WO2003100061A2

WO2003100061A2 - Three finger toxin fold proteins

Info

Publication number: WO2003100061A2
Application number: PCT/GB2003/002214
Authority: WO
Inventors: Mark Douglas Davies; Richard Joseph Fagan; Christopher Benjamin Phelps; Christine Power
Original assignee: Inpharmatica Limited
Priority date: 2002-05-23
Filing date: 2003-05-22
Publication date: 2003-12-04
Also published as: WO2003100061A3; AU2003227968A1; GB0211903D0

Abstract

This invention relates to novel proteins, termed INSPO62 and INSPO63, herein identified as members of the three finger toxin fold family and to the use of these proteins and nucleic acid sequences from the encoding genes in the diagnosis, prevention and treatment of disease.

Description

Three Finger Toxin Fold Proteins

This invention relates to proteins, termed INSP062 and INSP063, herein identified as members of the three finger toxin fold family and to the use of these proteins and nucleic acid sequences from the encoding genes 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.

Introduction to Secreted Proteins

The ability of cells to make and secrete extracellular proteins is central to many biological processes. Enzymes, growth factors, extracellular matrix proteins and signalling molecules are all secreted by cells. This is through fusion of a secretory vesicle with the plasma membrane. In most cases, but not all, proteins are directed to the endoplasmic reticulum and into secretory vesicles by a signal peptide. Signal peptides are cis-acting sequences that affect the transport of polypeptide chains from the cytoplasm to a membrane bound compartment such as a secretory vesicle. Polypeptides that are targeted to the secretory vesicles are either secreted into the extracellular matrix or are retained in the plasma membrane. The polypeptides that are retained in the plasma membrane will have one or more transmembrane domains or will be anchored in the membrane via an attached lipid molecule. Examples of secreted proteins that play a central role in the functioning of a cell are cytokines, hormones, extracellular matrix proteins (adhesion molecules), proteases, and growth and differentiation factors.

Introduction to the Three Finger Toxin Fold

The three finger toxin fold was originally identified in snake venom toxin polypeptides. These snake toxins exhibit a wide variety of pharmacological effects via interaction with postsynaptic membranes, and include the α-neurotoxins that block acetylcholine receptors, cardiotoxins that damage cell membranes, antiplatelet toxins, fasciculins that inhibit acetylcholine esterase and L-type calcium-channel blockers. For example, bucandin is a neurotoxic polypeptide isolated from the venom of the Malayan krait (Biochem. J., 2001, 360(Part 3):539-48).

These snake venom polypeptides are found to contain between 60 and 75 amino acid residues and between 4 and 5 disulphide bridges (formed between 8 or 10 cysteine residues with a characteristic spacing that is conserved across members of the three finger toxin fold family). They share a common structural fold which consists of a central core containing most of the disulphide bridges from which three antiparallel β-stranded loops extend like fingers (Tseπioglou et al. , Proc. Natl. Acad. Sci. U.S.A, 1977, 74:971 -974).

Although the snake venom toxins remain by far the most prominent subset of the three finger toxin fold family, it has been found since the initial characterisation of three finger toxic proteins that this fold is also shared by several larger mammalian glycosylphosphatidylinositol-anchored (GPI)-anchored cell-surface glycoproteins. Examples of these three finger toxin fold-containing GPI-linked polypeptides include urokinase-type plasminogen activator receptor, squid glycoprotein 2 (sgp-2), bone morphogenetic protein (BMP) and activin receptors (types I and II), CD59, and lymphocyte antigens (for example, Sea 2, the Thy-l/Ly-6 multigene family, thymocyte B cell antigen and prostate stem cell antigen). A post-translational cleavage of a C-terminal 'anchor signal' (a short string of hydrophobic residues) in these polypeptides allows attachment of a glycosylphosphatidylinositol (GPI) moiety to the polypeptide, leading to protein retention and function at the cell surface. The analysis of the C-terminal amino acid sequences of known GPI-anchored proteins by multiple alignment has allowed for the accurate prediction of GPI-anchor positions in other proteins (Mao et al, Proc. Natl. Acad. Sci. U.S.A., 1996, 93(12):5910-4; Behrendt et al, J. Biol. Chem., 1991, 266:7842-7847; Plough et al, J. Biol. Chem., 1993, 268:17539-17546). The physiological and pathological roles of some of these GPI-anchored mammalian members of the three finger toxin fold will now be described.

The Urokinase-type Plasminogen Activator Receptor

The urokinase-type plasminogen activator receptor (u-PAR), also known as CD87, is involved in the regulation of cell-surface plasminogen activation in several physiological and pathological conditions. Recent evidence suggests that the uPAR is also involved in processes that are not related to plasminogen activation, including cell adhesion and transmission of extracellular signals across the plasma membrane. The u-PAR binds to urokinase-type plasminogen activator (u-PA) and is responsible for its proteolysis- independent signal transduction activation effects (Mohanam et al, Frontiers in Biosci., 1999, 4:178-187). Investigations in cancer patients indicate that uPAR can be valuable as a prognostic marker, and the expression level of u-PAR has been shown to be increased in glioblastoma cell lines and in brain tumor tissues. The use of antisense molecules to down- regulate uPAR expression in glioblastoma cells has been shown to decrease both migration and invasion in vitro as well as tumor formation in nude mice (Mohanam et al, Frontiers in Biosci., 1999, 4:178-187). Therefore, the u-PAR is an important multifunctional protein implicated in variety of physiological and pathological conditions.

Bone Morphogenetic Protein and Activin Receptors

Bone morphogenetic protein (BMP) and Activin receptors are closely related and bind members of the TGF-β superfamily. Members of the TGF-β superfamily interact with two distinct transmembrane receptor serine kinases (type I and type II, (Greenwald et al, Nature Struc. Biol., 1999, 6(l):18-22). Activin (a member of the TGF-β superfamily of growth factors) elicits diverse biological responses upon binding to its receptors, such as cell growth, differentiation, and pattern formation during development. Surprisingly, the type II activin receptor (ActRII) was found to possess the three finger toxin fold. Notably, ActRII was the first three finger toxin fold protein found that is produced and functions within the same organism (Greenwald et al, Nature Struc. Biol., 1999, 6(l):18-22), in contrast to the snake venom polypeptides. The bone morphogenetic protein (BMP) family forms another category of TGF-β molecules, and is a diverse family of secreted signalling molecules whose members are involved in controlling a variety of developmental processes.

