WO2002029023A1

WO2002029023A1 - Nicastrin protein

Info

Publication number: WO2002029023A1
Application number: PCT/GB2001/004390
Authority: WO
Inventors: Richard Joseph Fagan; John Paul Overington; Mark Basil Swindells; Malcolm Weir
Original assignee: Inpharmatica Limited
Priority date: 2000-10-02
Filing date: 2001-10-02
Publication date: 2002-04-11
Also published as: WO2002029023A8; AU2001292059A1; GB0024086D0

Abstract

The invention relates to a protein termed Nicastrin, herein idenfified as an aminopeptidase and to the use of this protein and nucleic acis seqences from the encoding gene in the diagnosis, prevention and treatment of disease.

Description

Nicastrin protein

This invention relates to a protein termed Nicastrin, herein identified as an aminopeptidase and to the use of this protein and nucleic acid sequences from the encoding gene in the diagnosis, prevention and treatment of disease. All publications, patents and patent applications cited herein are incorporated in full by reference.

BACKGROUND

The process of drug discovery is presently undergoing a fundamental revolution as the era of functional genomics comes of age. The term "functional genomics" applies to an approach utilising bioinformatics tools to ascribe function to protein sequences of interest. Such tools are becoming increasingly necessary as the speed of generation of sequence data is rapidly outpacing the ability of research laboratories to assign functions to these protein sequences.

As bioinformatics tools increase in potency and in accuracy, these tools are rapidly replacing the conventional techniques of biochemical characterisation. Indeed, the advanced bioinformatics tools used in identifying the present invention are now capable of outputting results in which a high degree of confidence can be placed.

Various institutions and commercial organisations are examining sequence data as they become available and significant discoveries are being made on an on-going basis. However, there remains a continuing need to identify and characterise further genes and the polypeptides that they encode, as targets for research and for drug discovery.

Recently, a remarkable tool for the evaluation of sequences of unknown function has been developed by the Applicant for the present invention. This tool is a database system, termed the Biopendium™ search database, that is the subject of co-pending United Kingdom Patent Application No. PCT/GBOl/01105. This database system consists of an integrated data resource created using proprietary technology and containing information generated from an all-by-all comparison of all available protein or nucleic acid sequences.

The aim behind the integration of these sequence data from separate data resources is to combine as much data as possible, relating both to the sequences themselves and to information relevant to each sequence, into one integrated resource. All the available data relating to each sequence, including data on the three-dimensional structure of the encoded protein, if this is available, are integrated together to make best use of the information that is known about each sequence and thus to allow the most educated predictions to be made from comparisons of these sequences. The annotation that is generated in the database and which accompanies each sequence entry imparts a biologically-relevant context to the sequence information.

This data resource has made possible the accurate prediction of protein function from sequence alone. Using conventional technology, this is only possible for proteins that exhibit a high degree of sequence homology (above about 20 to 30% homology) to other proteins in the same functional family. Accurate predictions are not possible for proteins that exhibit a very low degree of sequence homology to other related proteins of known function.

Alzheimer's disease is a condition that affects millions of individuals around the world, across all races and ethnic backgrounds. After onset of the disease, life expectancy ranges from only five up to twenty years, and during this period, the patient requires a high degree of supervision and care. The number of sufferers of Alzheimer's disease is expected to expand markedly as the proportion of the aged in the population increases (Plum, 1979). There is presently no treatment that will arrest the progression of this disease. Although clinical trials are on-going involving the use of agents from such diverse pharmaceutical classes such as cerebral vasodilators, CNS stimulants, neuroleptics, nootropics, receptor stimulants, neuropeptides, aminergic enhancers and cholinergic enhancers, the results of these trials have been disappointing.

Alzheimer's disease is accompanied by a characteristic physical and biochemical pathology, notably prominent neuropathologic lesions, such as neurofibrillary tangles (NFTs), neuropil threads (NT) and amyloid-rich senile plaques (SP). These lesions are associated with massive loss of populations of CNS neurones and their development invariably accompanies the clinical dementia that is associated with AD. It is considered that proteolytic processing of proteins plays a key role in the pathogenesis of Alzheimer's disease (De Stooper, B., et al, J Cell Sci (2000), 113; 1857-1870). The brains of patients suffering from Alzheimer's disease are characterised by the progressive deposition of protein fragments in amyloid plaques. Amyloid plaques are deposits of amyloid beta peptides. Amyloid beta is the product of a precursor protein, beta amyloid precursor protein (B-APP). This protein is a transmembrane protein that undergoes several cleavage steps.

The first cleavage occurs extracellularly and is carried out by one of two proteases, alpha- secretase or beta-secretase (De Stooper, B., et al, J Cell Sci (2000), 113; 1857-1870). The identity of the beta-secretase at the molecular level has been identified independently by four groups and is termed BACE (beta-site APP-cleaving enzyme, Hussain, L, et al, Mol Cell Neurosci (1999) 14, 419-427). This protease contains the classical sequence characteristic of an aspartyl protease at its catalytic domain. The identity of the alpha secretase is unknown, but is hypothesised to be a metalloprotease (De Stooper, B., et al, J Cell Sci (2000), 113; 1857-1870).

After the first cleavage reaction, an intramembrane proteolysis occurs, carried out by a gamma secretase. There is growing experimental evidence that the presenilin 1 and 2 proteins provide the function of this gamma secretase (Wolfe, M., et al, Biochemistry (1999) 38; 11223-11230, and Li, Y.M., et al, Nature (2000) 405; 689-694).

There are two isoforms of the amyloid beta peptide generated by the cleavages discussed above. These isoforms differ by two amino acids on the C-terminus. These isoforms result from the different positions of cleavage on the B-APP depending upon whether the alpha or beta secretase carries out the first cleavage reaction. Cleavage of the B-APP with the beta secretase followed by the gamma secretase leads to the production of the amyloid beta peptide that form the amyloid plagues responsible for the pathology of Alzheimer's. Cleavage of B-APP with the alpha-secretase followed by the gamma secretase leads to the production of a non-pathogenic isoform.

Recent published work indicates that there are a number of proteins that reside in a complex with presenilin 1 (Yu, G., et al, Nature (2000) 407; 48-54), the protein thought to function as the gamma secretase responsible for production of the pathogenic form of amyloid beta peptide. The fact that other proteins are found as part of a complex with presenilin 1 implies that they play a role in the processing of the B-APP. One of these molecules has been named Nicastrin (Genebank nucleotide accession number AF240468, GeneBank protein accession number AAG11412). This protein was previously known as KIAA0253, (NCBI Genebank nucleotide accession number D87442 and a Genebank protein accession number BAA13383, SwissProt database accession number, Q92542), a hypothetical protein to which no biological function has been ascribed.

The biological function of Nicastrin is presently unclear. The gene encodes a 709 amino acid protein with a putative amino terminal signal peptide, an amino terminal long hydrophilic domain, a transmembrane domain, and a short hydrophilic carboxy terminus of 20 residues. Nicastrin has been shown to interact with both presenilin 1 and 2, as well as with the B-APP and with the alpha- and beta-cleaved version of B-APP. As Nicastrin is in the presenilin/B-APP complex and directly interacts with both proteins, it has been hypothesised that this protein may play a role in B-APP processing, although no mechanism or function has yet been suggested that explains the role of this protein in the pathology of Alzheimer's disease.

The paper identifying Nicastrin as a component of the presenilin/B-APP complex (Yu et al, 2000) was unable to functionally annotate the gene, despite using the most advanced publicly-available methods. What the paper demonstrates is that the alteration of the ability of Nicastrin to interact with the B-APP alters the ability to form amyloid beta peptide, hence having direct implications on the pathogenesis of Alzheimer's disease.

It is envisaged that direct inhibition of the interaction of Nicastrin and the beta-secretase cleaved version of the B-APP, or inhibition of the interaction of Nicastrin with presenilin, for example, by small molecule intervention, would provide an effective means of treatment for Alzheimer's disease. Indeed, it is quite possible that there will be other proteins in the Nicastrin/presenilin/B-APP complex that Nicastrin interacts with or modulates. Their presence may play a crucial role in the activity of the complex and, as such, modulation of their interaction with Nicastrin would provide another means for treatment of Alzheimer's disease. In view of the prevalence of diseases such as Alzheimer's disease, there remains a great need for agents that are effective in its treatment and diagnosis. The inventors have now characterised the function of the Nicastrin protein, thus paving the way for the identification of agents that are effective in the treatment of Alzheimer's disease and other diseases in which this protein is implicated. THE INVENTION

According to the invention, there is provided a method of treatment of Alzheimer's disease in a patient comprising administering an aminopeptidase inhibitor to the patient. The invention is based on the discovery that the Nicastrin protein functions as an aminopeptidase. Using the Biopendium™ search database, the inventors have discovered that Nicastrin contains a protein fold that is similar to that of the Transferrin receptor/aminopeptidase fold. As a member of the aminopeptidase family, Nicastrin has the ability to bind peptides and to cleave them, provided that certain sequence constraints are met. The inventors consider that Nicastrin directly binds to the B-APP protein and contributes to its proteolytic processing. A motif identified as being important for the Presenilin/Nicastrin interaction, D336-S340, is encompassed within the identified aminopeptidase domain, further implicating it in protein-protein interactions. According to a second aspect of the invention, there is provided an aminopeptidase inhibitor, for use in the treatment or diagnosis of Alzheimer's disease. Preferably, such an aminopeptidase inhibitor is effective to inhibit the aminopeptidase activity of the Nicastrin polypeptide. In this respect, the aminopeptidase inhibitor may interact with the Nicastrin polypeptide. The invention also provides for the use of an aminopeptidase inhibitor in the manufacture of a medicament for the treatment or diagnosis of Alzheimer's disease.

According to a third aspect of the invention, there is provided a polypeptide consisting of the aminopeptidase domain of the Nicastrin polypeptide. The putative aminopeptidase domain of Nicastrin is presented in SEQ ID NO:2, whilst the encoding sequence is given in SEQ ID NO:l. This aminopeptidase domain is considered to comprise residues 206 to 503 of the Nicastrin polypeptide sequence. However, as the skilled reader will appreciate, polypeptides that are extended or truncated versions of this sequence will also be useful in the present invention. Preferably, the aminopeptidase domain of Nicastrin according to the invention comprises residues 206 to 503 of the Nicastrin polypeptide sequence +/- approximately 30 amino acid residues at either one of or at both the N-terminus and C- termini of the peptide, more preferably, +/- 20 amino acid residues, most preferably +/- 10 amino acid residues or less.

In a preferred embodiment of this aspect of the invention, there is provided a polypeptide which: (i) has the amino acid sequence as recited in SEQ ID NO:2; (ii) is a fragment thereof having activity as an aminopeptidase or having an antigenic determinant in common with the polypeptide of (i); or

(iii) is a functional equivalent of (i) or (ii).

According to the invention, a polypeptide which is a functional equivalent according to (iii) above, is homologous to the amino acid sequence as recited in SEQ ID NO:2 and has activity as an aminopeptidase.

Site-directed mutants of the polypeptides of the third aspect of the invention also form a part of the invention. Such mutant proteins, mutated at positions in the sequence that are critical for the aminopeptidase function of the protein, may be useful in order to elucidate the mechanism of action of the protein and in the design of ligand molecules that are effective to alter this activity. Preferred mutant polypeptides according to this aspect of the invention comprise the Nicastrin polypeptide, mutated at one or more of the following positions: Asp 284; Glu 354; Pro 293; Glu 333; His 449. These amino acid residues are implicated herein as important residues in the aminopeptidase domain of the Nicastrin protein.

In a fourth aspect, the invention provides a purified nucleic acid molecule which encodes a polypeptide of the third aspect of the invention. Preferably, the purified nucleic acid molecule has the nucleic acid sequence as recited in SEQ ID NO:l (encoding the Nicastrin aminopeptidase domain) or is a redundant equivalent or fragment of either of these sequences.

Of particular use in generating the aminopeptidase domain by recombinant means is a nucleic acid construct derived from the GeneBank sequence AF240468, SEQ ID NO:3, using the naturally occurring restriction endonucleases Bbsl, which cuts at nucleotide base pair 661, and Aapl which cuts at nucleotide base pair 1959. The skilled reader will appreciate that any other restriction endonuclease sites occurring in the sequence that enable cloning of the aminopeptidase domain as set out in SEQ ID NO: 2 will also be useful in this respect.

In a further embodiment, the invention provides a purified nucleic acid molecule which hydridizes under high stringency conditions with a nucleic acid molecule of the fourth aspect of the invention. In a fifth aspect, the invention provides a vector, such as an expression vector, that contains a nucleic acid molecule according to the fourth aspects of the invention.

