., RNA polymerase which binds to the DNA molecule at the control sequences t~n~crihes the coding sequence). Mo-iifiç~tion of the coding sequences may be desirable to achieve this end. For example, in some cases it may be necessslry to lS modify the sequence so that it may be attached to the control sequences with the appropriate orientation; i.e., to maint~in the reading frame. The control sequences and other regulatory sequences may be ligated to the coding sequence prior to insertion into a vector, such as the cloning vectors described above. ~ltPrnatively, the coding sequence can be cloned directly into an expression vector which already 20 contains the control sequences and an appropriate restriction site. Morlific~tion of the coding sequences may also be performed to alter codon usage to suit the chosen host cell, for enh~nce-l expression.
Generally, recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin 2s resictanre gene of E. coli and S. cerevisiae TRP1 gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence.
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 tr~n~l~tPd protein into the periplasmic space or 30 extracellular medium. Optionally, the heterologous sequence can encode a fusion protein including an N-terminal identificS~tion peptide imparting desired chs~racter-istics, e.g., stabilization or simplified purification of expressed recombinant product.
The vector containing the appropriate DNA sequence as hereinabove described, as well as an appropriate promoter or control sequence, may be employed 3s to transform an appropriate host to permit the host to express the protein.
Examples of recombinant DNA vectors for cloning and host cells which they can transform include the bacteriophage ~ (E. coli), pBR322 (E. coli), pACYC177 (E. coli), pKT230 (gram-negative bacteria), pGVl 106 (gram-negative bacteria), CA 02233296 l998-03-27 W O 97/1298~ PCT/GB95/02320 pLAFRl (gram-negative bacteria), pME290 (non-E. coli grarn-negative bacteria), pHV14 (E. coli and Bacillus subtilis), pBD9 (Rnci~ ), pIJ61 (Streptomyces), pUC6(Streptom~ces), YIp5 (Saccharomyces), a baculovirus insect cell system,, YCpl9 (Saccharomyces). See, generally, "DNA Cloning": Vols. I & II, Glover et al. ed.
s IRL Press Oxford (1985) (1987) and; T. Maniatis et al. ("Molecular Cloning" Cold Spring Harbor Laboratory (1982).
In some cases, it may be desirable to add sequences which cause the secretion of the polypeptide from the host org~ni~m, with subsequent cleavage of the secretory signal.
Yeast e,~ ession vectors are also known in the art. See, e.g., U.S. Patent Nos. 4,446,235; 4,443,539; 4,430,428; see also European Patent Applir~tion.~
103,409; 100,561; 96,491. pSV2neo (as described in J. Mol. Appl. Genet. 1:327-341) which uses the SV40 late promoter to drive ~x},l~:ssion in m~mm~ n cells or pCDNAlneo, a vector derived from pCDNAl(Mol. Cell Biol. 7:4125-29) which uses 1S the CMV promoter to drive expression. Both these latter two vectors can be employed for transient or stable(using G418 resistance) expression in m~mm~ n cells. Insect cell ~pl~;ssion systems, e.g., Drosophila, are also useful, see for example, PCT applications WO 90/06358 and WO 92/06212 as well as EP 290,261-Bl.
Polypeptides can be expressed in host 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 constructs of the present invention.
Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook, et al., Molecular Cloning: A Laboratory Manual,2s Second Edition, Cold Spring Harbor, N.Y., (1989), the disclosure of which is hereby incorporated by reference.
Transcription of the 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 10 to 300 bp that act on a promoter to increase its transcription. Examples including the SV40 çlnhs~nrer on the late side of the replication origin bp 100 to 270, a cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirls enh~ncer~
In a further aspect, the present invention relates to host cells con~ining the above-described vectors. The host cell can be a higher eukaryotic cell, such as a m~mm~ n cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. As representative examples of appropriate hosts, there may be mentioned: prokaryotes for example bacterial cells, such as E.