CD59

The expression of CD59 (also called H19, HRF20, MACIF, MIRL, P-18 or protectin) at the cell surface of human erythrocytes and leukocytes serves to inhibit formation of the membrane attack complex (MAC) at the surface of those cells, protecting those cells from lysis by the complement system. CD59 also plays a transmembrane signalling role in T cell activation via the GPI-anchor that attaches the polypeptide to the cell membrane. Genetic defects in CD59 expression or function, including defects in GPI-anchor attachment that cause a reduction or loss of CD59 cell-surface expression, produce the symptoms of the disease paroxysmal nocturnal hemoglobinuria (PNH). This CD59 deficiency in humans causes haemolytic anaemia and thrombosis leading to cerebral infarction (Wiedmer et al, Blood, 1993, 82(4): 1192-6).

The LY-6 Family

The LY-6 family of genes encodes a group of small GPI-anchored cysteine-rich cell surface proteins of the three-finger toxin fold. This multigene family maps to the 8q24 chromosomal region in humans. Members of the LY-6 family are differentially expressed in several hematopoietic lineages, especially T-lymphocytes, suggesting that Ly-6 molecules may be involved in the development and homeostasis of hematopoietic cells. Members of the LY-6 family appear to function in signal transduction and cell activation (Rock et al, Immunol. Rev., 1989, 111:195-224; Shevach et al, Immunol. Today, 1989, 10:195-200; Fleming et al, J. Immunol., 150:5379-5390; MacNeil et al, J. Immunol., 151:6913-6923; Classon et al, Proc. Natl. Acad. Sci. U.S.A., 91:5296-5300). At least 18 genes have been discovered in mice to be members of the LY-6 multigene family (Kamiura et al, Genomics, 1992, 12:89-105) and several members have now been discovered in humans including RIG-E (Mao et al, Proc. Natl. Acad. Sci. U.S.A., 1996, 93(12):5910-4) and LY-6H (Horie et al, Genomics, 1998, 53(3):365-8). Notably, LY6H was revealed to be highly expressed in particular subdivisions of human brain as well as in MOLT-3 and MOLT-4 acute lymphoblastic leukemia cells. Thus, members of the LY-6 multigene family in humans may function in both the central nervous system and the immune system. The product of the RIG-E gene shares sequence homology with CD59 (described above) and with a number of growth factor receptors, but shows higher sequence homology to the murine LY-6 multigene family. Expression of RIG-E is not limited to myeloid differentiation, since it is also expressed in thymocytes and a variety of other tissues. For example, the highest expression levels of RIG-E were found in T-acute lymphoblastic leukemia cells and in ovary cells. Numerous diseases and disease types have been linked to the chromosome 8q24 region, including neuropathies, epilepsies and Mai de Meleda disease. Mai de Meleda disease is not only linked to the chromosome 8q24 region, but is also specifically linked to the lymphocyte antigen family.

Prostate Stem Cell Antigen

Prostate stem cell antigen (PSCA), which also maps to chromosome 8q24, is a homologue of the Ly-6 family of lymphocyte cell surface antigens. The PSCA polypeptide is a 123 amino acid glycoprotein with ~30% identity to stem cell antigen 2 (Sea 2), a cell surface marker of immature thymic lymphocytes (Antica et al, Immunol. Letts., 1997, 55:47-51). PSCA mRNA is expressed in the basal cells of the normal prostate and in more that 80% of prostate cancers (Gu et al, Oncogene, 2000, 19:1288-1296). It has also been shown by Reiter et al that PSCA may be involved in the progression of prostate cancer. As a result PSCA is a potential target in the treatment and diagnosis of prostate cancer. Prostate cancer was recently calculated to be the most common cancer diagnosis and the second most prominent cause of cancer-related death in American men (Lalani et al, Cancer Metas. Rev., 1997, 16:29-66), and therefore the identification of protein related to PSCA is of great importance.

The polypeptides described above illustrate the diverse physiological and pathological pathways in which the mammalian members of the three finger toxin fold family are implicated. Notably, these polypeptides are retained at the cell membrane by a GPI-anchor moiety, which is essential for the polypeptide' s biological function at the cell surface. Therefore, the identification of further mammalian members of the three finger toxin fold family, and especially those which are held at the cell surface by a GPI-anchor, is of extreme importance in increasing the understanding of the underlying pathways that lead to the disease states and associated disease states mentioned above, and in developing more effective gene and/or drug therapies to treat these disorders.

THE INVENTION

The invention is based on the discovery that the INSP062 and INSP063 proteins are members of the three finger toxin fold family.

In one embodiment of the first aspect of the invention, there is provided a polypeptide which:

(i) comprises the amino acid sequence as recited in SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:28 and/or SEQ ID NO:30; (ii) is a fragment thereof which is a member of the three finger toxin fold family, or has an antigenic determinant in common with the polypeptides of (i); or

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

Preferably, the polypeptide according to this first aspect of the invention:

(i) comprises the amino acid sequence as recited in SEQ ID NO: 18, SEQ ID NO:28 or SEQ IDNO:30;

(ii) is a fragment thereof which is a member of the three finger toxin fold family, or has an antigenic determinant in common with the polypeptides of (i); or

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

According to a second embodiment of this first aspect of the invention, there is provided a polypeptide which:

(i) consists of the amino acid sequence as recited in SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:28 and/or SEQ ID NO:30;

(ii) is a fragment thereof which is a member of the three finger toxin fold family, or having an antigenic determinant in common with the polypeptides of (i); or (iii) is a functional equivalent of (i) or (ii).

The polypeptide having the sequence recited in SEQ ID NO: 12 is referred to hereafter as "INSP062 exon 1 polypeptide". The polypeptide having the sequence recited in SEQ ID NO: 14 is referred to hereafter as "INSP062 exon 2 polypeptide". The polypeptide having the sequence recited in SEQ ID NO: 16 is referred to hereafter as "INSP062 exon 3 polypeptide". The polypeptide having the sequence recited in SEQ ID NO: 18 is referred to hereafter as the "INSP062 polypeptide".

Although the Applicant does not wish to be bound by this theory, it is postulated that the first 16 amino acids of the INSP062 exon 1 polypeptide form a signal peptide, as shown in the schematic representation below:

>INSP062

|SAGCATTPARNLSCYQCFKVSSWTECPPTWCSPLDQVCISNEW

VSFK SVRVLLSKRCAPRCPNDNMKFEWSPAPMVQGVITRRCCSWA CNRALTPQEGRA

LRGGLLLQVGLSLLRALL

The INSP062 exon 1 and full length INSP062 polypeptide sequences without this postulated signal sequence are recited in SEQ ID NO:27 and SEQ ID NO:28, respectively.

It is also considered by the Applicant that the first 21 amino acids of the INSP062 exon 1 polypeptide may form a signal peptide, as shown in the schematic representation below:

>INSP062

fTTPARNLSCYQCFKVSSWTECPPTWCSPLDQVCISNE

VSFKWSVRVLLSKRCAPRCPNDN KFE SPAPMVQGVITRRCCS ALCNR-_-[-TPQEGRWA

LRGGLLLQVGLSLLRALL

The INSP062 exon 1 and full length INSP062 polypeptide sequences without this postulated signal sequence are recited in SEQ ID NO:29 and SEQ ID NO:30, respectively.