In a sixth aspect, the invention provides a host cell transformed with a vector of the fifth aspect of the invention. In a seventh aspect, the invention provides a method of identifying a candidate ligand for the treatment of Alzheimer's disease comprising testing the ability of an aminopeptidase inhibitor to bind to the Nicastrin polypeptide or to a polypeptide according to the third aspect of the invention and selecting as a candidate agent, an aminopeptidase inhibitor that effectively inhibits the biological activity of the polypeptide. According to a preferred form of such a method, a library of aminopeptidase inhibitors may be tested in order to identify a candidate ligand that is effective to bind to the Nicastrin polypeptide or to a polypeptide according to the third aspect of the invention.

Of particular use when screening metalloproteases for small molecule inhibitors are the chemical series containing, but not limited to, the hydroxymates, carboxylates, enolate anion, acidic sulphonamides such as aryl sulphonamide and flurosulphonamide derivatives, and thiol containing compounds such as thiadiazol sulphodiamides.

The teaching of the invention confers a significant advance on the knowledge of the pathogenesis of Alzheimer's disease and of other diseases that are thought to involve the Nicastrin protein, such as CNS disease, inflammation, oncology, or cardiovascular disease. For example, by expression and purification of the polypeptides of the third aspect of the invention, these polypeptides may be used to configure numerous assays and screens. The knowledge that Nicastrin contains an aminopeptidase domain allows appropriate conditions to be taken into account when designing assays and screens involving the Nicastrin protein, such as the inclusion of metal ions, in particular zinc, in the assay buffer. Furthermore, knowledge of the function of the protein allows site directed mutants to be created of the identified critical residues in the aminopeptidase domain.

Preferably, ligands identified according to the method of the seventh aspect of the invention are effective to prevent the activity of the polypeptide as an aminopeptidase. Such ligands may inhibit the interaction of the aminopeptidase domain of Nicastrin with a naturally-occurring peptide, such as the full length B-APP, the beta-secretase cleaved version of the B-APP, the alpha-secretase cleaved version of B-APP, presenilin 1, presenilin 2, or a member of the Notch protein family. In an eighth aspect, the invention provides a ligand identified by a method according to the seventh aspect of the invention. Such ligands will preferably bind specifically to, and more preferably inhibit the aminopeptidase activity of, or the binding of, a naturally-occurring peptide, such as the full length B-APP, the beta-secretase cleaved version of the B-APP, the alpha-secretase cleaved version of B-APP, presenilin 1, presenilin 2, or a member of the Notch protein family, to a polypeptide of the third aspect of the invention.

Ligands according to the eighth aspect of the invention may be natural or modified substrates, ligands, enzymes, receptors or structural or functional mimetics that possess affinity for the aminopeptidase domain of the Nicastrin protein. Such ligands may also be peptide mimetics, small drug molecules, or antibodies. Preferably, ligands according to the eighth aspect of the invention inhibit the aminopeptidase activity of the Nicastrin polypeptide or of a polypeptide according to the third aspect of the invention. This inhibition preferably occurs without inducing any of the biological effects of the polypeptide. According to a ninth aspect of the invention, there is provided a ligand according to the eighth aspect of the invention, for use in the treatment or diagnosis of Alzheimer's disease. The invention also provides for the use of a ligand as described above in the manufacture of a medicament for the treatment or diagnosis of Alzheimer's disease.

According to a tenth aspect of the invention, there is provided a method of diagnosing the susceptibility of a patient to Alzheimer's disease comprising examining the Nicastrin polypeptide or gene sequence in said patient or in tissue from said patient and diagnosing as susceptible those patients in which a mutation is contained in a region of the sequence that is responsible for aminopeptidase activity in the full length protein. A similar diagnostic method involves examining the Nicastrin polypeptide or gene sequence in said patient or in tissue from said patient and comparing said level of expression or activity to a control level, wherein a level that is different to said control level is indicative of disease.

Such a method will preferably be carried out in vitro. Similar methods may be used for monitoring the therapeutic treatment of disease in a patient, wherein altering the level of expression or activity of a polypeptide or nucleic acid molecule over the period of time towards a control level is indicative of regression of disease. Particular diseases in which an aminopeptidase like protein according to the first aspect of the invention is implicated include Alzheimer's disease, CNS, inflammation, oncology, or cardiovascular disease.

A preferred method for detecting Nicastrin polypeptides of the third aspect of the invention comprises the steps of: (a) contacting a ligand, such as an antibody, of the eighth aspect of the invention with a biological sample under conditions suitable for the formation of a ligand-polypeptide complex; and (b) detecting said complex.

A number of different such methods according to the tenth aspect of the invention exist, as the skilled reader will be aware, such as methods of nucleic acid hybridization with short probes, point mutation analysis, polymerase chain reaction (PCR) amplification and methods using antibodies to detect aberrant protein levels. Similar methods may be used on a short or long term basis to allow therapeutic treatment of a disease to be monitored in a patient. The invention also provides kits that are useful in these methods for diagnosing disease. In an eleventh aspect, the invention provides for the use of the Nicastrin polypeptide, or a polypeptide of the third aspect of the invention as an aminopeptidase.

In an twelfth aspect, the invention provides a pharmaceutical composition comprising a polypeptide of the third aspect of the invention, or a nucleic acid molecule of the fourth aspect of the invention, or a vector of the fifth aspect of the invention, or a ligand of the eighth aspect of the invention, in conjunction with a pharmaceutically-acceptable carrier.

In a thirteenth aspect, the present invention provides a polypeptide of the third aspect of the invention, or a nucleic acid molecule of the fourth aspect of the invention, or a vector of the fifth aspect of the invention, or a ligand of the eighth aspect of the invention, for use in the manufacture of a medicament for the diagnosis or treatment of a disease, such as Alzheimer's Disease, inflammation, oncology, or cardiovascular disease.

In a fourteenth aspect, the invention provides a method of treating a disease in a patient comprising administering to the patient a polypeptide of the third aspect of the invention, or a nucleic acid molecule of the fourth aspect of the invention, or a vector of the fifth aspect of the invention, or a ligand of the eighth aspect of the invention. For diseases in which the expression of a natural gene encoding Nicastrin, or in which the aminopeptidase activity of Nicastrin is lower in a diseased patient when compared to the level of expression or activity in a healthy patient, the polypeptide, nucleic acid molecule, ligand or compound administered to the patient should be an agonist. Conversely, for diseases in which the expression of the natural gene or aminopeptidase activity of Nicastrin is higher in a diseased patient when compared to the level of expression or activity in a healthy patient, the polypeptide, nucleic acid molecule, ligand or compound administered to the patient should be an antagonist. Examples of such antagonists include antisense nucleic acid molecules, ribozymes and ligands, such as antibodies.

In a fifteenth aspect, the invention provides transgenic or knockout non-human animals that have been transformed to express higher, lower or absent levels Nicastrin. Such transgenic animals are very useful models for the study of disease and may also be using in screening regimes for the identification of compounds that are effective in the treatment or diagnosis of such a disease.

According to a sixteenth aspect of the invention, there is provided a method for screening for a candidate agent effective to treat disease, by contacting a non-human transgenic animal according to the fifteenth aspect of the invention with a candidate compound and determining the effect of the compound on the disease of the animal.

A summary of standard techniques and procedures which may be employed in order to utilise the invention is given below. It will be understood that this invention is not limited to the particular methodology, protocols, cell lines, vectors and reagents described. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and it is not intended that this terminology should limit the scope of the present invention. The extent of the invention is limited only by the terms of the appended claims.

Standard abbreviations for nucleotides and amino acids are used in this specification. The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA technology and immunology, which are within the skill of the those working in the art.

Such techniques are explained fully in the literature. Examples of particularly suitable texts for consultation include the following: Sambrook Molecular Cloning; A Laboratory Manual, Second Edition (1989); DNA Cloning, Volumes I and II (D.N Glover ed. 1985); Oligonucleotide Synthesis (M.J. Gait ed. 1984); Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins eds. 1984); Transcription and Translation (B.D. Hames & S J. Higgins eds. 1984); Animal Cell Culture (R.I. Freshney ed. 1986); Immobilized Cells and Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide to Molecular Cloning (1984); the Methods in Enzymology series (Academic Press, Inc.), especially volumes 154 & 155; Gene Transfer Vectors for Mammalian Cells (J.H. Miller and M.P. Calos eds. 1987, Cold Spring Harbor Laboratory); Immunochemical Methods in Cell and Molecular Biology (Mayer and Walker, eds. 1987, Academic Press, London); Scopes, (1987) Protein Purification: Principles and Practice, Second Edition (Springer-Verlag, N.Y.); and Handbook of Experimental Immunology, Volumes I-IV (DM.. Weir and C. C. Blackwell eds. 1986).

As used herein, the term "polypeptide" includes any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e. peptide isosteres. This term refers both to short chains (peptides and oligopeptides) and to longer chains (proteins).

The polypeptide of the present invention may be in the form of a mature protein or may be a pre-, pro- or prepro- protein that can be activated by cleavage of the pre-, pro- or prepro- portion to produce an active mature polypeptide. In such polypeptides, the pre-, pro- or prepro- sequence may be a leader or secretory sequence or may be a sequence that is employed for purification of the mature polypeptide sequence.

The polypeptide of the third aspect of the invention may form part of a fusion protein. For example, it is often advantageous to include one or more additional amino acid sequences which may contain secretory or leader sequences, pro-sequences, sequences which aid in purification, or sequences that confer higher protein stability, for example during recombinant production. Alternatively or additionally, the mature polypeptide may be fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol). Polypeptides may contain amino acids other than the 20 gene-encoded amino acids, modified either by natural processes, such as by post-translational processing or by chemical modification techniques which are well known in the art. Among the known modifications which may commonly be present in polypeptides of the present invention are glycosylation, lipid attachment, sulphation, gamma-carboxylation, for instance of glutamic acid residues, hydroxylation and ADP-ribosylation. Other potential modifications include acetylation, acylation, amidation, covalent attachment of flavin, covalent attachment of a haeme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulphide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, GPI anchor formation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.

Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. In fact, blockage of the amino or carboxyl terminus in a polypeptide, or both, by a covalent modification is common in naturally-occurring and synthetic polypeptides and such modifications may be present in polypeptides of the present invention.

The modifications that occur in a polypeptide often will be a function of how the polypeptide is made. For polypeptides that are made recombinantly, the nature and extent of the modifications in large part will be determined by the post-translational modification capacity of the particular host cell and the modification signals that are present in the amino acid sequence of the polypeptide in question. For instance, glycosylation patterns vary between different types of host cell.

The polypeptides of the present invention can be prepared in any suitable manner. Such polypeptides include isolated naturally-occurring polypeptides (for example purified from cell culture), recombinantly-produced polypeptides (including fusion proteins), synthetically-produced polypeptides or polypeptides that are produced by a combination of these methods.

The functionally-equivalent polypeptides of the third aspect of the invention may be polypeptides that are homologous to the Nicastrin polypeptides. Two polypeptides are said to be "homologous", as the term is used herein, if the sequence of one of the polypeptides has a high enough degree of identity or similarity to the sequence of the other polypeptide. "Identity" indicates that at any particular position in the aligned sequences, the amino acid residue is identical between the sequences. "Similarity" indicates that, at any particular position in the aligned sequences, the amino acid residue is of a similar type between the sequences. Degrees of identity and similarity can be readily calculated (Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing. Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). Homologous polypeptides therefore include natural biological variants (for example, allelic variants or geographical variations within the species from which the polypeptides are derived) and mutants (such as mutants containing amino acid substitutions, insertions or deletions) of the Nicastrin polypeptide. Such mutants may include polypeptides 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 acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code. Typical such substitutions are among Ala, Val, Leu and lie; among Ser and Thr; among the acidic residues Asp and Glu; among Asn and Gin; among the basic residues Lys and Arg; or among the aromatic residues Phe and Tyr. Particularly preferred are variants in which several, i.e. between 5 and 10, 1 and 5, 1 and 3, 1 and 2 or just 1 amino acids are substituted, deleted or added in any combination. Especially preferred are silent substitutions, additions and deletions, which do not alter the properties and activities of the protein. Also especially preferred in this regard are conservative substitutions.

Such mutants also include polypeptides in which one or more of the amino acid residues includes a substituent group;

Typically, greater than 30% identity between two polypeptides is considered to be an indication of functional equivalence. Preferably, functionally equivalent polypeptides of the first aspect of the invention have a degree of sequence identity with the Nicastrin polypeptide, or with active fragments thereof, of greater than 30%. More preferred polypeptides have degrees of identity of greater than 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99%, respectively.

The functionally-equivalent polypeptides of the third aspect of the invention may also be polypeptides which have been identified using one or more techniques of structural alignment. For example, the Inpharmatica Genome Threader technology that forms one aspect of the search tools used to generate the Biopendium™ search database may be used (see co-pending International patent application PCT/GBOl/01105) to identify polypeptides of presently-unknown function which, while having low sequence identity as compared to the Nicastrin polypeptide, are predicted to have aminopeptidase activity, by virtue of sharing significant structural homology with the Nicastrin polypeptide sequences. By "significant structural homology" is meant that the Inpharmatica Genome Threader predicts two proteins to share structural homology with a certainty of at least 10% more preferably, at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and above.