coli, Streptomyces, Salmonella typhimllrium and eukaryotes for example fungal cells, such as yeast, insect cells such as Drosophila and Spodoptera frugiperda, m~mm~ n cells such as CHO, COS or Bowes melanoma, plant cells, etc. The selection of an appropriate host is deemed to be within the scope of those skilled in the art from the tPachingc herein.
Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-Dextran m~ rPcl transfection, or electroporation.
(Davis, L., Dibner, M., Battey, I., Basic ~ethods in Molecular Biology, (1986)).Following transformation of a suitable host strain and growth of the host lo strain to an appropriate cell density, the selected promoter is induced by appropriate means (e.g., temperature shift or chPmic~l induction) and cells are cultured for an additional period.
Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for funher purification.
Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents, such methods are well know to those skilled in the an.
Various m~mm~ n cell culture systems can also be employed to express recombinant protein. Examples of m~mm;~ n expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Glll7m~n, 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. M~mm~ n expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any n~.ce ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking nontranscribed sequences.DNA sequences derived from the SV40 splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.
Depending on the expression system and host selected, the polypeptide of the present invention may be produced by growing host cells transformed by an expression vector described above under conditions whereby the polypeptide of interest is expressed. The polypeptide is then isolated from the host cells and purified. If the expression system secretes the polypeptide into growth media, the polypeptide can be purified directly from the media. If the polypeptide is not secreted, it is isolated from cell lysates or recovered from the cell membrane fraction.
Where the polypeptide is localized to the cell surface, whole cells or isolated membranes can be used as an assayable source of the desired gene product.
Polypeptide expressed in bacterial hosts such as E. coli may require isolation from g ~ - ~