The polypeptide having the sequence recited in SEQ ID NO:27 is referred to hereafter as "the INSP062 exon 1 mature polypeptide A". The polypeptide having the sequence recited in SEQ ID NO:28 is referred to hereafter as "the INSP062 mature polypeptide A". The polypeptide having the sequence recited in SEQ ID NO:29 is referred to hereafter as "the INSP062 exon 1 mature polypeptide B". The polypeptide having the sequence recited in SEQ ID NO:30 is referred to hereafter as "the INSP062 mature polypeptide B".

The term "INSP062 polypeptides" as used herein includes polypeptides comprising the INSP062 exon 3 polypeptide, the INSP062 polypeptide, the 1NSP062 mature polypeptide A and the INSP062 mature polypeptide B.

In a third embodiment of the first aspect of the invention, there is provided a polypeptide which:

(i) comprises the amino acid sequence as recited in SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO: 31 and/or SEQ ID NO:32;

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

Preferably, the polypeptide according to this embodiment of the first aspect of the invention:

(i) comprises the amino acid sequence as recited in SEQ ID NO:26 or SEQ ID NO:32;

(iii) is a functional equivalent of (i) or (ii). According to a fourth embodiment of this first aspect of the invention, there is provided a polypeptide which:

(i) consists of the amino acid sequence as recited in SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO: 31 and/or SEQ ID NO:32;

(ii) is a fragment thereof which is a member of the three finger toxin fold family, or having an antigenic determinant in common with the polypeptides of (i); or

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

The polypeptide having the sequence recited in SEQ ID NO:20 is referred to hereafter as "INSP063 exon 1 polypeptide". The polypeptide having the sequence recited in SEQ ID NO:22 is referred to hereafter as "INSP063 exon 2 polypeptide". The polypeptide having the sequence recited in SEQ ID NO:24 is referred to hereafter as "INSP063 exon 3 polypeptide". The polypeptide having the sequence recited in SEQ ID NO:26 is referred to hereafter as the "INSP063 polypeptide".

Although the Applicant does not wish to be bound by this theory, it is postulated that the first 16 amino acids of the INSP063 polypeptide form a signal peptide, as shown in the schematic representation below:

>INSP063

im m mmsMm |ECFQCYGVLDPSLCHPVSYPMQAQSCPSSVVTGTIDGES VGPS

VCRQGRSSATLARAVAPLCAQI NLTHPVVPGGSYPTEIEDRLIDSKIEKLDMTCCENSL

RNKAATVRRGLWCQAVRELLLSLSPFLWALL

Due to the short length of the INSP063 exon 1 polypeptide, this signal sequence is postulated to direct the cleavage of the INSP063 polypeptide between the third and fourth amino acids of the INSP063 exon 2 polypeptide. The INSP063 exon 2 and full length INSP063 polypeptide sequences without the postulated signal sequence are recited in SEQ ID NO:31 and SEQ ID NO:32, respectively. The polypeptide having the sequence recited in SEQ ID NO:31 is referred to hereafter as "the INSP063 exon 2 mature polypeptide". The polypeptide having the sequence recited in SEQ ID NO:32 is referred to hereafter as "the INSP063 mature polypeptide".

The term "INSP063 polypeptides" as used herein includes polypeptides comprising the INSP063 exon 1 polypeptide, the INSP063 exon 2 polypeptide, the INSP063 exon 2 mature polypeptide, the 1NSP063 exon 3 polypeptide, the INSP063 polypeptide and the INSP063 mature polypeptide.

In a second aspect, the invention provides a purified nucleic acid molecule which encodes a polypeptide of the first aspect of the invention. Preferably, the purified nucleic acid molecule comprises the nucleic acid sequence as recited in SEQ ID NO: 15 (encoding the INSP062 exon 3 polypeptide), SEQ ID NO: 17 (encoding the INSP062 polypeptide), SEQ ID NO: 19 (encoding the INSP063 exon 1 polypeptide), SEQ ID NO:21 (encoding the INSP063 exon 2 polypeptide), SEQ ID NO:23 (encoding the INSP063 exon 3 polypeptide) and/or SEQ ID NO:25 (encoding the INSP063 polypeptide) or is a redundant equivalent or fragment of any one of these sequences.

The invention further provides that the purified nucleic acid molecule consists of the nucleic acid sequence as recited in SEQ ID NO: 15 (encoding the INSP062 exon 3 polypeptide), SEQ ID NO: 17 (encoding the INSP062 polypeptide), SEQ ID NO: 19 (encoding the INSP063 exon 1 polypeptide), SEQ ID NO:21 (encoding the INSP063 exon 2 polypeptide), SEQ ID NO:23 (encoding the LNSP063 exon 3 polypeptide) and/or SEQ ID NO:25 (encoding the INSP063 polypeptide) or is a redundant equivalent or fragment of any one of these sequences.

In a third aspect, the invention provides a purified nucleic acid molecule which hybridizes under high stringency conditions with a nucleic acid molecule of the second aspect of the invention.

In a fourth aspect, the invention provides a vector, such as an expression vector, that contains a nucleic acid molecule of the second or third aspect of the invention.

In a fifth aspect, the invention provides a host cell transformed with a vector of the fourth aspect of the invention. In a sixth aspect, the invention provides a ligand wliich binds specifically to members of the three finger toxin fold family of the first aspect of the invention.

In a seventh aspect, the invention provides a compound that is effective to alter the expression of a natural gene which encodes a polypeptide of the first aspect of the invention or to regulate the activity of a polypeptide of the first aspect of the invention. A compound of the seventh aspect of the invention may either increase (agonise) or decrease (antagonise) the level of expression of the gene or the activity of the polypeptide.

Importantly, the identification of the function of the INSP062 and INSP063 polypeptides allows for the design of screening methods capable of identifying compounds that are effective in the treatment and/or diagnosis of disease. Ligands and compounds according to the sixth and seventh aspects of the invention may be identified using such methods. These methods are included as aspects of the present invention.