The polypeptides of the third aspect of the invention also include fragments of the Nicastrin polypeptide and fragments of the functional equivalents of the Nicastrin polypeptide, provided that those fragments retain zinc aminopeptidase activity or have an antigenic determinant in common with the Nicastrin polypeptide. As used herein, the term "fragment" refers to a polypeptide having an amino acid sequence that is the same as part, but not all, of the Nicastrin polypeptide or one of its functional equivalents. The fragments should comprise at least n consecutive amino acids from the sequence and, depending on the particular sequence, n preferably is 7 or more (for example, 8, 10, 12, 14, 16, 18, 20 or more). Small fragments may form an antigenic determinant.

Such fragments may be "free-standing", i.e. not part of or fused to other amino acids or polypeptides, or they may be comprised within a larger polypeptide of which they form a part or region. When comprised within a larger polypeptide, the fragment of the invention most preferably forms a single continuous region. For instance, certain preferred embodiments relate to a fragment having a pre - and/or pro- polypeptide region fused to the amino terminus of the fragment and/or an additional region fused to the carboxyl terminus of the fragment. However, several fragments may be comprised within a single larger polypeptide.

The polypeptides of the present invention or their immunogenic fragments (comprising at least one antigenic determinant) can be used to generate ligands, such as polyclonal or monoclonal antibodies, that are immunospecific for the polypeptides. Such antibodies may be employed to isolate or to identify clones expressing the polypeptides of the invention or to purify the polypeptides by affinity chromatography. The antibodies may also be employed as diagnostic or therapeutic aids, amongst other applications, as will be apparent to the skilled reader.

The term "immunospecific" means that the antibodies have substantially greater affinity for the polypeptides of the invention than their affinity for other related polypeptides in the prior art. As used herein, the term "antibody" refers to intact molecules as well as to fragments thereof, such as Fab, F(ab')₂ and Fv, which are capable of binding to the antigenic determinant in question. Such antibodies thus bind to the polypeptides of the third aspect of the invention. If polyclonal antibodies are desired, a selected mammal, such as a mouse, rabbit, goat or horse, may be immunised with Nicastrin or with a polypeptide of the third aspect of the invention. The polypeptide used to immunise the animal can be derived by recombinant DNA technology or can be synthesized chemically. If desired, the polypeptide can be conjugated to a carrier protein. Commonly used carriers to which the polypeptides may be chemically coupled include bovine serum albumin, thyroglobulin and keyhole limpet haemocyanin. The coupled polypeptide is then used to immunise the animal. Serum from the immunised animal is collected and treated according to known procedures, for example by immunoaffinity chromatography.

Monoclonal antibodies to the Nicastrin or to polypeptides of the third aspect of the invention can also be readily produced by one skilled in the art. The general methodology for making monoclonal antibodies using hybridoma technology is well known (see, for example, Kohler, G. and Milstein, C, Nature 256: 495-497 (1975); Kozbor et al, Immunology Today 4: 72 (1983); Cole et al, 77-96 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985). Panels of monoclonal antibodies produced against the polypeptides of the third aspect of the invention can be screened for various properties, i.e., for isotype, epitope, affinity, etc. Monoclonal antibodies are particularly useful in purification of the individual polypeptides against which they are directed. Alternatively, genes encoding the monoclonal antibodies of interest may be isolated from hybridomas, for instance by PCR techniques known in the art, and cloned and expressed in appropriate vectors.

Chimeric antibodies, in which non-human variable regions are joined or fused to human constant regions (see, for example, Liu et al, Proc. Natl. Acad. Sci. USA, 84, 3439 (1987)), may also be of use.

The antibody may be modified to make it less immunogenic in an individual, for example by humanisation (see Jones et al, Nature, 321, 522 (1986); Verhoeyen et al, Science, 239,

1534 (1988); Kabat et al, J. Immunol., 147, 1709 (1991); Queen et al, Proc. Natl Acad.

Sci. USA, 86, 10029 (1989); Gorman et al, Proc. Natl Acad. Sci. USA, 88, 34181 (1991); and Hodgson et al., Bio/Technology, 9, 421 (1991)). The term "humanised antibody", as used herein, refers to antibody molecules in which the CDR amino acids and selected other amino acids in the variable domains of the heavy and/or light chains of a non-human donor antibody have been substituted in place of the equivalent amino acids in a human antibody. The humanised antibody thus closely resembles a human antibody but has the binding ability of the donor antibody.

In a further alternative, the antibody may be a "bispecific" antibody, that is an antibody having two different antigen binding domains, each domain being directed against a different epitope. Phage display technology may be utilised to select genes which encode antibodies with binding activities towards the polypeptides of the invention either from repertoires of PCR amplified V-genes of lymphocytes from humans screened for possessing the relevant antibodies, or from naive libraries (McCafferty, J. et al, (1990), Nature 348, 552-554; Marks, J. et al, (1992) Biotechnology 10, 779-783). The affinity of these antibodies can also be improved by chain shuffling (Clackson, T. et al, (1991) Nature 352, 624-628).

Antibodies generated by the above techniques, whether polyclonal or monoclonal, have additional utility in that they may be employed as reagents in immunoassays, radioimmunoassays (RIA) or enzyme-linked immunosorbent assays (ELISA). In these applications, the antibodies can be labelled with an analytically-detectable reagent such as a radioisotope, a fluorescent molecule or an enzyme.

Preferred nucleic acid molecules of the fourth aspect of the invention are those which encode the polypeptide sequences recited in SEQ ID NO:2, and functionally equivalent polypeptides. These nucleic acid molecules may be used in the methods and applications described herein. The nucleic acid molecules of the invention preferably comprise at least n consecutive nucleotides from the sequences disclosed herein where, depending on the particular sequence, n is 10 or more (for example, 12, 14, 15, 18, 20, 25, 30, 35, 40 or more).

The nucleic acid molecules of the invention also include sequences that are complementary to nucleic acid molecules described above (for example, for antisense or probing purposes).

Nucleic acid molecules of the present invention may be in the form of RNA, such as mRNA, or in the form of DNA, including, for instance cDNA, synthetic DNA or genomic DNA. Such nucleic acid molecules may be obtained by cloning, by chemical synthetic techniques or by a combination thereof. The nucleic acid molecules can be prepared, for example, by chemical synthesis using techniques such as solid phase phosphoramidite chemical synthesis, from genomic or cDNA libraries or by separation from an organism. RNA molecules may generally be generated by the in vitro or in vivo transcription of DNA sequences.

The nucleic acid molecules may be double-stranded or single-stranded. Single-stranded DNA may be the coding strand, also known as the sense strand, or it may be the non- coding strand, also referred to as the anti-sense strand. The term "nucleic acid molecule" also includes analogues of DNA and RNA, such as those containing modified backbones, and peptide nucleic acids (PNA). The term "PNA", as used herein, refers to an antisense molecule or an anti-gene agent which comprises an oligonucleotide of at least five nucleotides in length linked to a peptide backbone of amino acid residues, which preferably ends in lysine. The terminal lysine confers solubility to the composition. PNAs may be pegylated to extend their lifespan in a cell, where they preferentially bind complementary single stranded DNA and RNA and stop transcript elongation (Nielsen, P.E. et al. (1993) Anticancer Drug Des. 8:53-63).

A nucleic acid molecule which encodes the polypeptide of SEQ ID NO:2 may be identical to the coding sequence of the nucleic acid molecule shown in SEQ JD NO:l. These molecules also may have a different sequence which, as a result of the degeneracy of the genetic code, encodes a polypeptide of SEQ ID NO:2. The nucleic acid molecules of the fourth aspect of the invention encode the fragments or the functional equivalents of the polypeptides and fragments of the third aspect of the invention. Such a nucleic acid molecule may be a naturally-occurring variant such as a naturally-occurring allelic variant, or the molecule may be a variant that is not known to occur naturally. Such non-naturally occurring variants of the nucleic acid molecule may be made by mutagenesis techniques, including those applied to nucleic acid molecules, cells or organisms.

Among variants in this regard are variants that differ from the aforementioned nucleic acid molecules by nucleotide substitutions, deletions or insertions. The substitutions, deletions or insertions may involve one or more nucleotides. The variants may be altered in coding or non-coding regions or both. Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or insertions. The nucleic acid molecules of the invention can also be engineered, using methods generally known in the art, for a variety of reasons, including modifying the cloning, processing, and/or expression of the gene product (the polypeptide). DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides are included as techniques which may be used to engineer the nucleotide sequences. Site-directed mutagenesis may be used to insert new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, introduce mutations and so forth.

Nucleic acid molecules which encode a polypeptide of the third aspect of the invention may be ligated to a heterologous sequence so that the combined nucleic acid molecule encodes a fusion protein. Such combined nucleic acid molecules are included within the fourth aspects of the invention. For example, to screen peptide libraries for inhibitors of the aminopeptidase activity of the polypeptide, it may be useful to express, using such a combined nucleic acid molecule, a fusion protein that can be recognised by a commercially-available antibody. A fusion protein may also be engineered to contain a cleavage site located between the sequence of the polypeptide of the invention and the sequence of a heterologous protein so that the polypeptide may be cleaved and purified away from the heterologous protein.

The nucleic acid molecules of the invention also include antisense molecules that are partially complementary to nucleic acid molecules encoding polypeptides of the present invention and that therefore hybridize to the encoding nucleic acid molecules (hybridization). Such antisense molecules, such as oligonucleotides, can be designed to recognise, specifically bind to and prevent transcription of a target nucleic acid encoding a polypeptide of the invention, as will be known by those of ordinary skill in the art (see, for example, Cohen, J.S., Trends in Pharm. Sci., 10, 435 (1989), Okano, J. Neurochem. 56, 560 (1991); O'Connor, J. Neurochem 56, 560 (1991); Lee et al, Nucleic Acids Res 6, 3073 (1979); Cooney et al, Science 241, 456 (1988); Dervan et al, Science 251, 1360 (1991).

The term "hybridization" as used here refers to the association of two nucleic acid molecules with one another by hydrogen bonding. Typically, one molecule will be fixed to a solid support and the other will be free in solution. Then, the two molecules may be placed in contact with one another under conditions that favour hydrogen bonding. Factors that affect this bonding include: the type and volume of solvent; reaction temperature; time of hybridization; agitation; agents to block the non-specific attachment of the liquid phase molecule to the solid support (Denhardt's reagent or BLOTTO); the concentration of the molecules; use of compounds to increase the rate of association of molecules (dextran sulphate or polyethylene glycol); and the stringency of the washing conditions following hybridization (see Sambrook et al. {supra]).

The inhibition of hybridization of a completely complementary molecule to a target molecule may be examined using a hybridization assay, as known in the art (see, for example, Sambrook et al [supra]). A substantially homologous molecule will then compete for and inhibit the binding of a completely homologous molecule to the target molecule under various conditions of stringency, as taught in Wahl, G.M. and S.L. Berger (1987; Methods Enzymol. 152:399-407) and Kimmel, A.R. (1987; Methods Enzymol. 152:507- 511).

"Stringency" refers to conditions in a hybridization reaction that favour the association of very similar molecules over association of molecules that differ. High stringency hybridisation conditions are defined as overnight incubation at 42°C in a solution comprising 50% formamide, 5XSSC (150mM NaCl, 15mM trisodium citrate), 50mM sodium phosphate (pH7.6), 5x Denhardts solution, 10% dextran sulphate, and 20 microgram/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1X SSC at approximately 65°C. Low stringency conditions involve the hybridisation reaction being carried out at 35°C (see Sambrook et al. [supra]). Preferably, the conditions used for hybridization are those of high stringency.

Preferred embodiments of this aspect of the invention are nucleic acid molecules that are at least 70% identical over their entire length to a nucleic acid molecule encoding the Nicastrin polypeptide (SEQ ID NO: 2) and nucleic acid molecules that are substantially complementary to such nucleic acid molecules. Preferably, a nucleic acid molecule according to this aspect of the invention comprises a region that is at least 80% identical over its entire length to the nucleic acid molecule having the sequence given in SEQ ID NO: 1 or a nucleic acid molecule that is complementary thereto. In this regard, nucleic acid molecules at least 90%, preferably at least 95%, more preferably at least 98% or 99% identical over their entire length to the same are particularly preferred. Preferred embodiments in this respect are nucleic acid molecules that encode polypeptides which retain substantially the same biological function or activity as the Nicastrin polypeptide. The invention also provides a process for detecting a nucleic acid molecule of the invention, comprising the steps of: (a) contacting a nucleic probe according to the invention with a biological sample under hybridizing conditions to form duplexes; and (b) detecting any such duplexes that are formed. As discussed additionally below in connection with assays that may be utilised according to the invention, a nucleic acid molecule as described above may be used as a hybridization probe for RNA, cDNA or genomic DNA, in order to isolate full-length cDNAs and genomic clones encoding the Nicastrin polypeptide and to isolate cDNA and genomic clones of homologous or orthologous genes that have a high sequence similarity to the gene encoding this polypeptide.