inclusion bodies and refolding. The selection of the appropriate growth conditions and recovery methods are within the skill of the art.
The polypeptide can be recovered and purified from recombin~nt cell cultures by methods including ammonium sulfate or ethanol precipitation, acid s extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography hydroxylapatite chromatography and lectin chromatography. Protein refolding steps can be used, as necess~ry~ in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final pllri~ tion steps.
Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. Polypeptides of the invention may also include an initial methionine amino acid residue.
The polypeptide of the present invention is also useful for identifying other molecules which have similar biological activity. An example of a screen for this is isolating the coding region of the lipase gene by using the known DNA sequence to synthPci7f~ an oligonucleotide probe or as a probe itself. Labeled oligonucleotides having a sequence complementary to that of the gene of the present invention areused to screen a library of human cDNA, genomic DNA or mRNA to determine which members of the library the probe hybridizes to.
The polypeptides may also be employed in accordance with the present invention by expression of such polypeptides in vivo, which is often referred to as "gene therapy."
Thus, for example, cells from a patient may be çngin~ered with a polynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with the engineered cells then being provided to a patient to be treated with the polypeptide. Such methods are well-known in the art. For ex~mplç, cells may be engineered by procedures known in the art by use of a retroviral particle cont~ining RNA encoding a polypeptide of the present invention.
Similarly, cells may be engineered in vivo for expression of a polypeptide in vivo by, for example, procedures known in the art. As known in the art, a producer cell for producing a retroviral particle cont~ining RNA encoding the polypeptide of the present invention may be ~dministered to a patient for engineering cells in vivo and expression of the polypeptide i~ vivo. These and other methods for ,~riminict~ring a polypeptide of the present invention by such method should be apparent to those skilled in the art from the teachings of the present invention. For example, thee~plession vehicle for engineering cells may be other than a retrovirus, for exarnple, CA 02233296 l99X-03-27 an adenovirus which may be used to engineer cells in vivo after combination with a suitable delivery vehicle.
"Recombinant" polypeptides refer to polypeptides produced by recombinant DNA techniques; i.e., produced from cells transformed by an exogenous DNA
5 construct encoding the desired polypeptide. "Synthetic" polypeptides are those prepared by chemi~l synthesis.
A "replicon" is any genetic element (e.g., plasmid, chromosome, virus) that fun-~tion.c as an autonomous unit of DNA replication in vivo; i.e., capable of replication under its own control.
A "vector" is a replicon, such as a plasmid, phage, or cosmid, to which another DNA segment may be attached so as to bring about the replication of the ~,tt~h.~d segment.
A "double-stranded DNA molecule" refers to the polymeric form of deoxyribonucleotides (bases ~deninç. guanine, thymine, or cytosine) in a double-stranded helix, both relaxed and supercoiled. This term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linearDNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes. In (liscucsing the structure of particular double-stranded DNA molecules, sequences may be described herein according to the norrnal convention of giving only the sequence in the 5' to 3' direction along the sense strand of DNA.
A DNA "coding sequence of" or a "nucleotide sequence encoding" a particular protein, is a DNA sequence which is transcribed and tr~lnsl~tf d into a polypeptide when placed under the control of appropriate regulatory sequences.
A "promoter sequence" is a DNA regulatory region capable of binding RNA
polymerase in a cell and initi~ting transcription of a downstream (3' direction) coding sequence. Within the promoter sequence will be found a transcription initiation site (conveniently defined by mapping with nuclease S1), as well as protein binding dom~inc (consensus sequences) responsible for the binding of RNA polymerase.
Eukaryotic promoters will often, but not always, contain "TATA" boxes and "CAT"
boxes.
DNA "control sequences" refers collectively to promoter sequences, ribosome binding sites, polyadenylation signals, transcription termination sequences, upstream regulatory dom~ins, enhancers, and the like, which collectively provide for the expression (i.e., the transcription and translation) of a coding sequence in a host cell.
A control sequence "directs the expression" of a coding sequence in a cell when RNA polymerase will bind the promoter sequence and transcribe the coding WO 97/12984 PCT/GB9JJ~2320 sequence into mRNA, which is then translated into the polypeptide encoded by thecoding sequence.
A "host cell" is a cell which has been transformed or ~n.~fectetl, or is capable of transformation or transfection by an exogenous DNA sequence.
A cell has been "transformed" by exogenous DNA when such exogenous DNA has been introduced inside the cell membrane. Exogenous DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell. In prokaryotes and yeasts, for example, the exogenous DNA may be m~int~inPd on an episomal element, such as a pl~cmi~l With respect to eukaryoticlo cells, a stably transformed or transfected cell is one in which the exogenous DNA has become integrated into the chromosome so that it is inherit~od by ~ hter cells through chromosome replication. This stability is demon~tr~qtf~d by the ability of the eukaryodc cell to establish cell lines or clones comprised of a population of d~ughter cell co~t~ining the exogenous DNA.
A "clone" is a population of cells derived from a single cell or common ancestor by mitocic. A "cell line" is a clone of a primary cell that is capable of stable growth in vitro for many generations.