In an eighth aspect, the invention provides a polypeptide of the first aspect of the invention, or a nucleic acid molecule of the second or third aspect of the invention, or a vector of the fourth aspect of the invention, or a ligand of the sixth aspect of the invention, or a compound of the seventh aspect of the invention, for use in therapy or diagnosis of diseases in which members of the three finger toxin fold family are implicated. Such diseases may include cell proliferative disorders, including neoplasm, melanoma, lung, colorectal, breast, pancreas, head and neck and other solid tumours; myeloproliferative disorders, such as leukemia, non-Hodgkin lymphoma, leukopenia, thrombocytopenia, angiogenesis disorder, Kaposis' sarcoma; autoimmune/inflammatory disorders, including allergy, inflammatory bowel disease, arthritis, psoriasis and respiratory tract inflammation, asthma, and organ transplant rejection; cardiovascular disorders, including hypertension, oedema, angina, atherosclerosis, thrombosis, sepsis, shock, reperfusion injury, and ischemia; neurological disorders including central nervous system disease, Alzheimer's disease, brain injury, amyotrophic lateral sclerosis, and pain; developmental disorders; metabolic disorders including diabetes mellitus, osteoporosis, and obesity, AIDS and renal disease; infections including viral infection, bacterial infection, fungal infection and parasitic infection and other pathological conditions. Preferably, the diseases are those in which lymphocyte antigens are implicated. These molecules may also be used in the manufacture of a medicament for the treatment of such diseases. These moieties of the first, second, third, fourth, fifth, sixth or seventh aspect of the invention may also be used in the manufacture of a medicament for the treatment of such diseases.

In a ninth aspect, 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 of the first aspect of the invention or the activity of a polypeptide of the first aspect of the invention 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.

A preferred method for detecting polypeptides of the first aspect of the invention comprises the steps of: (a) contacting a ligand, such as an antibody, of the sixth 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 ninth 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.

Preferably, the disease diagnosed by a method of the ninth aspect of the invention is a disease in which members of the three finger toxin fold family are implicated, as described above.

In a tenth aspect, the invention provides for the use of a polypeptide of the first aspect of the invention as a three finger toxin fold protein. Suitable uses of the polypeptides of the invention as three finger toxin fold proteins include use as a regulator of cellular growth, metabolism or differentiation, use as part of a receptor/ligand pair and use as a diagnostic marker for a physiological or pathological condition.

In an eleventh aspect, the invention provides a pharmaceutical composition comprising a polypeptide of the first aspect of the invention, or a nucleic acid molecule of the second or third aspect of the invention, or a vector of the fourth aspect of the invention, or a host cell of the fifth aspect of the invention, or a ligand of the sixth aspect of the invention, or a compound of the seventh aspect of the invention, in conjunction with a pharmaceutically- acceptable carrier.

In a twelfth aspect, the present invention provides a polypeptide of the first aspect of the invention, or a nucleic acid molecule of the second or third aspect of the invention, or a vector of the fourth aspect of the invention, or a host cell of the fifth aspect of the invention, or a ligand of the sixth aspect of the invention, or a compound of the seventh aspect of the invention, for use in the manufacture of a medicament for the diagnosis or treatment of a disease. Preferably, the disease is a disease in which members of the three finger toxin fold family are implicated, as described above.

In a thirteenth aspect, the invention provides a method of treating a disease in a patient comprising administering to the patient a polypeptide of the first aspect of the invention, or a nucleic acid molecule of the second or third aspect of the invention, or a vector of the fourth aspect of the invention, or a host cell of the fifth aspect of the invention or a ligand of the sixth aspect of the invention, or a compound of the seventh aspect of the invention.

For diseases in which the expression of a natural gene encoding a polypeptide of the first aspect of the invention, or in which the activity of a polypeptide of the first aspect of the invention, is lower in a diseased patient when compared to the level of expression or activity in a healthy patient, the polypeptide, nucleic acid molecule, vector, host cell, ligand or compound administered to the patient should be an agonist. Conversely, for diseases in which the expression of the natural gene or activity of the polypeptide is higher in a diseased patient when compared to the level of expression or activity in a healthy patient, the polypeptide, nucleic acid molecule, vector, host cell, 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.

Preferably, the disease is a disease in which members of the three finger toxin fold family are implicated, as described above.

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

Preferably, the disease is a disease in which members of the three finger toxin fold family are implicated, as described above. 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 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 & SJ. Higgins eds. 1984); Transcription and Translation (B.D. Hames & SJ. 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 (D.M. 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 first 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 first aspect of the invention may be polypeptides that are homologous to the INSP062 and INSP063 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 wliich the polypeptides are derived) and mutants (such as mutants containing amino acid substitutions, insertions or deletions) of the INSP062 and INSP063 polypeptides. 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 He; 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 INSP062 or INSP063 polypeptides, or with active fragments thereof, of greater than 80%. More preferred polypeptides have degrees of identity of greater than 85%, 90%, 95%, 98% or 99%, respectively. The predicted translation product of the AAX19982 EST, whose sequence is recited in SEQ ID NO: 10, is specifically excluded from the scope of this aspect of the invention.

The functionally-equivalent polypeptides of the first 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 PCT application WO 01/69507) to identify polypeptides of presently-unknown function which, while having low sequence identity as compared to the INSP062 and INSP063 polypeptides, are predicted to be members of the three finger toxin fold family, by virtue of sharing significant structural homology with the INSP062 and INSP063 polypeptide sequences. By "significant structural homology" is meant that the Inpharmatica Genome Threader predicts two proteins to share structural homology with a certainty of 10% and above.

The polypeptides of the first aspect of the invention also include fragments of the INSP062 and INSP063 polypeptides and fragments of the functional equivalents of the INSP062 and INSP063 polypeptides, provided that those fragments are members of the three finger toxin fold family or have an antigenic determinant in common with the INSP062 and INSP063 polypeptides.

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 amino acid sequence of the INSP062 and INSP063 polypeptides or one of their 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 first aspect of the invention.

By "substantially greater affinity" we mean that there is a measurable increase in the affinity for a polypeptide of the invention as compared with the affinity for known cell- surface receptors. Preferably, the affinity is at least 1.5-fold, 2-fold, 5-fold 10-fold, 100-fold, 10³-fold, 10⁴- fold, 10⁵-fold or 10⁶-fold greater for a polypeptide of the invention than for known cell- surface receptor polypeptides.

If polyclonal antibodies are desired, a selected mammal, such as a mouse, rabbit, goat or horse, may be immunised with a polypeptide of the first 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 polypeptides of the first 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 first 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 wliich 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 second and third aspects of the invention are those which encode a polypeptide sequence as recited in SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID

NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:31 and SEQ ID NO:32 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 a polypeptide of this invention may be identical to the coding sequence of one or more of the nucleic acid molecules disclosed herein. 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:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:31 or SEQ ID NO:32. Such nucleic acid molecules may include, but are not limited to, the coding sequence for the mature polypeptide by itself; the coding sequence for the mature polypeptide and additional coding sequences, such as those encoding a leader or secretory sequence, such as a pro-, pre- or prepro- polypeptide sequence; the coding sequence of the mature polypeptide, with or without the aforementioned additional coding sequences, together with further additional, non-coding sequences, including non- coding 5' and 3' sequences, such as the transcribed, non-translated sequences that play a role in transcription (including termination signals), ribosome binding and mRNA stability. The nucleic acid molecules may also include additional sequences which encode additional amino acids, such as those which provide additional functionalities.