In this regard, the following techniques, among others known in the art, may be utilised and are discussed below for purposes of illustration. Methods for DNA sequencing and analysis are well known and are generally available in the art and may, indeed, be used to practice many of the embodiments of the invention discussed herein. Such methods may employ such enzymes as the Klenow fragment of DNA polymerase I, Sequenase (US Biochemical Corp, Cleveland, OH), Taq polymerase (Perkin Elmer), thermostable T7 polymerase (Amersham, Chicago, IL), or combinations of polymerases and proof-reading exonucleases such as those found in the ELONGASE Amplification System marketed by Gibco/BRL (Gaithersburg, MD). Preferably, the sequencing process may be automated using machines such as the Hamilton Micro Lab 2200 (Hamilton, Reno, NV), the Peltier Thermal Cycler (PTC200; MJ Research, Watertown, MA) and the ABI Catalyst and 373 and 377 DNA Sequencers (Perkin Elmer).

One method for isolating a nucleic acid molecule encoding a polypeptide with an equivalent function to that of the Nicastrin polypeptide is to probe a genomic or cDNA library with a natural or artificially-designed probe using standard procedures that are recognised in the art (see, for example, "Current Protocols in Molecular Biology", Ausubel et al. (eds). Greene Publishing Association and John Wiley Interscience, New York, 1989,1992). Probes comprising at least 15, preferably at least 30, and more preferably at least 50, contiguous bases that correspond to, or are complementary to, nucleic acid sequences from the appropriate encoding gene (SEQ ID NO:l), are particularly useful probes. Such probes may be labelled with an analytically-detectable reagent to facilitate their identification. Useful reagents include, but are not limited to, radioisotopes, fluorescent dyes and enzymes that are capable of catalysing the formation of a detectable product. Using these probes, the ordinarily skilled artisan will be capable of isolating complementary copies of genomic DNA, cDNA or RNA polynucleotides encoding proteins of interest from human, mammalian or other animal sources and screening such sources for related sequences, for example, for additional members of the family, type and/or subtype.

In many cases, isolated cDNA sequences will be incomplete, in that the region encoding the polypeptide will be cut short, normally at the 5' end. Several methods are available to obtain full length cDNAs, or to extend short cDNAs. Such sequences may be extended utilising a partial nucleotide sequence and employing various methods known in the art to detect upstream sequences such as promoters and regulatory elements. For example, one method which may be employed is based on the method of Rapid Amplification of cDNA Ends (RACE; see, for example, Frohman et al, PNAS USA 85, 8998-9002, 1988). Recent modifications of this technique, exemplified by the Marathon™ technology (Clontech Laboratories Inc.), for example, have significantly simplified the search for longer cDNAs. A slightly different technique, termed "restriction-site" PCR, uses universal primers to retrieve unknown nucleic acid sequence adjacent a known locus (Sarkar, G. (1993) PCR Methods Applic. 2:318-322). Inverse PCR may also be used to amplify or to extend sequences using divergent primers based on a known region (Triglia, T. et al. (1988) Nucleic Acids Res. 16:8186). Another method which may be used is capture PCR which involves PCR amplification of DNA fragments adjacent a known sequence in human and yeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCR Methods Applic, 1, 111-119). Another method which may be used to retrieve unknown sequences is that of Parker, J.D. et al. (1991); Nucleic Acids Res. 19:3055-3060). Additionally, one may use PCR, nested primers, and PromoterFinder™ libraries to walk genomic DNA (Clontech, Palo Alto, CA). This process avoids the need to screen libraries and is useful in finding intron/exon junctions.

When screening for full-length cDNAs, it is preferable to use libraries that have been size- selected to include larger cDNAs. Also, random-primed libraries are preferable, in that they will contain more sequences that contain the 5' regions of genes. Use of a randomly primed library may be especially preferable for situations in which an oligo d(T) library does not yield a full-length cDNA. Genomic libraries may be useful for extension of sequence into 5' non-transcribed regulatory regions. In one embodiment of the invention, the nucleic acid molecules of the present invention may be used for chromosome localisation. In this technique, a nucleic acid molecule is specifically targeted to, and can hybridize with, a particular location on an individual human chromosome. The mapping of relevant sequences to chromosomes according to the present invention is an important step in the confirmatory correlation of those sequences with the gene-associated disease. 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 in, for example, V. McKusick, Mendelian Inheritance in Man (available on-line through Johns Hopkins University Welch Medical Library). The relationships between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheritance of physically adjacent genes). This provides valuable information to investigators searching for disease genes using positional cloning or other gene discovery techniques. Once the disease or syndrome has been crudely localised by genetic linkage to a particular genomic region, any sequences mapping to that area may represent associated or regulatory genes for further investigation. The nucleic acid molecule may also be used to detect differences in the chromosomal location due to translocation, inversion, etc. among normal, carrier, or affected individuals.

The nucleic acid molecules of the present invention are also valuable for tissue localisation. Such techniques allow the determination of expression patterns of the polypeptide in tissues by detection of the mRNAs that encode them. These techniques include in situ hybridization techniques and nucleotide amplification techniques, such as PCR. Results from these studies provide an indication of the normal functions of the polypeptide in the organism. In addition, comparative studies of the normal expression pattern of mRNAs with that of mRNAs encoded by a mutant gene provide valuable insights into the role of mutant polypeptides in disease. Such inappropriate expression may be of a temporal, spatial or quantitative nature.

The vectors of the present invention comprise nucleic acid molecules of the invention and may be cloning or expression vectors. The host cells of the invention, which may be transformed, transfested or transduced with the vectors of the invention may be prokaryotic or eukaryotic. The polypeptides of the invention may be prepared in recombinant form by expression of their encoding nucleic acid molecules in vectors contained within a host cell. Such expression methods are well known to those of skill in the art and many are described in detail by Sambrook et al (supra) and Fernandez & Hoeffler (1998, eds. "Gene expression systems. Using nature for the art of expression". Academic Press, San Diego, London, Boston, New York, Sydney, Tokyo, Toronto).

Generally, any system or vector that is suitable to maintain, propagate or express nucleic acid molecules to produce a polypeptide in the required host may be used. The appropriate nucleotide sequence may be inserted into an expression system by any of a variety of well- known and routine techniques, such as, for example, those described in Sambrook et al, (supra). Generally, the encoding gene can be placed under the control of a control element such as a promoter, ribosome binding site (for bacterial expression) and, optionally, an operator, so that the DNA sequence encoding the desired polypeptide is transcribed into RNA in the transformed host cell. Examples of suitable expression systems include, for example, chromosomal, episomal and virus-derived systems, including, for example, vectors derived from: bacterial plasmids, bacteriophage, transposons, yeast episomes, insertion elements, yeast chromosomal elements, viruses such as baculoviruses, papova viruses such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, or combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, including cosmids and phagemids. Human artificial chromosomes (HACs) may also be employed to deliver larger fragments of DNA than can be contained and expressed in a plasmid.

Particularly suitable expression systems include microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (for example, baculovirus); plant cell systems transformed with virus expression vectors (for example, cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (for example, Ti or pBR322 plasmids); or animal cell systems. Cell-free translation systems can also be employed to produce the polypeptides of the invention.

Introduction of nucleic acid molecules encoding a polypeptide of the present invention into host cells can be effected by methods described in many standard laboratory manuals, such as Davis et al, Basic Methods in Molecular Biology (1986) and Sambrook et al, [supra]. Particularly suitable methods include calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection (see Sambrook et al, 1989 [supra]; Ausubel et al, 1991 [supra]; Spector, Goldman & Leinwald, 1998). In eukaryotic cells, expression systems may either be transient (for example, episomal) or permanent (chromosomal integration) according to the needs of the system.

The encoding nucleic acid molecule may or may not include a sequence encoding a control sequence, such as a signal peptide or leader sequence, as desired, for example, for secretion of the translated polypeptide into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular environment. These signals may be endogenous to the polypeptide or they may be heterologous signals. Leader sequences can be removed by the bacterial host in post-translational processing. In addition to control sequences, it may be desirable to add regulatory sequences that allow for regulation of the expression of the polypeptide relative to the growth of the host cell. Examples of regulatory sequences are those which cause the expression of a gene to be increased or decreased in response to a chemical or physical stimulus, including the presence of a regulatory compound or to various temperature or metabolic conditions. Regulatory sequences are those non-translated regions of the vector, such as enhancers, promoters and 5' and 3' untranslated regions. These interact with host cellular proteins to carry out transcription and translation. Such regulatory sequences may vary in their strength and specificity. Depending on the vector system and host utilised, any number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used. For example, when cloning in bacterial systems, inducible promoters such as the hybrid lacZ promoter of the Bluescript phagemid (Stratagene, LaJolla, CA) or pSportlTM plasmid (Gibco BRL) and the like may be used. The baculovirus polyhedrin promoter may be used in insect cells. Promoters or enhancers derived from the genomes of plant cells (for example, heat shock, RUBISCO and storage protein genes) or from plant viruses (for example, viral promoters or leader sequences) may be cloned into the vector. In mammalian cell systems, promoters from mammalian genes or from mammalian viruses are preferable. If it is necessary to generate a cell line that contains multiple copies of the sequence, vectors based on SV40 or EBV may be used with an appropriate selectable marker.

An expression vector is constructed so that the particular nucleic acid coding sequence is located in the vector with the appropriate regulatory sequences, the positioning and orientation of the coding sequence with respect to the regulatory sequences being such that the coding sequence is transcribed under the "control" of the regulatory sequences, i.e., RNA polymerase which binds to the DNA molecule at the control sequences transcribes the coding sequence. In some cases it may be necessary to modify the sequence so that it may be attached to the control sequences with the appropriate orientation; i.e., to maintain the reading frame.

The control sequences and other regulatory sequences may be ligated to the nucleic acid coding sequence prior to insertion into a vector. Alternatively, the coding sequence can be cloned directly into an expression vector that already contains the control sequences and an appropriate restriction site. For long-term, high-yield production of a recombinant polypeptide, stable expression is preferred. For example, cell lines which stably express the polypeptide of interest may be transformed using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for 1-2 days in an enriched media before they are switched to selective media. The purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells that successfully express the introduced sequences. Resistant clones of stably transformed cells may be proliferated using tissue culture techniques appropriate to the cell type. Mammalian cell lines available as hosts for expression are known in the art and include many immortalised cell lines available from the American Type Culture Collection (ATCC) including, but not limited to, Chinese hamster ovary (CHO), HeLa, baby hamster kidney (BHK), monkey kidney (COS), C127, 3T3, BHK, HEK 293, Bowes melanoma and human hepatocellular carcinoma (for example Hep G2) cells and a number of other cell lines.

In the baculovirus system, the materials for baculovirus/insect cell expression systems are commercially available in kit form from, inter alia, Invitrogen, San Diego CA (the "MaxBac" kit). These techniques are generally known to those skilled in the art and are described fully in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987). Particularly suitable host cells for use in this system include insect cells such as Drosophila S2 and Spodoptera Sf9 cells. There are many plant cell culture and whole plant genetic expression systems known in the art. Examples of suitable plant cellular genetic expression systems include those described in US 5,693,506; US 5,659,122; and US 5,608,143. Additional examples of genetic expression in plant cell culture has been described by Zenk, Phytochemistry 30, 3861-3863 (1991). In particular, all plants from which protoplasts can be isolated and cultured to give whole regenerated plants can be utilised, so that whole plants are recovered which contain the transferred gene. Practically all plants can be regenerated from cultured cells or tissues, including but not limited to all major species of sugar cane, sugar beet, cotton, fruit and other trees, legumes and vegetables. Examples of particularly preferred bacterial host cells include streptococci, staphylococci, E. coli, Streptomyces and Bacillus subtilis cells.

Examples of particularly suitable host cells for fungal expression include yeast cells (for example, S. cerevisiae) and Aspergillus cells.

Any number of selection systems are known in the art that may be used to recover transformed cell lines. Examples include the herpes simplex virus thymidine kinase (Wigler, M. et al. (1977) Cell 11:223-32) and adenine phosphoribosyltransferase (Lowy, I. et al. (1980) Cell 22:817-23) genes that can be employed in tk- or aprt± cells, respectively.

Also, antimetabolite, antibiotic or herbicide resistance can be used as the basis for selection; for example, dihydrofolate reductase (DHFR) that confers resistance to methotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. 77:3567-70); npt, which confers resistance to the aminoglycosides neomycin and G-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol. 150:1-14) and als or pat, which confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively. Additional selectable genes have been described, examples of which will be clear to those of skill in the art. Although the presence or absence of marker gene expression suggests that the gene of interest is also present, its presence and expression may need to be confirmed. For example, if the relevant sequence is inserted within a marker gene sequence, transformed cells containing the appropriate sequences can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a sequence encoding a polypeptide of the invention under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as well.