Two DNA or polypeptide sequences are "subst~nti~lly homologous" or "substantially the same" when at least about 85% (preferably at least about 90%, and most preferably at least about 95%) of the nucleotides or amino acids match over a deflned length of the molecule and includes allelic variations. As used herein, substantially homologous also refers to sequences showing identity to the specified DNA or polypeptide sequence. DNA sequences that are subst~nti~lly homologous can be i-lentified in a Southern hybri~ tion e~pe~ ent under, for example, stringent 2s conditions, as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., "Current Protocols in Mol. Bio~."
Vol. 1 & II, Wiley In~erscience. Ausbel et al. (ed.) (19g2). Protein sequences that are subst~nti~lly the same can be i(lentified by proteolytic digestion, gel electrophoresis and microsequencing.
The term "functionally equivalent" intends that the amino acid sequence of the subject protein is one that will exhibit enzymadc actdvity of the same kind as that of the lipase.
A "heterologous" region of a DNA construct is an identifi~ble segment of DNA within or attached to another DNA molecule that is not found in association -with the other molecule in nature.
The polypeptides and polynucleotides of the present invendon may be employed in combination with a suitable pharrn~l~eutic~l carrier. Such composidons comprise a therapeutically effective amount of the actdve agent, and a -CA 02233296 l99X-03-27 ph~rm~ceutically acceptable carrier or excipient. Such a carrier includes but is not limited to saline. buffered saline, dextrose, water, glycerol, ethanol. and combinations thereof. The formulation should suit the mode of ~-iminictration.
The invention also provides a pharmaceutical pack or kit comprising one or s more cont~in-ors filled with one or more of the ingredients of the ph~rm~eutical compositions of the invention. Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the m~nllf~chlre~ use or sale of ph~rm~ceuticals or biological products, which notice reflects approval by theagency of m~nuf~eture, use or sale for human ?l-lminictration. In addition, the polypeptides of the present invention may be employed in conjunction with other therapeutic compounds.
The pharmaceutical compositions may be ~dminictered in a convenient manner such as by the oral, topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal routes. The polypeptides or polynucleotides of the present invention is ~minictoted in an amount which is effective for treatment and/or prophylaxis of the specific indication. The amounts and dosage regimens of active agent ~rlminictered to a subject will depend on a number of factors such as the mode of ~rlmini~ctration~ the nature of the condition being treated and the judgment of the prescribing physician.
The sequences of the present invention are also valuable for chromosome idPntifir~tion The sequence is specifically targeted to and can hybridi~ with a particular location on an individual human chromosome. Moreover, there is a current need for identifying particular sites on the chromosome. Chromosome marking reagents based on actual sequence data (repeat polymorphisms) are presently available for m~rking 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 disease.
Briefly, sequences can be mapped to chromosomes by preparing PCR
primers (preferably 15-25 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 &agment.
3s PCR mapping of somatic cell hybrids is a rapid procedure for :~c~igning 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.
s Fluorescence in situ hybrifli~tion (FISH) of a cDNA clones 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. F~SHrequires use of the clones 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 nece.cs~ry 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 position of the sequence on the chromosome can be correlated with genetic map data. Such data are found, for example, in V. McKusick, Mendelian Tnh~rit~nce in Man (available on line through Johns Hopkins University Welch Medical Library).
The relationship between genes and ~iice~çs that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheritance of physically adjacent genes).
Next, it is necess~ry 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 2s 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 from chromosome spreads or detectable using PCR
based on that cDNA sequence. Ultimately, complete sequencing of genes from several individuals is required to confrm the presence of a mutation and to distinguish mutations from polymorphisms.
The polypeptides of the invention or cells expressing them can be used as an immunogen to produce antibodies thereto. These antibodies can be, for example, polyclonal or monoclonal antibodies. The present invention also includes chimeric, single chain, and hum~ni7Pd antibodies, as well as Fab fr~gment.c, or the product of an Fab expression library. Various procedures known in the art may be used for the production of such antibodies and fr~g,m.ont.c.
Antibodies generated against the polypeptides of the present invention can be s obtained by direct injection of the polypeptides into an animal or by ~rlminictering the polypeptides to an animal, preferably a nnnhllm~n The antibody so obtained will then bind the polypeptides itself. In this manner, even a sequence encoding only a fragment of the polypeptides can be used to generate antibodies binding the whole native polypeptides. Such antibodies 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., 1983, Immunology Today 4:72), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole, et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
Techniques described for the production of single chain antibodies (U.S.
Patent 4,946,778) can be adapted to produce single chain antibodies to immunogenic polypeptide products of this invention.
This invention further provides a method of screening compounds to identify those compounds which inhibit the polypeptide comprising contacting isolated polypeptide with a test compound and measuring the rate of turnover of an enzymesubstrate as compared with the rate of turnover in the absence of test compound. The 2s invention also relates to compounds identified thereby.