The nucleic acid molecules of the second and third aspects of the invention may also encode the fragments or the functional equivalents of the polypeptides and fragments of the first 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 first 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 second or third aspects of the invention. For example, to screen peptide libraries for inhibitors of the 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 NaCI, 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 INSP062 and INSP063 polypeptides 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 such coding sequences, or is 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%, 99% or more 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 INSP062 and INSP063 polypeptides. Nucleic acid molecules that encode the predicted translation product of the AAX19982 EST, whose sequence is recited in SEQ ID NO: 10, are specifically excluded from the scope of this aspect of the invention. 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 INSP062 and INSP063 polypeptides 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 INSP062 and INSP063 polypeptides 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 l, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23 and SEQ ID NO:25), 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 wliich 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 wliich 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, N. 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 mRΝAs 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 mRΝAs with that of mRΝAs 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.

Gene silencing approaches may also be undertaken to down-regulate endogenous expression of a gene encoding a polypeptide of the invention. RΝA interference (R Ai) (Elbashir, SM et al, Nature 2001, 411, 494-498) is one method of sequence specific post- transcriptional gene silencing that may be employed. Short dsRNA oligonucleotides are synthesised in vitro and introduced into a cell. The sequence specific binding of these dsRNA oligonucleotides triggers the degradation of target mRNA, reducing or ablating target protein expression.

Efficacy of the gene silencing approaches assessed above may be assessed through the measurement of polypeptide expression (for example, by Western blotting), and at the RNA level using TaqMan-based methodologies.

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, transfected 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 pSportl™ 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 subtϊlis 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 mRNA 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, wliich 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 IMAC (immobilised metal ion affinity chromatography as described in Porath, J. et al. (1992), Prot. Exp. Purif. 3: 263-281) while the thioredoxm 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 first aspect of the invention or to regulate the activity of a polypeptide of the first aspect of the invention.

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.

A preferred method for identifying an agonist or antagonist compound of a polypeptide of the present invention comprises:

(a) contacting a cell expressing on the surface thereof the polypeptide according to the first aspect of the invention, the polypeptide being associated with a second component capable of providing a detectable signal in response to the binding of a compound to the polypeptide, with a compound to be screened under conditions to permit binding to the polypeptide; and

(b) determining whether the compound binds to and activates or inhibits the polypeptide by measuring the level of a signal generated from the interaction of the compound with the polypeptide.

A further preferred method for identifying an agonist or antagonist of a polypeptide of the invention comprises:

(a) contacting a cell expressing on the surface thereof the polypeptide, the polypeptide being associated with a second component capable of providing a detectable signal in response to the binding of a compound to the polypeptide, with a compound to be screened under conditions to permit binding to the polypeptide; and (b) determining whether the compound binds to and activates or inhibits the polypeptide by comparing the level of a signal generated from the interaction of the compound with the polypeptide with the level of a signal in the absence of the compound.

In further preferred embodiments, the general methods that are described above may further comprise conducting the identification of agonist or antagonist in the presence of labelled or unlabelled ligand for the polypeptide.

In another embodiment of the method for identifying an agonist or antagonist of a polypeptide of the present invention comprises: determining the inhibition of binding of a ligand to cells which have a polypeptide of the invention on the surface thereof, or to cell membranes containing such a polypeptide, in the presence of a candidate compound under conditions to permit binding to the polypeptide, and determining the amount of ligand bound to the polypeptide. A compound capable of causing reduction of binding of a ligand is considered to be an agonist or antagonist. Preferably the ligand is labelled.

More particularly, a method of screening for a polypeptide antagonist or agonist compound comprises the steps of: (a) incubating a labelled ligand with a whole cell expressing a polypeptide according to the invention on the cell surface, or a cell membrane containing a polypeptide of the invention,

(b) measuring the amount of labelled ligand bound to the whole cell or the cell membrane;

(c) adding a candidate compound to a mixture of labelled ligand and the whole cell or the cell membrane of step (a) and allowing the mixture to attain equilibrium; (d) measuring the amount of labelled ligand bound to the whole cell or the cell membrane after step (c); and

(e) comparing the difference in the labelled ligand bound in step (b) and (d), such that the compound which causes the reduction in binding in step (d) is considered to be an agonist or antagonist.

The 1NSP062 and INSP063 polypeptides of the present invention may modulate cellular growth and differentiation. Thus, the biological activity of the INSP062 and INSP063 polypeptides can be examined in systems that allow the study of cellular growth and differentiation such as organ culture assays or in colony assay systems in agarose culture. Stimulation or inhibition of cellular proliferation may be measured by a variety of assays.

For example, for observing cell growth inhibition, one can use a solid or liquid medium. In a solid medium, cells undergoing growth inhibition can easily be selected from the subject cell group by comparing the sizes of colonies formed. In a liquid medium, growth inhibition can be screened by measuring culture medium turbity or incorporation of labelled thymidine in DNA. Typically, the incorporation of a nucleoside analog into newly synthesised DNA may be employed to measure proliferation (i. e., active cell growth) in a population of cells. For example, bromodeoxyuridine (BrdU) can be employed as a DNA labelling reagent and anti-BrdU mouse monoclonal antibodies can be employed as a detection reagent. This antibody binds only to cells containing DNA which has incorporated bromodeoxyuridine. A number of detection methods may be used in conjunction with this assay including immunofluorescence, immunohistochemical, ELISA, and colorimetric methods. Kits that include bromodeoxyuridine (BrdU) and anti-BrdU mouse monoclonal antibody are commercially available from Boehringer Mannheim (Indianapolis, IN).

The effect of the INSP062 and INSP063 polypeptides upon cellular differentiation can be measured by contacting stem cells or embryonic cells with various amounts of the INSP062 or INSP063 polypeptides and observing the effect upon differentiation of the stem cells or embryonic cells. Tissue-specific antibodies and microscopy may be used to identify the resulting cells.

The INSP062 and INSP063 polypeptides may also be found to modulate immune and/or nervous system cell proliferation and differentiation in a dose-dependent manner in the above-described assays. Thus, the "functional equivalents" of the INSP062 and INSP063 polypeptides include polypeptides that exhibit any of the same growth and differentiation regulating activities in the above-described assays in a dose-dependent manner. Although the degree of dose-dependent activity need not be identical to that of the INSP062 and INSP063 polypeptides, preferably the "functional equivalents" will exhibit substantially similar dose-dependence in a given activity assay compared to the INSP062 and INSP063 polypeptides.

In certain of the embodiments described above, simple binding assays may 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.