Alternatively, host cells that contain a nucleic acid sequence encoding a polypeptide of the invention and which express said polypeptide may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA- DNA or DNA-RNA hybridizations and protein bioassays, for example, fluorescence activated cell sorting (FACS) or immunoassay techniques (such as the enzyme-linked immunosorbent assay [ELISA] and radioimmunoassay [RIA]), that include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein (see Hampton, R. et al (1990) Serological Methods, a Laboratory Manual, APS Press, St Paul, MN) and Maddox, D.E. et al. (1983) J. Exp. Med, 158, 1211-1216).

A wide variety of labels and conjugation techniques are known by those skilled in the art and may be used in various nucleic acid and amino acid assays. Means for producing labelled hybridization or PCR probes for detecting sequences related to nucleic acid molecules encoding polypeptides of the present invention include oligolabelling, nick translation, end-labelling or PCR amplification using a labelled polynucleotide. Alternatively, the sequences encoding the polypeptide of the invention may be cloned into a vector for the production of an niRNA probe. Such vectors are known in the art, are commercially available, and may be used to synthesise RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3 or SP6 and labelled nucleotides. These procedures may be conducted using a variety of commercially available kits (Pharmacia & Upjohn, (Kalamazoo, MI); Promega (Madison WI); and U.S. Biochemical Corp., Cleveland, OH)).

Suitable reporter molecules or labels, which may be used for ease of detection, include radionuclides, enzymes and fluorescent, chemiluminescent or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles, and the like.

Nucleic acid molecules according to the present invention may also be used to create transgenic animals, particularly rodent animals. Such transgenic animals form a further aspect of the present invention. This may be done locally by modification of somatic cells, or by germ line therapy to incorporate heritable modifications. Such transgenic animals may be particularly useful in the generation of animal models for drug molecules effective as modulators of the polypeptides of the present invention. The polypeptide can be recovered and purified from recombinant cell cultures by well- known methods including ammonium sulphate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. High performance liquid chromatography is particularly useful for purification. Well known techniques for refolding proteins may be employed to regenerate an active conformation when the polypeptide is denatured during isolation and or purification.

Specialised vector constructions may also be used to facilitate purification of proteins, as desired, by joining sequences encoding the polypeptides of the invention to a nucleotide sequence encoding a polypeptide domain that will facilitate purification of soluble proteins. Examples of such purification-facilitating domains include metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilised metals, protein A domains that allow purification on immobilised immunoglobulin, and the domain utilised in the FLAGS extension/affinity purification system (Immunex Corp., Seattle, WA). The inclusion of cleavable linker sequences such as those specific for Factor XA or enterokinase (Invitrogen, San Diego, CA) between the purification domain and the polypeptide of the invention may be used to facilitate purification. One such expression vector provides for expression of a fusion protein containing the polypeptide of the invention fused to several histidine residues preceding a thioredoxin or an enterokinase cleavage site. The histidine residues facilitate purification by LMAC (immobilised metal ion affinity chromatography as described in Porath, J. et al. (1992), Prot. Exp. Purif. 3: 263-281) while the thioredoxin or enterokinase cleavage site provides a means for purifying the polypeptide from the fusion protein. A discussion of vectors which contain fusion proteins is provided in Kroll, D.J. et al. (1993; DNA Cell Biol. 12:441-453). If the polypeptide is to be expressed for use in screening assays, generally it is preferred that it be produced at the surface of the host cell in which it is expressed. In this event, the host cells may be harvested prior to use in the screening assay, for example using techniques such as fluorescence activated cell sorting (FACS) or immunoaffinity techniques. If the polypeptide is secreted into the medium, the medium can be recovered in order to recover and purify the expressed polypeptide. If polypeptide is produced intracellularly, the cells must first be lysed before the polypeptide is recovered. The polypeptide of the invention can be used to screen libraries of compounds in any of a variety of drug screening techniques. Such compounds may activate (agonise) or inhibit (antagonise) the level of expression of the gene or the activity of the polypeptide of the invention and form a further aspect of the present invention. Preferred compounds are effective to alter the expression of a natural gene which encodes a polypeptide of the third aspect of the invention or to regulate the activity of a polypeptide of the third aspect of the invention, particularly the aminopeptidase activity.

Agonist or antagonist compounds may be isolated from, for example, cells, cell-free preparations, chemical libraries or natural product mixtures. These agonists or antagonists may be natural or modified substrates, ligands, enzymes, receptors or structural or functional mimetics. For a suitable review of such screening techniques, see Coligan et al, Current Protocols in Immunology l(2):Chapter 5 (1991).

Compounds that are most likely to be good antagonists are molecules that bind to the polypeptide of the invention without inducing the biological effects of the polypeptide upon binding to it. Potential antagonists include small organic molecules, peptides, polypeptides and antibodies that bind to the polypeptide of the invention and thereby inhibit or extinguish its activity. In this fashion, binding of the polypeptide to normal cellular binding molecules may be inhibited, such that the normal biological activity of the polypeptide is prevented.

The polypeptide of the invention that is employed in such a screening technique may be free in solution, affixed to a solid support, borne on a cell surface or located intracellularly. In general, such screening procedures may involve using appropriate cells or cell membranes that express the polypeptide that are contacted with a test compound to observe binding, or stimulation or inhibition of a functional response. The functional response of the cells contacted with the test compound is then compared with control cells that were not contacted with the test compound. Such an assay may assess whether the test compound results in a signal generated by activation of the polypeptide, using an appropriate detection system. Inhibitors of activation are generally assayed in the presence of a known agonist and the effect on activation by the agonist in the presence of the test compound is observed. In view of the aminopeptidase activity of the polypeptides of the invention, when configuring the assay conditions for the identification of ligands effective to affect this activity, metal ions, in particular zinc, should generally be included in the assay buffer.

Simple binding assays may also be used, in which the adherence of a test compound to a surface bearing the polypeptide is detected by means of a label directly or indirectly associated with the test compound or in an assay involving competition with a labelled competitor. In another embodiment, competitive drug screening assays may be used, in which neutralising antibodies that are capable of binding the polypeptide specifically compete with a test compound for binding. In this manner, the antibodies can be used to detect the presence of any test compound that possesses specific binding affinity for the polypeptide.

Assays may also be designed to detect the effect of added test compounds on the production of mRNA encoding the polypeptide in cells. For example, an ELISA may be constructed that measures secreted or cell-associated levels of polypeptide using monoclonal or polyclonal antibodies by standard methods known in the art, and this can be used to search for compounds that may inhibit or enhance the production of the polypeptide from suitably manipulated cells or tissues. The formation of binding complexes between the polypeptide and the compound being tested may then be measured.

Another technique for drug screening which may be used provides for high throughput screening of compounds having suitable binding affinity to the polypeptide of interest (see International patent application WO84/03564). In this method, large numbers of different small test compounds are synthesised on a solid substrate, which may then be reacted with the polypeptide of the invention and washed. One way of immobilising the polypeptide is to use non-neutralising antibodies. Bound polypeptide may then be detected using methods that are well known in the art. Purified polypeptide can also be coated directly onto plates for use in the aforementioned drag screening techniques.

The polypeptide of the invention may be used to identify membrane-bound or soluble receptors, through standard receptor binding techniques that are known in the art, such as ligand binding and crosslinking assays in which the polypeptide is labelled with a radioactive isotope, is chemically modified, or is fused to a peptide sequence that facilitates its detection or purification, and incubated with a source of the putative receptor (for example, a composition of cells, cell membranes, cell supernatants, tissue extracts, or bodily fluids). The efficacy of binding may be measured using biophysical techniques such as surface plasmon resonance and spectroscopy. Binding assays may be used for the purification and cloning of the receptor, but may also identify agonists and antagonists of the polypeptide, that compete with the binding of the polypeptide to its receptor. Standard methods for conducting screening assays are well understood in the art.

The invention also includes a screening kit useful in the methods for identifying agonists, antagonists, ligands, receptors, substrates, enzymes, that are described above. The invention includes the agonists, antagonists, ligands, receptors, substrates and enzymes, and other compounds which modulate the activity or antigenicity of the polypeptide of the invention discovered by the methods that are described above.

The invention also provides pharmaceutical compositions comprising a polypeptide, nucleic acid, ligand or compound of the invention in combination with a suitable pharmaceutical carrier. These compositions may be suitable as therapeutic or diagnostic reagents, as vaccines, or as other immunogenic compositions, as outlined in detail below.

According to the terminology used herein, a composition containing a polypeptide, nucleic acid, ligand or compound [X] is "substantially free of impurities [herein, Y] when at least 85% by weight of the total X+Y in the composition is X. Preferably, X comprises at least about 90% by weight of the total of X+Y in the composition, more preferably at least about 95%, 98% or even 99% by weight.

The pharmaceutical compositions should preferably comprise a therapeutically effective amount of the polypeptide, nucleic acid molecule, ligand, or compound of the invention. The term "therapeutically effective amount" as used herein refers to an amount of a therapeutic agent needed to treat, ameliorate, or prevent a targeted disease or condition, or to exhibit a detectable therapeutic or preventative effect. For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays, for example, of neoplastic cells, or in animal models, usually mice, rabbits, dogs, or pigs. The animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans. The precise effective amount for a human subject will depend upon the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. This amount can be determined by routine experimentation and is within the judgement of the clinician. Generally, an effective dose will be from 0.01 mg/kg to 50 mg/kg, preferably 0.05 mg/kg to 10 mg/kg. Compositions may be administered individually to a patient or may be administered in combination with other agents, drugs or hormones.

A pharmaceutical composition may also contain a pharmaceutically acceptable carrier, for administration of a therapeutic agent. Such carriers include antibodies and other polypeptides, genes and other therapeutic agents such as liposomes, provided that the carrier does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity. Suitable carriers may be large, slowly metabolised macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers and inactive virus particles.

Pharmaceutically acceptable salts can be used therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulphates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. A thorough discussion of pharmaceutically acceptable carriers is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991).

Pharmaceutically acceptable carriers in therapeutic compositions may additionally contain liquids such as water, saline, glycerol and ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such compositions. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.

Once formulated, the compositions of the invention can be administered directly to the subject. The subjects to be treated can be animals; in particular, human subjects can be treated.

The pharmaceutical compositions utilised in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra- arterial, intramedullary, intrathecal, intraventricular, transdermal or transcutaneous applications (for example, see WO98/20734), subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, intravaginal or rectal means. Gene guns or hyposprays may also be used to administer the pharmaceutical compositions of the invention. Typically, the therapeutic compositions may be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared.

Direct delivery of the compositions will generally be accomplished by injection, subcutaneously, intraperitoneally, intravenously or intramuscularly, or delivered to the interstitial space of a tissue. The compositions can also be administered into a lesion. Dosage treatment may be a single dose schedule or a multiple dose schedule.

If the activity of the polypeptide of the invention is in excess in a particular disease state, several approaches are available. One approach comprises administering to a subject an inhibitor compound (antagonist) as described above, along with a pharmaceutically acceptable carrier in an amount effective to inhibit the function of the polypeptide, such as by blocking the binding of ligands, substrates, enzymes, receptors, or by inhibiting a second signal, and thereby alleviating the abnormal condition. Preferably, such antagonists are antibodies. Most preferably, such antibodies are chimeric and/or humanised to minimise their immunogenicity, as described previously. In another approach, soluble forms of the polypeptide that retain binding affinity for the ligand, substrate, enzyme, receptor, in question, may be administered. Typically, the polypeptide may be administered in the form of fragments that retain the relevant portions.

In an alternative approach, expression of the gene encoding the polypeptide can be inhibited using expression blocking techniques, such as the use of antisense nucleic acid molecules (as described above), either internally generated or separately administered. Modifications of gene expression can be obtained by designing complementary sequences or antisense molecules (DNA, RNA, or PNA) to the control, 5' or regulatory regions (signal sequence, promoters, enhancers and introns) of the gene encoding the polypeptide. Similarly, inhibition can be achieved using "triple helix" base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules. Recent therapeutic advances using triplex DNA have been described in the literature (Gee, J.E. et al. (1994) In: Huber, B.E. and B.I. Carr, Molecular and Immunologic Approaches, Futura Publishing Co., Mt. Kisco, NY). The complementary sequence or antisense molecule may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes. Such oligonucleotides may be administered or may be generated in situ from expression in vivo.

In addition, expression of the polypeptide of the invention may be prevented by using ribozymes specific to its encoding mRNA sequence. Ribozymes are catalytically active RNAs that can be natural or synthetic (see for example Usman, N, et al, Curr. Opin. Struct. Biol (1996) 6(4), 527-33). Synthetic ribozymes can be designed to specifically cleave mRNAs at selected positions thereby preventing translation of the mRNAs into functional polypeptide. Ribozymes may be synthesised with a natural ribose phosphate backbone and natural bases, as normally found in RNA molecules. Alternatively the ribozymes may be synthesised with non-natural backbones, for example, 2'-O-methyl RNA, to provide protection from ribonuclease degradation and may contain modified bases.