This invention also provides transgenic non-human ~nim~lc comprising a polynucleotide encoding a polypeptide of the invention. Also provided are methods for use of said transgenic ~nim~lc as models for mutation and SAR (structure/activity relationship) evaluation as well as in drug screens.
The present invention is also directed to inhibitor molecules of the polypeptides of the present invention, and their use in reducing or elimin~ting the function of the polypeptide.
An example of an inhibitor is an antibody or in some cases, an oligonucleotide which binds to the polypeptide.
3s An example of an inhibitor is an antisense construct prepared using ~nticence technology. Antisense technology can be used to control gene expression through triple-helix formation or ~ntisence DNA or RNA, both of which methods are based on binding of a polynucleotide to DNA or RNA. For example, the 5' coding portion of the polynucleotide sequence, which encodes for the polypeptides of the present invention, is used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length. A DNA oligonucleotide is designed to be complPm~nt~ry to aregion of the gene involved in transcription (triple helix -see Lee et al., Nucl. Acids s 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 polypeptide. The ~nticçnce RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into the polypeptide (Okano, J.
Neurochem., 56:~60 (1991); Oligodeoxynucleotides as .AnticenSe Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988)). The oligonucleotides described above can also be delivered to cells such that the ~ntic.once RNA or DNA may be expressed in vivo to inhibit production of polypeptide.
Another example of an inhibitor is a small molecule which binds to and occupies the catalytic site of the polypeptide thereby making the catalytic sitein:~c-ocsjhle to substrate such that normal biological activity is prevented. Examples of small molecules include but are not limited to small peptides or peptide-likemolecules.
When used in therapy, the inhibitors of the invention are formulated in accordance with standard pharmaceutical practice.
The inhibitors which are active when given orally can be formulated as liquids, for example syrups, suspçncions or emulsions, tablets, capsules and, lozenges.
A liquid formulation will generally consist of a suspension or solution of the compound or pharmaceutically acceptable salt in a suitable liquid carrier(s) forexample, ethanol, glycerine, non-aqueous solvent, for example polyethylene glycol, 2s oils, or water with a suspending agent, preservative, flavouring or colouring agent.
A composition in the form of a tablet can be prepared using any suitable pharmaceutical carrier(s) routinely used for preparing solid formulations. Examples of such carriers include m~"nesillm stearate, starch, lactose, sucrose and cellulose.
A composition in the form of a capsule can be prepared using routine çnc~ps~ tion procedures. For example, pellets containing the active ingredient can be prepared using standard carriers and then filled into a hard gelatin capsule;alternatively, a dispersion or suspension can be prepared using any suitable pharrnaceutical carrier(s), for example aqueous gums, celluloses, silicates or oils and the dispersion or suspension then filled into a soft gelatin capsule.
3s Typical parenteral compositions consist of a solution or suspension of the compound or pharmaceutically acceptable salt in a sterile aqueous carrier or parenterally acceptable oil, for example polyethylene glycol, polyvinyl pyrrolidone, leeithin, arachis oil or sesame oil. Alternatively, the solution ean be lyophilised and then reeonstituted with a suitable solvent just prior to ~lminictration.
A typieal suppository formulation comprises a compound of formula (I) or a ph~rm~eutically acceptable salt thereof whieh is aetive when ~tlminictered in this s way, with a binding and/or lubric~ting agent sueh as polymeric glyeols, gelatins or eoeoa butter or other low melting vegetable or synthetic waxes or fats.
Preferably the composition is in unit dose form such as a tablet or capsule.
Each dosage unit for oral ~dminictration contains preferably from 1 to 250 mg (and for parenteral ~-lminictration contains preferably from 0.1 to 25 mg) of an inhibitor of the invention.
The daily dosage regimen for an adult patient may be, for example, an oral dose of between 1 mg and 500 mg, preferably between 1 mg and 250 mg, or an intravenous, subcutaneous, or intramuscular dose of between 0.1 mg and 100 mg, preferably between 0.1 mg and 25 mg, of the eompound of the formula (I) or a pharrn~eutically acceptable salt thereof calculated as the free base, the compound being ~riminictPred 1 to 4 times per day. Suitably the eompounds will be ~dminict~red for a period of continuous 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 occurring methods and/or terms will be described.
"Plasmids" are design~ttod by a lower case p preceded and/or followed by 2s capital letters and/or numbers. The starting plasmids herein are either commercially available, publicly available 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 skilled artisan.
"Oligonucleotides" refers to either a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands which may be chemieally synthf~ci7Prl Sueh synthetic oligonucleotides have no 5' phosphate and thus will not ligate to another oligonueleotide without adding a phosphate with an ATP in the presence of a kinase. A synthetic oligonucleotide will ligate to a fragment that has 3s not been dephosphorylated.
"Ligation" refers to the process of forming phosphodiester bonds between two double stranded nucleic acid fragments (M~ni~tic, T., et al., Id., p. 146). Unless otherwise provided, ligation may be accomplished using known buffers and , conditions with l0 units to T4 DNA ligase ("ligase") per 0.5 ,ug of approximately equimolar amounts of the DNA fra~ments to be ligated.