Assay methods that are also included within the terms of the present invention are those that involve the use of the genes and polypeptides of the invention in overexpression or ablation assays. Such assays involve the manipulation of levels of these genes/polypeptides in cells and assessment of the impact of this manipulation event on the physiology of the manipulated cells. For example, such experiments reveal details of signalling and metabolic pathways in which the particular genes/polypeptides are implicated, generate information regarding the identities of polypeptides with which the studied polypeptides interact and provide clues as to methods by which related genes and proteins are regulated. 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 drug 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 (supplied by Biacore AB, Uppsala, Sweden) 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., NJ. 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, intrameduUary, 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) J-n: 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 virus (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 immunogen 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 antioxidants, 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.

Genetic delivery of antibodies that bind to polypeptides according to the invention may also be effected, for example, as described in International patent application WO98/55607.

The technology referred to as jet injection (see, for example, www.powderject.com) may also be useful in the formulation of vaccine compositions.

A number of suitable methods for vaccination and vaccine delivery systems are described in International patent application WO00/29428.

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 Brunt, 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); c)and 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, UN, 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 mRΝA. Decreased or increased expression can be measured at the RΝA 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, RΝase 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 in members of the three finger toxin fold family are implicated. Such diseases may include cell proliferative disorders, including neoplasm, melanoma, lung, colorectal, breast, pancreas, head and neck and other solid tumours; myeloproliferative disorders, such as leukemia, non-Hodgkin lymphoma, leukopenia, thrombocytopenia, angiogenesis disorder, Kaposis' sarcoma; autoimmune/inflammatory disorders, including allergy, inflammatory bowel disease, arthritis, psoriasis and respiratory tract inflammation, asthma, and organ transplant rejection; cardiovascular disorders, including hypertension, oedema, angina, atherosclerosis, thrombosis, sepsis, shock, reperfusion injury, and ischemia; neurological disorders including central nervous system disease, Alzheimer's disease, brain injury, amyotrophic lateral sclerosis, and pain; developmental disorders; metabolic disorders including diabetes mellitus, osteoporosis, and obesity, AIDS and renal disease; infections including viral infection, bacterial infection, fungal infection and parasitic infection and other pathological conditions. Preferably, the diseases are those in which lymphocyte antigens are implicated.

Various aspects and embodiments of the present invention will now be described in more detail by way of example, with particular reference to the INSP062 and INSP063 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: Top ten results from BLAST against NCBI non-redundant database using SEQ ID NO: 18 (TNSP062 polypeptide sequence).

Figure 2: Alignment generated by BLAST between SEQ ID NO:18 (INSP062 polypeptide sequence) and the top two biochemically annotated hits, the Ly-6.1 protein precursor (Mus musculus) and the lymphocyte antigen 6 complex, locus A (Mus musculus). Figure 3: Top ten results from BLAST against NCBI non-redundant database using SEQ ID NO:26 (INSP063 polypeptide sequence). Figure 4: Alignment generated by BLAST between SEQ ID NO:26 (INSP063 polypeptide sequence) and the top two hits, the Lymphocyte antigen Ly-6I precursor (Mus musculus) and Ly6I.l (Mus musculus).

Figure 5: Multiple alignment showing the INSP062 polypeptide aligned with other lymphocyte antigen family members and related sequences in addition to the AAXl 9982 EST full-length translation product. The predicted GPI-linkage site (asparagine 109) is highlighted with an arrow on the alignment.

Figure 6: Multiple alignment showing the INSP062 polypeptide aligned with other lymphocyte antigen family members and related sequences. The predicted GPI-linkage site (asparagine 109) is highlighted with an arrow on the alignment.

Figure 7: Multiple alignment showing the INSP063 polypeptide aligned with other lymphocyte antigen family members and related sequences. The predicted GPI-linkage site (asparagine 122) is highlighted with an arrow on the alignment.

Figure 8: Schematic representation highlighting the difference between the INSP062 polypeptide (SEQ ID NO: 18) and the predicted translation product of the AAXl 9982 EST sequence (SEQ ID NO: 10). This figure illustrates that the difference between the AAXl 9982 sequence and the INSP062 sequence resides in the final C-terminal exon. For INSP062 (SEQ ID NO: 18), the third exon is the final exon. In the case of SEQ ID: 10 (AAXl 9982) the third exon is spliced out of the transcript before the stop codon utilised in SEQ ID: 18, also removing the GPI-anchor attachment signal.

Figure 9: INSP062 coding exon organization in genomic DNA and position of PCR primers.

Figure 10: Nucleotide sequence and translation of cloned INSP062.

Figure 11: Map of pENTR-INSP062-6HIS plasmid. Figure 12: Map of pEAK12d-INSP062-6HIS plasmid.

Figure 13: INSP063 coding exon organization in genomic DNA and position of PCR primers.

Figure 14: Nucleotide sequence and translation of INSP063 cloned by exon assembly.

Figure 15: Map of pENTR-LNSP063-6HIS plasmid. Figure 16: Map of pEAK12d-INSP063-6HIS plasmid

Examples

Example 1 : INSP062 Protein BLAST Results The INSP062 polypeptide sequence, shown in SEQ ID NO: 18, was used as a BLAST query against the NCBI non-redundant sequence database. Nine of the ten top hits are annotated as members of the LY-6 lymphocyte antigen multigene family. The top ten hits all align to the query sequence with highly significant E-values (3e^"04 to le^"52) (Figure 1). Figure 2 shows the alignment of the INSP062 polypeptide query sequence to the sequence of the top two biochemically annotated hits, the Mus musculus Ly6.1 protein precursor and the Mus musculus lymphocyte antigen complex, locus A. These results indicate that the INSP062 polypeptide is a member of the three finger toxin fold family, and moreover, is a member of the lymphocyte antigen family.