RNA molecules may be modified to increase intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends of the molecule or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the backbone of the molecule. This concept is inherent in the production of PNAs and can be extended in all of these molecules by the inclusion of non-traditional bases such as inosine, queosine and butosine, as well as acetyl-, methyl-, thio- and similarly modified forms of adenine, cytidine, guanine, thymine and uridine which are not as easily recognised by endogenous endonucleases.

For treating abnormal conditions related to an under-expression of the polypeptide of the invention and its activity, several approaches are also available. One approach comprises administering to a subject a therapeutically effective amount of a compound that activates the polypeptide, i.e., an agonist as described above, to alleviate the abnormal condition. Alternatively, a therapeutic amount of the polypeptide in combination with a suitable pharmaceutical carrier may be administered to restore the relevant physiological balance of polypeptide.

Gene therapy may be employed to effect the endogenous production of the polypeptide by the relevant cells in the subject. Gene therapy is used to treat permanently the inappropriate production of the polypeptide by replacing a defective gene with a corrected therapeutic gene.

Gene therapy of the present invention can occur in vivo or ex vivo. Ex vivo gene therapy requires the isolation and purification of patient cells, the introduction of a therapeutic gene and introduction of the genetically altered cells back into the patient. In contrast, in vivo gene therapy does not require isolation and purification of a patient's cells.

The therapeutic gene is typically "packaged" for administration to a patient. Gene delivery vehicles may be non-viral, such as liposomes, or replication-deficient viruses, such as adenovirus as described by Berkner, K.L., in Curr. Top. Microbiol. Immunol., 158, 39-66 (1992) or adeno-associated viras (AAV) vectors as described by Muzyczka, N., in Curr. Top. Microbiol. Immunol, 158, 97-129 (1992) and U.S. Patent No. 5,252,479. For example, a nucleic acid molecule encoding a polypeptide of the invention may be engineered for expression in a replication-defective retroviral vector. This expression construct may then be isolated and introduced into a packaging cell transduced with a retroviral plasmid vector containing RNA encoding the polypeptide, such that the packaging cell now produces infectious viral particles containing the gene of interest. These producer cells may be administered to a subject for engineering cells in vivo and expression of the polypeptide in vivo (see Chapter 20, Gene Therapy and other Molecular Genetic-based Therapeutic Approaches, (and references cited therein) in Human Molecular Genetics (1996), T Strachan and A P Read, BIOS Scientific Publishers Ltd).

Another approach is the administration of "naked DNA" in which the therapeutic gene is directly injected into the bloodstream or muscle tissue.

In situations in which the polypeptides or nucleic acid molecules of the invention are disease-causing agents, the invention provides that they can be used in vaccines to raise antibodies against the disease causing agent.

Vaccines according to the invention may either be prophylactic (ie. to prevent infection) or therapeutic (ie. to treat disease after infection). Such vaccines comprise immunising antigen(s), immunogen(s), polypeptide(s), protein(s) or nucleic acid, usually in combination with pharmaceutically-acceptable carriers as described above, which include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition. Additionally, these carriers may function as immunostimulating agents ("adjuvants"). Furthermore, the antigen or irnmunogen may be conjugated to a bacterial toxoid, such as a toxoid from diphtheria, tetanus, cholera, H. pylori, and other pathogens.

Since polypeptides may be broken down in the stomach, vaccines comprising polypeptides are preferably administered parenterally (for instance, subcutaneous, intramuscular, intravenous, or intradermal injection). Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti- oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the recipient, and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents. The vaccine formulations of the invention may be presented in unit-dose or multi-dose containers. For example, sealed ampoules and vials and may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier immediately prior to use. The dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation. This invention also relates to the use of nucleic acid molecules according to the present invention as diagnostic reagents. Detection of a mutated form of the gene characterised by the nucleic acid molecules of the invention which is associated with a dysfunction will provide a diagnostic tool that can add to, or define, a diagnosis of a disease, or susceptibility to a disease, which results from under-expression, over-expression or altered spatial or temporal expression of the gene. Individuals carrying mutations in the gene may be detected at the DNA level by a variety of techniques.

Nucleic acid molecules for diagnosis may be obtained from a subject's cells, such as from blood, urine, saliva, tissue biopsy or autopsy material. The genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR, ligase chain reaction (LCR), strand displacement amplification (SDA), or other amplification techniques (see Saiki et al, Nature, 324, 163-166 (1986); Bej, et al, Crit. Rev. Biochem. Molec. Biol., 26, 301-334 (1991); Birkenmeyer et al, J. Virol. Meth., 35, 117-126 (1991); Van Brant, J., Bio/Technology, 8, 291-294 (1990)) prior to analysis.

In one embodiment, this aspect of the invention provides a method of diagnosing a disease in a patient, comprising assessing the level of expression of a natural gene encoding a polypeptide according to the invention and comparing said level of expression to a control level, wherein a level that is different to said control level is indicative of disease. The method may comprise the steps of: a) contacting a sample of tissue from the patient with a nucleic acid probe under stringent conditions that allow the formation of a hybrid complex between a nucleic acid molecule of the invention and the probe; b) contacting a control sample with said probe under the same conditions used in step a); and c) detecting the presence of hybrid complexes in said samples; wherein detection of levels of the hybrid complex in the patient sample that differ from levels of the hybrid complex in the control sample is indicative of disease.

A further aspect of the invention comprises a diagnostic method comprising the steps of: a) obtaining a tissue sample from a patient being tested for disease; b) isolating a nucleic acid molecule according to the invention from said tissue sample; and c) diagnosing the patient for disease by detecting the presence of a mutation in the nucleic acid molecule which is associated with disease.

To aid the detection of nucleic acid molecules in the above-described methods, an amplification step, for example using PCR, may be included.

Deletions and insertions can be detected by a change in the size of the amplified product in comparison to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to labelled RNA of the invention or alternatively, labelled antisense DNA sequences of the invention. Perfectly-matched sequences can be distinguished from mismatched duplexes by RNase digestion or by assessing differences in melting temperatures. The presence or absence of the mutation in the patient may be detected by contacting DNA with a nucleic acid probe that hybridises to the DNA under stringent conditions to form a hybrid double-stranded molecule, the hybrid double-stranded molecule having an unhybridised portion of the nucleic acid probe strand at any portion corresponding to a mutation associated with disease; and detecting the presence or absence of an unhybridised portion of the probe strand as an indication of the presence or absence of a disease-associated mutation in the corresponding portion of the DNA strand. Such diagnostics are particularly useful for prenatal and even neonatal testing.

Point mutations and other sequence differences between the reference gene and "mutant" genes can be identified by other well-known techniques, such as direct DNA sequencing or single-strand conformational polymorphism, (see Orita et al, Genomics, 5, 874-879 (1989)). For example, a sequencing primer may be used with double-stranded PCR product or a single-stranded template molecule generated by a modified PCR. The sequence determination is performed by conventional procedures with radiolabelled nucleotides or by automatic sequencing procedures with fluorescent-tags. Cloned DNA segments may also be used as probes to detect specific DNA segments. The sensitivity of this method is greatly enhanced when combined with PCR. Further, point mutations and other sequence variations, such as polymorphisms, can be detected as described above, for example, through the use of allele-specific oligonucleotides for PCR amplification of sequences that differ by single nucleotides.

DNA sequence differences may also be detected by alterations in the electrophoretic mobility of DNA fragments in gels, with or without denaturing agents, or by direct DNA sequencing (for example, Myers et al, Science (1985) 230:1242). Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and SI protection or the chemical cleavage method (see Cotton et al, Proc. Natl. Acad. Sci. USA (1985) 85: 4397-4401). In addition to conventional gel electrophoresis and DNA sequencing, mutations such as microdeletions, aneuploidies, translocations, inversions, can also be detected by in situ analysis (see, for example, Keller et al, DNA Probes, 2nd Ed., Stockton Press, New York, N.Y., USA (1993)), that is, DNA or RNA sequences in cells can be analysed for mutations without need for their isolation and/or immobilisation onto a membrane. Fluorescence in situ hybridization (FISH) is presently the most commonly applied method and numerous reviews of FISH have appeared (see, for example, Trachuck et al, Science, 250, 559-562 (1990), and Trask et al, Trends, Genet., 7, 149-154 (1991)).

In another embodiment of the invention, an array of oligonucleotide probes comprising a nucleic acid molecule according to the invention can be constructed to conduct efficient screening of genetic variants, mutations and polymorphisms. Array technology methods are well known and have general applicability and can be used to address a variety of questions in molecular genetics including gene expression, genetic linkage, and genetic variability (see for example: M.Chee et al, Science (1996), Vol 274, pp 610-613).

In one embodiment, the array is prepared and used according to the methods described in PCT application WO95/11995 (Chee et al); Lockhart, D. J. et al (1996) Nat. Biotech. 14: 1675-1680); and Schena, M. et al (1996) Proc. Natl. Acad. Sci. 93: 10614-10619). Oligonucleotide pairs may range from two to over one million. The oligomers are synthesized at designated areas on a substrate using a light-directed chemical process. The substrate may be paper, nylon or other type of membrane, filter, chip, glass slide or any other suitable solid support. In another aspect, an oligonucleotide may be synthesized on the surface of the substrate by using a chemical coupling procedure and an ink jet application apparatus, as described in PCT application W095/251116 (Baldeschweiler et. al). In another aspect, a "gridded" array analogous to a dot (or slot) blot may be used to arrange and link cDNA fragments or oligonucleotides to the surface of a substrate using a vacuum system, thermal, UV, mechanical or chemical bonding procedures. An array, such as those described above, may be produced by hand or by using available devices (slot blot or dot blot apparatus), materials (any suitable solid support), and machines (including robotic instruments), and may contain 8, 24, 96, 384, 1536 or 6144 oligonucleotides, or any other number between two and over one million which lends itself to the efficient use of commercially-available instrumentation. In addition to the methods discussed above, diseases may be diagnosed by methods comprising determining, from a sample derived from a subject, an abnormally decreased or increased level of polypeptide or mRNA. Decreased or increased expression can be measured at the RNA level using any of the methods well known in the art for the quantitation of polynucleotides, such as, for example, nucleic acid amplification, for instance PCR, RT-PCR, RNase protection, Northern blotting and other hybridization methods.

Assay techniques that can be used to determine levels of a polypeptide of the present invention in a sample derived from a host are well-known to those of skill in the art and are discussed in some detail above (including radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays). This aspect of the invention provides a diagnostic method which comprises the steps of: (a) contacting a ligand as described above with a biological sample under conditions suitable for the formation of a ligand- polypeptide complex; and (b) detecting said complex.

Protocols such as ELISA, RIA, and FACS for measuring polypeptide levels may additionally provide a basis for diagnosing altered or abnormal levels of polypeptide expression. Normal or standard values for polypeptide expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, preferably humans, with antibody to the polypeptide under conditions suitable for complex formation The amount of standard complex formation may be quantified by various methods, such as by photometric means. Antibodies which specifically bind to a polypeptide of the invention may be used for the diagnosis of conditions or diseases characterised by expression of the polypeptide, or in assays to monitor patients being treated with the polypeptides, nucleic acid molecules, ligands and other compounds of the invention. Antibodies useful for diagnostic purposes may be prepared in the same manner as those described above for therapeutics. Diagnostic assays for the polypeptide include methods that utilise the antibody and a label to detect the polypeptide in human body fluids or extracts of cells or tissues. The antibodies may be used with or without modification, and may be labelled by joining them, either covalently or non-covalently, with a reporter molecule. A wide variety of reporter molecules known in the art may be used, several of which are described above. Quantities of polypeptide expressed in subject, control and disease samples from biopsied tissues are compared with the standard values. Deviation between standard and subject values establishes the parameters for diagnosing disease. Diagnostic assays may be used to distinguish between absence, presence, and excess expression of polypeptide and to monitor regulation of polypeptide levels during therapeutic intervention. Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials or in monitoring the treatment of an individual patient.

A diagnostic kit of the present invention may comprise:

(a) a nucleic acid molecule of the present invention;

(b) a polypeptide of the present invention; or (c) a ligand of the present invention. In one aspect of the invention, a diagnostic kit may comprise a first container containing a nucleic acid probe that hybridises under stringent conditions with a nucleic acid molecule according to the invention; a second container containing primers useful for amplifying the nucleic acid molecule; and instructions for using the probe and primers for facilitating the diagnosis of disease. The kit may further comprise a third container holding an agent for digesting unhybridised RNA.

In an alternative aspect of the invention, a diagnostic kit may comprise an array of nucleic acid molecules, at least one of which may be a nucleic acid molecule according to the invention. To detect polypeptide according to the invention, a diagnostic kit may comprise one or more antibodies that bind to a polypeptide according to the invention; and a reagent useful for the detection of a binding reaction between the antibody and the polypeptide.