E~AMPLE
GENE C~ONING AND EXPRESSION
cDNA Library construction Poly A+ (mRNA) was isolated from human prostate (benign possible hyperplasia) using standard methods (ref Maniatis et al). First strand cDNA was primed using an oligo dT primer. The cDNA library was constructed with the Stratagene ZAP-cDNA
synthesis kit, packaged with Gigpack 11 gold p~ck~ging extract and amplified in XLl-blue MRF bacterial cells. The cDNA inserts were cloned unidirectionally into thevector.
DNA Sequencing The phage clone containing the EST was excised from the ~ Unizap XR
bacteriophage vector into the Bluescript phagemid ~according to the Stratagene manual) for characterisation. The insert of 1823bp was m~n~l~lly sequenced on both strands (using the ~m~or~h~m -USB Sequenase 2.0 DNA sequencing kit) by primer walking (SEQ ID 2). The cDNA has an open reading frame with the potential to code for a polypeptide of 393 amino acids (SEQ ID 1). The predicted MW for the full reading frame is 44143Da.

Sequence Data:

MGVNQSVGFPPVTGPHLVGCGDVMEGQNLQGSFFRLFYPCQKAEETMEQPLWIPRYEYCTGLAEYLQFN
2s KRCAGACCSTWRWDLVACLLAGMAPFKTKDSGYPLIIFSHGLGAFRTLYSAFCMELASRGFVVAVPEPQ
DRSAATTYFCKQAPEENQPTNESLQEEWIPFRR~ K~ HVRNPQVHQRVSECLRVLKILQEVTAGQ
TVFNIFPGGLDLMTLKGNIDMSRVAVMGHSFGGATAILALAKETQFRCAVALDAWMFPLERDFYPKARG
PVFFINTEKFQTMESVNLMKKICAQHEQSRIITVLGSVHRSQTDFAFVTGNLIGKFFSTETRGSLDPYE
GQEVMVRAMLAFLQKHLDLKEDYNQWNNLIEGIGPSLTPGAPHHLSSL

GGCACGAGCT TCTGAGGAAT CAGCTTGACT GGCCAGCAAG TTCAGCTCCG
GCAAGTCATT TGATTCACCC GGTGATGAAA TGGGGGTCAA CCAGTCTGTG
GGCTTTCCAC CTGTCACAGG ACCCCACCTC GTAGGCTGTG GGGATGATGA
TGGAGGGGTC AGAATCTCCA GGGGAGCTTC TTTCGACTCT TCTACCCCTG
CCAAAAGGCA GAGGAGACCA TGGAGCAGCC CCTGTGGATT CCCCGCTATG
AGTACTGCAC TGGCCTGGCC GAGTACCTGC AGTTTAATAA GACGACTGCG
GGGGCTTGCT GTTCAACCTG GCGGTGGGAT CTTGTCGCCT GCCTGTTAGC

TGGAATGGCC CCCTTTAAGC ACMAAAGGAC TcTGGATAAc CCCATTGATC
AGTCTTCTCC CATGGCCTAG GAGCCTTCAG GACTTTGTAT TCAGCCTTCT
GCATGGAGCT GGCCTACACG TGGCTTTGTG ~ll~G~ GC CAGAGCCACA
GGACCGGTCA GCGGCAACCA CCTATTTCTG CAAGCAGGCC CCAGAAGAGA
ACCAGCCCAC CAATGAATCG CTGCAGGAGG AATGGATCCC TTTCCGTCGA
GTTGAGGAAG GGGAGAAGGA ATTTCATGTT CGGAATCCCC AGGTGCATCA
GCCGGGTAAG CGAGTGTTTA CGGGTGTTGA AGATCCTGCA AGAGGTCACT
GCTGGGCAGA CTGTCTTCAA CATCTTTCCT GGTGGCTTGG ATCTGATGAC
TTTGAAGGGC AACATTGACA TGAGCCGTGT GGCTGTGATG GGACATTCAT