Example 2: The EST AAXl 9982 Predicted Translation Product The nucleotide sequences shown in SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 and SEQ ID NO:9 represent the coding sequence for exons one to four of the AAX19982 expressed sequence tag (EST) (SEQ ID NO:25 of PCT publication WO 99/06439 A2). The amino acid sequences shown in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 and SEQ ID NO:10 represent the predicted translation products of the coding sequence for exons one to four of the AAX19982 EST. The Applicant proposes that the INSP062 polypeptide is a splice variant of the native mRNA from which the AAXl 9982 EST is derived. Furthermore, it is proposed that this splice variant is the 'normal' active splice variant, and that the AAXl 9982 protein is an inactive variant or a variant with altered function compared to the INSP062 polypeptide. The INSP062 and AAXl 9982 polypeptide sequences given in SEQ ID NO: 10 and SEQ ID NO: 18 were analysed by multiple alignment against members of the lymphocyte antigen family. Figure 5 shows the INSP062 polypeptide aligned with other lymphocyte antigen family members and also the AAXl 9982 EST predicted translation product. Figure 6 shows the INSP062 polypeptide aligned with other lymphocyte antigen family members and related sequences, this time without the AAXl 9982 EST predicted translation product. These Figures show the position of the conserved cysteine residues and their characteristic spacing, providing further evidence that the INSP062 polypeptide is a member of the three finger toxin fold. Figure 5 shows that the C-terminal region of the AAXl 9982 translation product does not align correctly with that of other lymphocyte antigen family members, and that the alignment of the C-terminal sequence of the lymphocyte antigens is disrupted by the C-terminal sequence of the AAXl 9982 translation product. In contrast, the alignment shown in Figure 6, in which the AAXl 9982 translation product is not included, shows that the INSP062 polypeptide does not disrupt the alignment of the C-terminal sequence of the lymphocyte antigens, and also aligns well over the entire length of the other lymphocyte antigens shown. The lymphocyte antigens shown in Figures 5 and 6 each contain a sequence of hydrophobic residues at their C-terminus. These sequences are the signals for GPI anchor attachment, and are absent from the AAX19982 polypeptide but are present for the INSP062 polypeptide. Figure 6 shows the predicted attachment site for the INSP062 GPI-anchor (position 109) after cleavage of the C-terminal hydrophobic sequence.

Thus, Figures 5 and 6 provide support for the assertion in Example 1 that the INSP062 polypeptide is a three finger toxin fold polypeptide and also a member of the lymphocyte antigen family. In addition, Figures 5 and 6 indicate that the INSP062 polypeptide contains a GPI-anchor attachment site, in agreement with the known members of the lymphocyte antigen family, and in contrast to the AAXl 9982 polypeptide.

Figure 8 shows an exon-by-exon alignment of the AAXl 9982 predicted translation product (SEQ ID NO: 10) against the INSP062 polypeptide (SEQ ID NO: 18), and illustrates the cause of these differing C-terminal sequences. The difference between the two polypeptides resides in the most 3' exon of each protein. The AAXl 9982 predicted polypeptide contains four exons, whilst the INSP063 polypeptide contains only three exons. This is accounted for by the alternative splicing of the 3' end of the transcript, resulting in inclusion or exclusion of in-frame coding sequence containing a 'stop' signal.

The AAXl 9982 splice site selection removes the 'stop' signal utilised by the INSP062 polypeptide and the GPI-anchor attachment signal and creates a fourth exon. In contrast, the LNSP062 splice site selection retains this important sequence of hydrophobic residues and has only three exons. The alternative splicing of mRNA transcripts to produce mature mRNAs with varying C-terminal sequences, especially C-terminal sequences with or without membrane retention properties, is well known, especially in the area of immune molecules. On the basis of these data, the Applicant proposes that the INSP062 polypeptide is a functionally active variant of the protein encoded by the AAXl 9982 EST, formed by alternative splicing of the native mRNA transcript. Importantly, due to the variation in the C-terminal amino acid sequence between INSP062 and AAXl 9982 (Figure 8), only the INSP062 polypeptide is predicted to contain a GPI-anchor attachment site. As described above, the presence of a GPI anchor attachment site allows the secreted protein to be retained at the cell membrane, enabling that polypeptide to carry out additional functions such as transmembrane signalling, which would otherwise not be possible. Thus, the finding that the INSP062 polypeptide, and not the AAXl 9982 polypeptide, contains a GPI anchor attachment site is of great functional importance, and the 1NSP062 polypeptide is very likely to possess altered biological functions compared to the AAXl 9982 predicted translation product. This is especially relevant in light of the fact that the other known mammalian three finger toxin fold proteins are GPI-anchored to the cellular membrane, suggesting that the INSP062 polypeptide, and not the AAX19982 polypeptide, is the 'normal' functional variant in humans.

Example 3: INSP063 Protein BLAST Results

The INSP063 polypeptide sequence, shown in SEQ ID NO:26, was used as a BLAST query against the NCBI non-redundant Sequence database. Nine of the top ten hits are members of the lymphocyte antigen family. The top ten hits all align to the query sequence with highly significant E-values (2.5 to 8e^"04) (Figure 3). Figure 4 shows the alignment of the INSP063 polypeptide query sequence to the sequence of the top two hits, the Mus musculus lymphocyte antigen Ly-6I precursor and the Mus musculus Ly6I.l protein. These results indicate that the INSP063 polypeptide is a member of the three finger toxin fold family, and moreover, is a member of the lymphocyte antigen family. Example 4: Multiple alignments of INSP063 against other lymphocyte antigen familv members

Figure 7 shows the INSP063 polypeptide aligned with other lymphocyte antigen family members and related sequences. The location of the conserved cysteine residues, and the predicted position for GPI-anchor attachment, are indicated. Figure 7 therefore provides further evidence in support of Example 3 that the INSP063 polypeptide is a three finger toxin fold polypeptide that is a member of the lymphocyte antigen family, and is also anchored to the membrane by a GPI-moiety. Example 5: Cloning of INSP062 by exon assembly

As described above, the coding sequence of INSP062 spans 3 exons (see Figure 8).

PCR amplification of exons encoding INSP062 from genomic DNA

PCR primers were designed to amplify exons 1, 2 and 3 individually (Table 1 and Figure 9). The reverse primer for exon 1 (ΪNSP062 - exonlR) has an overlap of 18 bases with exon 2 at its 5' end. The forward primer for exon 2 (INSP062 - exon2F) has an 18 bp overlap with exon 1 at its 5' end. The reverse primer for exon 2 (INSP062 - exon2R ) has an overlap of 18 bases with exon 3 at its 5' end. The forward primer for exon 3 (INSP062 - exon3F) contains an 18 bp overlap with exon 2 at its 5' end. The overlap between the primers is shown in Figure 9.

To generate 1NSP062 exon 1, the PCR reaction was performed in a final volume of 50 μl and contained 2 μl of 5 mM dNTPs (Amersham Pharmacia Biotech), 3 μl of INSP062- exonlF (10 μM), 3 μl of INSP062 -exonlR (10 μM) , 5 μl of 10X Amplitaq™ buffer and 0.5 μl of Amplitaq (5 U/μl) (Applied Biosystems cat. no. N808-0155). The PCR conditions were 94 °C for 2 min; 25 cycles of 94 °C for 30 s, 55 °C for 30 s and 72 °C for 30 s; an additional elongation cycle of 72 °C for 3 min; and a holding cycle of 4 °C. Reaction products were loaded onto a 1.5 % agarose gel (IX TAE) and PCR products of the correct size (91 bp) were gel- purified using a Qiaquick Gel Extraction Kit (Qiagen cat. no. 28704) and eluted in 50 μl of elution buffer (Qiagen).