Such kits will be of use in diagnosing a disease or susceptibility to disease, particularly inflammation, oncology, or cardiovascular disease. Various aspects and embodiments of the present invention will now be described in more detail by way of example, with particular reference to the AD1 and AD2 polypeptides. It will be appreciated that modification of detail may be made without departing from the scope of the invention.

Brief description of the Figures Figure 1: Front page of the Biopendium™. Search initiated using the Nicastrin SwissProt Accession number Q92542.

Figure 2: Redundant Sequence Display page for Q92542.

Figure 3: NCBI protein report for BAA13383, Q92542 (KIAA0253, Nicastrin)

Figure 4: PFAM search results forBAA13383, Nicastrin. Figure 5A: Inpharmatica Genome Threader results of search using Q92542, Nicastrin. The arrow points to 1XJO.

Figure 5B: PSI-Blast results from search using Q92542, Nicastrin.

Figure 6: Sequence alignment of Q92542 (Nicastrin) and 1XJO. Figure 7A: LigEye for XJO, which illustrates the sites of interaction of the zinc ion number

1 and IXJO.

Figure 7B: LigEye for XJO, which illustrates the sites of interaction of the zinc ion number

2 and IXJO. Figure 8: RasMol view of IXJO, illustrating the amino acid residues (ball stractures) that are conserved in the zinc binding domain of the S. griseus aminopeptidase and Nicastrin

Example 1: Nicastrin (092542. KIAA0253)

In order to identify if the Inpharmatica Biopendium™ is able to add functional annotation to the Nicastrin protein, the Biopendium™ is searched for the Nicastrin sequence Q92542 (Figure 1). The database annotates this sequence as KIAA0253, hypothetical protein.

The redundant Sequence Display page (Figure 2) indicates that there is a National Centre for Biotechnology Information (NCBI), GeneBank (this is the U.S. public domain database for protein and gene sequence deposition) protein database record for Nicastrin, sequence BAA13383, as well as the SwissProt protein database record, Q92542. The NCBI Genebank protein database is viewed to examine if there is any further information that is known in the public domain relating to Nicastrin (Figure 3). BAA13383, KIAA0235 was cloned by a group of scientists in Chiba, Japan (Nagase, T. et al, (1999) DNA Res. 6(1), 63-70). There is no further annotation for BAA13383 except that the BAA13383 gene was cloned from brain tissue. The public domain information for this gene does not contain any suggestion whatsoever for the function of this protein.

In order to identify whether any other public domain annotation vehicle is able to annotate Nicastrin, the BAA13383 protein sequence is searched against the Protein Family Database of Alignment and HMM's (PFAM) database (Figure 4). The results identify that Nicastrin has no identifiable PFAMs annotation. This indicates that there are no identifiable domains in Nicastrin that may help decipher its function.

A search of the Biopendium™ using Nicastrin, Q92542, as the query sequence returns 19 Inpharmatica Genome Threader results (selection given in Figure 5A) and 20 PSI-Blast results (selection in Figure 5B). The 19 Genome Threader results identify Nicastrin as having a stracture similar to the transferrin receptor/aminopeptidase/carboxypeptidase stracture (Mahadevan, D., et al, Protein Science (1999) 8; 2546-2549). This is of interest as Nicastrin was identified as being involved in the processing of the B-APP protein, and the Inpharmatica Genome Threader identifies Nicastrin as an aminopeptidase.

The Inpharmatica Genome Threader has thus identified a sequence, Q92542, Nicastrin, as having a stracture similar to the aminopeptidase family. Having a structure similar to this domain suggests that Nicastrin is a protein that functions in the proteolytic cleavage of other proteins. The Inpharmatica Genome Threader identifies this with 99% confidence.

PSI-Blast (Figure 5B) is unable to identify this relationship in the forward direction; it is only the Inpharmatica Biopendium™ all by all forward and reverse search that is able to identify the homology of Nicastrin with the aminopeptidase family in the negative 3 iteration.

Therefore it is only the Inpharmatica Biopendium™ that is able to identify that the Nicastrin protein may function as an aminopeptidase.

Among the structures that the Inpharmatica Genome Threader returns as being similar to Nicastrin is the aminopeptidase from the bacteria Streptomyces griseus. This family of zinc peptidases is known to bind two zinc molecules at the active site of the peptidase. The zinc molecules are necessary for catalytic activity. The area of metal ion interaction is well conserved in this family. Viewing the alignment (Figure 6) of the query protein against the proteins identified as being of a similar stracture helps to visualize the areas of homology. Figure 6 illustrates the point that the area of metal ion interaction in IXJO is partially conserved in Nicastrin, Q92542.

In order to ensure that the protein identified is a homologue of the query sequence, the visualisation program LigEye (Figure 7A, 7B) and RasMol (Figure 8) are used. These visualization tools identify the active site of known protein stractures by indicating the amino acids with which known small molecule inhibitors interact at the active site. These interactions are through either a direct hydrogen bond or hydrophobic interactions. In this manner one can see if the active site fold/structure is conserved between the identified homologue and the chosen protein of known stracture.

This visualisation is shown with IXJO, which illustrates the sites of interaction of the 2 zinc ions with the S. griseus aminopeptidase (Figure 7Aand 7B). Zinc ion number 1 sees 4 different amino acids in thelXJO. The 4 amino acids of IXJO (His85, Asp87, Asp97, and Asp 160) are partially conserved in Q92542 (Figure 8, ball stractures). In order to bind the zinc ion the His85 residue is expected to be conserved. In Nicastrin this residue is an arginine 281, suggesting that the Nicastrin molecule may not bind zinc 1. Of the three other amino acids in the zinc 1 binding site, two are conserved, Asp284 and Glu364 (a conservative substitution) corresponding to Asp87 and Aspl60 in IXJO. Figure 8B identifies the zinc 2 binding residues in IXJO, Asp 97, Glu 132, and His247. The corresponding amino acids in Nicastrin are Pro293, Glu333, and His449. This illustrates that the Zinc 2 site may be conserved in Nicastrin.

Figure 8 illustrates the residues of IXJO involved in biding of the two zinc ions that are conserved in the Nicastrin molecule. As there is not 100% conservation of these amino acids in Nicastrin, it may not be able to bind the two zinc molecules. Hence, Nicastrin may not be fully catalytically competent. Nicastrin may still be able to bind peptide though. A similar mechanism has occurred with the aminopeptidase domain of the transferrin receptor (Mahadevan, D., et al, Protein Science (1999) 8; 2546-2549).

A publication in a peer-reviewed journal since the initial filing of this invention further exemplifies the methods of the invention (Fagan et al, Trends Biochem Sci 2001 26(4):213-4).

As the skilled reader will appreciate, there are numerous methods available to investigate and further validate the functional annotation described herein that assigns aminopeptidase activity to Nicastrin. An assay that examines the peptide binding capacity and enzymatic activity of Nicastrin could easily be configured, for example, see Yu G. et al, (Nature 2000 407:48-54), and Chen F. et al, (Nat Cell Biol. 2001 3(8):751-4).

SEQUENCE LISTING

SEQ ID NO:1 (Nucleotide coding sequence for Nicastrin aminopeptidase domain) gcaccaacct tcccactatg

781 tgccatgcag ctcttttcac acatgcatgc tgtcatcagc actgccacct gcatgcggcg 841 cagctccatc caaagcacct tcagcatcaa cccagaaatc gtctgtgacc ccctgtctga 901 ttacaatgtg tggagcatgc taaagcctat aaatacaact gggacattaa agcctgacga 961 cagggttgtg gttgctgcca cccggctgga tagtcgttcc tttttctgga atgtggcccc 1021 aggggctgaa agcgcagtgg cttcctttgt cacccagctg gctgctgctg aagctttgca 1081 aaaggcacct gatgtgacca ccctgccccg caatgtcatg tttgtcttct ttcaagggga 1141 aacttttgac tacattggca gctcgaggat ggtctacgat atggagaagg gcaagtttcc 1201 cgtgcagtta gagaatgttg actcatttgt ggagctggga caggtggcct taagaacttc 1261 attagagctt tggatgcaca cagatcctgt ttctcagaaa aatgagtctg tacggaacca 1321 ggtggaggat ctcctggcca cattggagaa gagtggtgct ggtgtccctg ctgtcatcct 1381 caggaggcca aatcagtccc agcctctccc accatcttcc ctgcagcgat ttcttcgagc 1441 tcgaaacatc tctggcgttg ttctggctga ccactctggt gccttccata acaaatatta 1501 ccagagtatt tacgacactg ctgagaacat taatgtgagc tatcccgaat ggctgagccc 1561 tgaagaggac ctgaactttg taacagacac tgccaaggcc ctggcagatg tggccacggt 1621 gctgggacgt gctctgtatg agcttgcagg ag

SEQ ID NO:2 (Nicastrin Aminopeptidase Domain Protein)

206 aptf plcamglfsh m avistatc mrrssigstf

241 sinpeivcdp lsdynvwsml kpinttgtlk pddrvwaat rldsrsffwn vapgaesava

301 sfvtqlaaae algkapdvtt lprnvmfvff qgetfdyigs srmvydmekg kfpvqlenvd 361 sfvelgqval rtslelwmht dpvsqknesv rngvedllat leksgagvpa vilrrpnqsq

421 plppsslqrf Irarnisgw ladhsgafhn kyyqsiydta eninvsypew lspeedlnfv

481 tdtakaladv atvlgralye lag

SEQ ID NO:3 (Nucleotide coding sequence for Nicastrin, AF240468) 1 tctgcagaat tcggcttgcg cctggaaaca cgaacttccg gtctcttagg ctccgggcca 61 cagagacggt gtcagtggta gcctagagag gccgctaaca gacaggagcc gaacgggggc 121 ttccgctcag cagagaggca agatggctac ggcagggggt ggctctgggg ctgacccggg 181 aagtcggggt ctccttcgcc ttctgtcttt ctgcgtccta ctagcaggtt tgtgcagggg 241 aaactcagtg gagaggaaga tatatatccc cttaaataaa acagctccct gtgttcgcct 301 gctcaacgcc actcatcaga ttggctgcca gtcttcaatt agtggagaca caggggttat 361 ccacgtagta gagaaagagg aggacctaca gtgggtattg actgatggcc ccaacccccc 421 ttacatggtt ctgctggaga gcaagcattt taccagggat ttaatggaga agctgaaagg 481 gagaaccagc cgaattgctg gtcttgcagt gtccttgacc aagcccagtc ctgcctcagg 541 cttctctcct agtgtacagt gcccaaatga tgggtttggt gtttactcca attcctatgg 601 gccagagttt gctcactgca gagaaataca gtggaattcg ctgggcaatg gtttggctta 661 tgaagacttt agtttcccca tctttcttct tgaagatgaa aatgaaacca aagtcatcaa 721 gcagtgctat caagatcaca acctgagtca gaatggctca gcaccaacct tcccactatg 781 tgccatgcag ctcttttcac acatgcatgc tgtcatcagc actgccacct gcatgcggcg 841 cagctccatc caaagcacct tcagcatcaa cccagaaatc gtctgtgacc ccctgtctga 901 ttacaatgtg tggagcatgc taaagcctat aaatacaact gggacattaa agcctgacga 961 cagggttgtg gttgctgcca cccggctgga tagtcgttcc tttttctgga atgtggcccc 1021 aggggctgaa agcgcagtgg cttcctttgt cacccagctg gctgctgctg aagctttgca 1081 aaaggcacct gatgtgacca ccctgccccg caatgtcatg tttgtcttct ttcaagggga 1141 aacttttgac tacattggca gctcgaggat ggtctacgat atggagaagg gcaagtttcc 1201 cgtgcagtta gagaatgttg actcatttgt ggagctggga caggtggcct taagaacttc 1261 attagagctt tggatgcaca cagatcctgt ttctcagaaa aatgagtctg tacggaacca 1321 ggtggaggat ctcctggcca cattggagaa gagtggtgct ggtgtccctg ctgtcatcct 1381 caggaggcca aatcagtccc agcctctccc accatcttcc ctgcagcgat ttcttcgagc 1441 tcgaaacatc tctggcgttg ttctggctga ccactctggt gccttccata acaaatatta 1501 ccagagtatt tacgacactg ctgagaacat taatgtgagc tatcccgaat ggctgagccc 1561 tgaagaggac ctgaactttg taacagacac tgccaaggcc ctggcagatg tggccacggt 1621 gctgggacgt gctctgtatg agcttgcagg aggaaccaac ttcagcgaca cagttcaggc 1681 tgatccccaa acggttaccc gcctgctcta tgggttcctg attaaagcca acaactcatg 1741 gttccagtct atcctcaggc aggacctaag gtcctacttg ggtgacgggc ctcttcaaca 1801 ttacatcgct gtctccagcc ccaccaacac cacttatgtt gtacagtatg ccttggcaaa 1861 tttgactggc acagtggtca acctcacccg agagcagtgc caggatccaa gtaaagtccc 1921 aagtgaaaac aaggatctgt atgagtactc atgggtccag ggccctttgc attctaatga 1981 gacggaccga ctcccccggt gtgtgcgttc tactgcacga ttagccaggg ccttgtctcc 2041 tgcctttgaa ctgagtcagt ggagctctac tgaatactct acatggactg agagccgctg 2101 gaaagatatc cgtgcccgga tatttctcat cgccagcaaa gagcttgagt tgatcaccct 2161 gacagtgggc ttcggcatcc tcatcttctc cctcatcgtc acctactgca tcaatgccaa 2221 agctgatgtc cttttcattg ctccccggga gccaggagct gtgtcatact gagsaggacc 2281 scagcttttc ttgccagctc agcagttcac ttcctagagc atctgtccca ctgggacaca 2341 accactaatt tgtcactgga acctccctgg gcctgtctca gattgggatt aacataaaag 2401 agtggaacta tccaaaagag acagggagaa ataaataaat tgcctccctt cctccgctcc 2461 cctttcccat caccccttcc ccatttcctc ttccttctct actcatgcca gattttggga 2521 ttacaaatag aagcttcttg ctcctgttta actccctagt tacccaccct aatttgccct 2581 tcaggaccct tctacttttt ccttcctgcc ctgtacctct ctctgctcct cacccccacc 2641 cctgtaccca gccaccttcc tgactgggaa ggacataaaa ggtttaatgt cagggtcaaa 2701 ctacattgag cccctgagga caggggcatc tctgggctga gcctactgtc tccttcccac 2761 tgtoctttct ccaggccctc agatggcaca ttagggtggg cgtgctgcgg gtgggtatcc 2821 cacctccagc ccacagtgct cagttgtact ttttattaag ctgtaatatc tatttttgtt 2881 tttgtctttt tcctttattc tttttgtaaa tatatatata atgagtttca ttaaaataga 2941 ttatcccac