TCGTGTGCGG TGGCTCTGGA TGCTTGGATG TTTCCTCTGG AACGTGACTT
TTACCCCAAG GCCCGAGGAC ~'1~'1'~'1"1'~'1"1' TATCAATACT GAGAAATTCC
AGACAATGGA GAGTGTCAAT TTGATGAAGA AGATATGTGC CCAGCATGAA
CAGTCTAGGA TCATAACCGT TCTTGGTTCT GTTCATCGGA GTCAAACTGA
CTTTGCTTTT GTGACTGGCA ACTTGATTGG TAAATTCTTC TCCACTGAAA
CCCGTGGGAG CCTGGACCCC TATGAAGGGC AGGAGGTTAT GGTACGGGCC
ATGTTGGCCT TCCTGCAGAA GCACCTCGAC CTGAAAGAAG ACTATAATCA
ATGGAACAAC CTTATTGAAG GCATTGGACC GTCGCTCACC CCAGGGGCCC
CCCACCCATC TGTCCAGCCT GTAGGCGACA ACTGGCTCAT TTGTAAAGTC
ACTTCAGCCA AGCTTTTCAT TTGGGAGCTA CCCAAGGGCA CCCATGAGCT
CCTATCAAGA AGTGATCAAC GTGACCCCTT TTCACAGATT GAAAGGTGTA
ATCACACTGC TGCTTGGATA ACTGGGTACT TTGATCTTAG ATTTGATCTT
AAAATCACTT TGGGACTGGG ATCCCTTGCT GATTGACAAA CAGACTTTCT
GGGACCTTGA TGGAGTGGGG AACAAGCAGT AGAGTGGGAC TGGGGGAGAC
CCAGGCCCCG GGCTGAGCAC TGTGAGGCCT GGATGTGAAG ACTCAMCCCA
CGAACGCTCA TTCCCTTACC CCCGGCCAGT GCTGCTGCTT CAGTGGAAGA
GATGAAGCCA AAGGTAACAG AATGAAAAAT CCCTACCTTC AGAGACTCTA
GCCCAGCCCA ACACCATCTC TTCCTACCTC TCAGCCTTCT CCCTCCCCAG

GGCCACTTGT TGAGAAGTCT GAGCACTTTA TGTAAATTTC TAGGTGTGAG
CCGTGAAAAA AAAAAAAAAA AAAA

M is defined as either A or C, where the actual base is unclear from either DNA
strand.

Claims (22)

1. A polynucleotide encoding the polypeptide having the amino acid sequenceof SEQ ID NO 1 or a fragment, analog or derivative of said polypeptide.

2. The polynucleotide of claim 1 wherein the polynucleotide is DNA.

3. The polynucleotide of claim 1 wherein the polynucleotide is RNA.

4. The polynucleotide of claim 2 wherein the polynucleotide is genomic DNA.

5. The polynucleotide of any preceding claim wherein said polynucleotide encodes the polypeptide having the amino acid sequence of SEQ ID NO 1.

6. The polynucleotide of claim 2 having the DNA sequence as shown in SEQ ID NO 2.

7. The polynucleotide of any preceding claim in isolated form.

8. A vector containing the DNA of any one of claims 2, 4, 5, 6, or 7.

9. A host cell genetically engineered with the vector of claim 8.

10. A process for producing a polypeptide comprising: expressing from the host cell of claim 9 the polypeptide encoded by said DNA.

11. A process for producing cells capable of expressing a polypeptide comprising genetically engineering cells with the vector of claim 8.

12. A polynucleotide hybridizable to the polynucleotide of any one of claims 1 to 7 and encoding a polypeptide having substantially the same biological function or activity as the polypeptide of SEQ ID NO 1.

13. A polypeptide having the amino acid sequence of SEQ ID NO 1 and fragments, analogs and derivatives thereof.

14. The polypeptide of Claim 13 wherein the polypeptide has the amino acid sequence of SEQ ID NO 1.

15. The polypeptide of claim 13 or 14 in isolated form.

16. A method of screening compounds to identify those compounds which inhibit the polypeptide of claim 13 or 14 comprising contacting isolated polypeptide with a test compound and measuring the rate of turnover of an enzyme substrate as compared with the rate of turnover in the absence of test compound.

17. A compound identified by the method of claim 15.

18. An inhibitor of the polypeptide of claim 13 or 14.

19. An inhibitor according to claim 17 which is an antibody to the polypeptide of claim 13 or 14.

20. A pharmaceutical composition comprising the polynucleotide of claim 1 or 12, a polypeptide of claim 13, a compound of claim 16 or an inhibitor of claim 17 and a pharmaceutically acceptable carrier.

21. - A method for the treatment of a patient having need to inhibit the polypeptide of claim 13 or 14 comprising: administering to the patient a therapeutically effective amount of the compound of claim 16 or inhibitor of claim

22. The use of a compound of claim 16 or inhibitor of claim 17 for the manufacture of a medicament for use in therapy.