SEQ ID NO:4 (Nicastrin Protein)

1 matagggsga dpgsrgllrl Isfcvllagl crgnsverki yiplnktapc vrllnathgi 61 gcgssisgdt gvihwekee dlgwvltdgp nppymvlles khftrdlmek Ikgrtsriag 121 lavsltkpsp asgfspsvgc pndgfgvysn sygpefahcr eiqwnslgng layedfsfpi 181 flledenetk vikqcyqdhn lsqngsaptf plcamqlfsh mhavistatc mrrssigstf 241 sinpeivcdp lsdynvwsml kpinttgtlk pddrvwaat rldsrsffwn vapgaesava 301 sfvtglaaae algkapdvtt Iprnvmfvff ggetfdyigs srmvydmekg kfpvglenvd 361 sfvelggyal rtslelwmht dpvsgknesv rngvedllat leksgagvpa vilrrpngsg 421 plppsslqrf lrarnisgw ladhsgafhn kyyqsiydta eninvsypew lspeedlnfv 481 tdtakaladv atvlgralye laggtnfsdt vqadpqtvtr llygflikan nswfqsilrq 541 dlrsylgdgp lghyiavssp tnttywgya lanltgtwn ltregcgdps kvpsenkdly 601 eyswvggplh snetdrlprc vrstarlara Ispafelsqw ssteystwte srwkdirari 661 fliaskelel itltvgfgil ifslivtyci nakadvlfia prepgavsy

Claims

I. A method of treatment of Alzheimer's disease in a patient comprising administering an aminopeptidase inhibitor to the patient.

2. An aminopeptidase inhibitor, for use in the treatment or diagnosis of Alzheimer's disease.

3. An aminopeptidase inhibitor that is effective to inhibit the aminopeptidase activity of the Nicastrin polypeptide.

4. An aminopeptidase inhibitor according to claim 3, wherein said aminopeptidase inhibitor interacts with the Nicastrin polypeptide.

5. Use of an aminopeptidase inhibitor in the manufacture of a medicament for the treatment or diagnosis of Alzheimer's disease.

6. A polypeptide consisting of the aminopeptidase domain of the Nicastrin polypeptide.

7. A polypeptide according to claim 6, wherein said aminopeptidase domain comprises residues 206 to 503 of the Nicastrin polypeptide sequence presented in SEQ ID No:2.

8. A polypeptide according to claim 6 or claim 7, which polypeptide:

(i) has the amino acid sequence as recited in SEQ ID NO: 2

(ii) is a fragment thereof having activity as an aminopeptidase or having an antigenic determinant in common with the polypeptide of (i); or (iv) is a functional equivalent of (i) or (ii).

9. A polypeptide which is a functional equivalent according to claim l(iii), is homologous to the amino acid sequence as recited in SEQ J-D NO:2 and has activity as an aminopeptidase.

10. A purified nucleic acid molecule which encodes a polypeptide according to any one of claims 6-9.

II. A purified nucleic acid molecule according to claim 10, which has the nucleic acid sequence as recited in SEQ ID NO:l or is a redundant equivalent or fragment thereof.

12. A purified nucleic acid molecule which hydridizes under high stringency conditions with a nucleic acid molecule according to claim 10 or claim 11.

13. A vector comprising a nucleic acid molecule as recited in any one of claims 10-12.

14. A host cell transformed with a vector according to claim 13.

15. A method of identifying a candidate ligand for the treatment of Alzheimer's disease comprising testing the ability of an aminopeptidase inhibitor to bind to the Nicastrin polypeptide or to a polypeptide according to any either one of claims 6-9 and selecting as a candidate agent, an aminopeptidase inhibitor that effectively inhibits the biological activity of the polypeptide.

16. A method according to claim 15, wherein a library of aminopeptidase inhibitors is tested in order to identify a candidate ligand that is effective to bind to the Nicastrin polypeptide or to a polypeptide according to any one of claims 6-9.

17. A method according to claim 15 or claim 16, wherein said ligand prevents the activity of the polypeptide as an aminopeptidase.

18. A method according to any one of claims 15-17, wherein said ligand inhibits the interaction of the aminopeptidase domain of Nicastrin with a naturally-occurring peptide, such as the full length B-APP, the beta-secretase cleaved version of the B-

APP, the alpha-secretase cleaved version of B-APP, presenilin 1, presenilin 2, or a member of the Notch protein family.

19. A ligand identified according to the method of any one of claims 15-18.

20. A ligand according to claim 19, which is a natural or modified substrate, ligand, enzyme, receptor or structural or functional mimetic that possesses affinity for the aminopeptidase domain of the Nicastrin protein.

21. A ligand according to claim 20, which is a peptide mimetic, a small drug molecule, or an antibody.

22. A ligand according to any one of claims 19-21, which inhibits the aminopeptidase activity of a polypeptide according to any one of claims 6-9.

23. A ligand according to any one of claims 19-21, which binds specifically to a polypeptide according to any one of claim 6-9 without inducing any of the biological effects of the polypeptide.

24. A ligand according to any one of claim 19-23, wherein said agent inhibits the interaction of the aminopeptidase domain of Nicastrin with a naturally-occurring peptide, such as the full length B-APP, the beta-secretase cleaved version of the B- APP, the alpha-secretase cleaved version of B-APP, presenilin 1, presenilin 2, or a member of the Notch protein family.

25. A ligand according to any one of claims 19-24, for use in the treatment or diagnosis of Alzheimer's disease.

26. Use of a ligand according to according to any one of claims 19 to 25 in the manufacture of a medicament for the treatment or diagnosis of Alzheimer's disease.

27. A method of inhibiting the function of the Nicastrin polypeptide comprising bringing the polypeptide into contact with an aminopeptidase inhibitor.

28. A method of diagnosing the susceptibility of a patient to Alzheimer's disease comprising examining the Nicastrin polypeptide or gene sequence in said patient or in tissue from said patient and diagnosing as susceptible those patients in which a mutation is contained in a region of the sequence that is responsible for aminopeptidase activity in the full length protein.

29. A method according to claim 28, wherein said method involves examining the genotype of the patient.

30. A method of diagnosing Alzheimer's disease in a patient comprising examining the Nicastrin polypeptide or gene sequence in said patient or in tissue from said patient and comparing said level of expression or activity to a control level, wherein a level that is different to said control level is indicative of disease.

31. A method according to claim 30, which comprises the steps of: (a) contacting a ligand according to any one of claims 19-24 with a biological sample under conditions suitable for the formation of a ligand-polypeptide complex; and (b) detecting said complex.

32. A method according to any one of claims 28-30, comprising the steps of: a) contacting a sample of tissue from the patient with a nucleic acid probe under stringent conditions that allow the formation of a hybrid complex between a nucleic acid molecule according to any one of claims 10-12 and the probe; b) contacting a control sample with said probe under the same conditions used in step a); and c) detecting the presence of hybrid complexes in said samples; wherein detection of levels of the hybrid complex in the patient sample that differ from levels of the hybrid complex in the control sample is indicative of disease.

33. A method according to any one of claims 28-30, comprising: a) contacting a sample of nucleic acid from tissue of the patient with a nucleic acid primer under stringent conditions that allow the formation of a hybrid complex between a nucleic acid molecule according to any one of claims 10-12 and the primer; b) contacting a control sample with said primer under the same conditions used in step a); and c) amplifying the sampled nucleic acid; and d) detecting the level of amplified nucleic acid from both patient and control samples; wherein detection of levels of the amplified nucleic acid in the patient sample that differ significantly from levels of the amplified nucleic acid in the control sample is indicative of disease.

34. A method according to either one of claims 28-29 comprising: a) obtaining a tissue sample from a patient being tested for disease; b) isolating a nucleic acid molecule according to any one of claims 10-12 from said tissue sample; and c) diagnosing the patient for disease by detecting the presence of a mutation which is associated with disease in the nucleic acid molecule as an indication of the disease.

35. The method of claim 34, further comprising amplifying the nucleic acid molecule to form an amplified product and detecting the presence or absence of a mutation in the amplified product.

36. The method of any one of claims 28, 29, 34 or 35, wherein the presence or absence of the mutation in the patient is detected by contacting said nucleic acid molecule with a nucleic acid probe that hybridises to said nucleic acid molecule under stringent conditions to form a hybrid double-stranded molecule, the hybrid double-stranded molecule having an unhybridised portion of the nucleic acid probe strand at any portion corresponding to a mutation associated with disease; and detecting the presence or absence of an unhybridised portion of the probe strand as an indication of the presence or absence of a disease-associated mutation.

37. A method according to any one of claims 28-36 that is carried out in vitro.

38. A polypeptide according to any one of claim 6-9, a nucleic acid molecule according to any one of claims 10-12, a vector according to claim 13, or a ligand according to any one of claims 19-24, for use in the therapy or diagnosis of disease.

39. A polypeptide, nucleic acid molecule, vector, ligand, or compound according to claim 38, wherein said disease is inflammation, oncology, or cardiovascular disease.

40. Use of a polypeptide according to any one of claim 6-9, a nucleic acid molecule according to any one of claims 10-12, a vector according to claim 13, or a ligand according to any one of claims 19-24 in the manufacture of a medicament for the treatment of an inflammatory, oncology, or cardiovascular disease.

41. Use of a polypeptide according to any one of claims 6-9 as an aminopeptidase domain.

42. A pharmaceutical composition comprising a polypeptide according to any one of claim 6-9, a nucleic acid molecule according to any one of claims 10-12, a vector according to claim 13, or a ligand according to any one of claims 19-24.

43. A vaccine composition comprising a polypeptide according to any one of claims 6-9 or a nucleic acid molecule according to any one of claims 10-12.

44. A method of monitoring the therapeutic treatment of disease in a patient, comprising monitoring over a period of time the level of expression or activity of the Nicastrin polypeptide, or the level of expression of a nucleic acid molecule encoding the Nicastrin polypeptide in tissue from said patient, wherein altering said level of expression or activity over the period of time towards a control level is indicative of regression of said disease.

45. A kit useful for diagnosing disease comprising a first container containing a nucleic acid probe that hybridises under stringent conditions with a nucleic acid molecule according to any one of claims 10-12; a second container containing primers useful for amplifying said nucleic acid molecule; and instructions for using the probe and primers for facilitating the diagnosis of disease.

46. The kit of claim 45, further comprising a third container holding an agent for digesting unhybridised RNA.

47. A kit comprising an array of nucleic acid molecules, at least one of which is a nucleic acid molecule according to any one of claims 10-12.

48. A kit comprising one or more antibodies that bind to a polypeptide as recited in any one of claims 6-9; and a reagent useful for the detection of a binding reaction between said antibody and said polypeptide.

49. A transgenic or knockout non-human animal that has been transformed to express higher, lower or absent levels of a polypeptide according to any one of claims 6-9.

50. A method for screening for a candidate agent effective to treat disease, by contacting a non-human transgenic animal according to claim 49 with a candidate compound and determining the effect of the compound on the disease of the animal.