For amplification of exon 2, the PCR reaction was performed in a final volume of 100 μl and contained 1.5 μl of human genomic DNA (0.1 μg/μl, Novagen cat. no. 69237), 4 μl of 5 mM dNTPs (Amersham Pharmacia Biotech), 6 μl of INSP062-exon2F (10 μM), 6 μl of INSP062-exon2R (10 μM), 10 μl of 10X Amplitaq™ buffer and 0.5 μl of Amplitaq™ (5 U/μl). The PCR conditions were 94 °C for 2 min; 35 cycles of 94 °C for 30 s, 60 °C for 30 s and 72 °C for 30 s; an additional elongation cycle of 72 °C for 3 min; and a holding cycle of 4 °C. Reaction products were loaded onto a 1.5 % agarose gel (IX TAE) and PCR products of the correct size (153 bp) were gel-purified using a Qiaquick Gel Extraction Kit (Qiagen cat. no. 28704) and eluted in 30 μl of elution buffer (Qiagen).

To generate LNSP062 exon 3, the PCR reaction was performed in a final volume of 50 μl and contained 1.5 μl of human genomic DNA (0.1 μg/μl, Novagen cat. no. 69237), 2 μl of 5 mM dNTPs (Amersham Pharmacia Biotech), 3 μl of INSP062-exon3F (10 μM), 3 μl of INSP062-exon3R (10 μM), 5 μl of 10X Amplitaq™ buffer and 0.5 μl of Amplitaq (5 U/μl). The PCR conditions were 94 °C for 2 min; 30 cycles of 94 °C for 30 s, 66 °C for 30 s and 72 °C for 30 s; an additional elongation cycle of 72 °C for 3 min; and a holding cycle of 4 °C. Reaction products were loaded onto a 2 % agarose gel (IX TBE) and PCR products of the correct size (245 bp) were gel-purified using a Qiaquick Gel Extraction Kit (Qiagen cat. no. 28704) and eluted in 50 μl of elution buffer (Qiagen).

Table 1

Primer Sequence (5' -3')

GCP Forward G GGG ACA AGT TTG TAG AAA AAA GCA GGC TTC GCC ACC

GCP Reverse GGG GAC CAC TTT GTA CAA GAA AGC TGG GTT TCA ATG GTG ATG GTG ATG GTG

INSP062-exonlF ATG GAG AGG CTC GTC CTA ACC CTG TGC ACC CTC CCG CTG GCT GTG GCG TCT GCT G

INSP062-exonlF2 ATG GAG AGG CTC GTC CTA ACC CTG TG

INSP062-exonlR AGC AGC TCA GGT TGC GAG CTG GCG TCG TGG CGC AGC CAG CAG ACG CCA CAG CCA

INSP062- exon2F GCT GCG CCA CGA CGC CAG CTC GCA ACC TGA GCT GCT AC

INSP062- exon2R GGA CGC GTA CAC TCC ATT TAA AAG AGA CGA CCA CCT CG

INSP062- exon3F AGG TGG TCG TCT CTT TTA AAT GGA GTG TAG GCG TCC TG

INSP062- exon3R TCA CAA CAG GGC CCT GAG GAG GCT GA

INSP062-EX1 GCA GGC TTC GCC ACC ATG GAG AGG CTC GTC CTA AC

INSP062-EX2 GTG ATG GTG ATG GTG CAA CAG GGC CCT GAG GAG GCT GA pEAK12-F GCC AGC TTG GCA CTT GAT GT pEAK12-R GAT GGA GGT GGA CGT GTC AG pENTR-Fl TCG CGT TAA CGC TAG CAT GGA TCT C pENTR-Rl GTA ACA TCA GAG ATT TTG AGA CAC Underlined sequence = Koza sequence Bold = Stop codon

Italic sequence = His tag Sequence in grey = overlap with adjacent exon

Assembly of exons 1-3 encoding the ORF of INSP062

Exons 1, 2 and 3 were assembled in a 100 μl PCR reaction containing 3 μl of purified exon 1, 3 μl of purified exon 2, 5 μl of purified exon 3, 4 μl of 5 mM dNTPs, 6 μl of INSP062- exonlF2 (10 μM), 6 μl of INSP062-exon3R (10 μM), 10 μl of 10X Amplitaq™ buffer, and 0.5 μl of Amplitaq™ (5 U/μl). The reaction conditions were: 94 °C, 4 min; 10 cycles of 94°C for 30 s, 48 °C for 30 s and 70 °C for 2 min; 25 cycles of 94 °C for 30 s, 52 °C, for 30 s and 70°C for 2 min ; an additional elongation step of 70 °C for 10 min; and a holding cycle at 4 °C. Reaction products were analysed on a 1.5 % agarose gel (IX TAE). PCR products of the correct size (417 bp) were gel purified using a Qiaquick Gel Extraction Kit (Qiagen cat. no. 28704) and eluted in 50 μl of elution buffer (Qiagen). The resultant PCR product contains the ORF of INSP062 (see Figure 10).

Subcloning of the INSP062 ORF into pDONR201

The INSP062 ORF was subcloned into pDONR201 Gateway™ cloning system (Invitrogen). The first stage of the Gateway cloning process involves a two step PCR reaction which generates the ORF of INSP062 flanked at the 5' end by an attBl recombination site and Kozak sequence, and flanked at the 3' end by a sequence encoding an in-frame 6 histidine (6HIS) tag, a stop codon and the attB2 recombination site (Gateway compatible cDNA). The first PCR reaction contained 5 μl of gel purified INSP062 ORF, 2 μl of 5 mM dNTPs (Amersham Pharmacia Biotech), 3 μl of INSP062-EX1 (10 μM), 3 μl of LNSP062-EX2 (10 μM), 5 μl of Amplitaq™ buffer and 0.5 μl of Amplitaq™ (5 U/μl) in a final volume of 50 μl. The PCR conditions were 94 °C for 2 min; 30 cycles of 94 °C for 30 sec; 55 °C for 30 sec and 72 °C for 1 min; an additional elongation step of 72 °C for 3 min and a holding cycle of 4 °C. Reaction products were analysed on a 1.5 % agarose gel (IX TAE) and PCR products of the correct size (partial attBl -INSP062-5HIS) were gel purified using a Qiaquick Gel Extraction Kit (Qiagen cat. no. 28704) and eluted in 50 μl of elution buffer (Qiagen). The 2^nd PCR reaction contained 5 μl of gel purified partial attBl - INSP062-5HIS, 2 μl of 5 mM dNTPs (Amersham Pharmacia Biotech), 3 μl of GCP-F (10 μM), 3 μl of GCP-R (10 μM), 5 μl of Amplitaq™ buffer and 0.5 μl of Amplitaq™ (5 U/μl) in a final volume of 50 μl. The PCR conditions were 94 °C for 2 min; 30 cycles of 94 °C for 30 sec; 55 °C for 30 sec and 72 °C for 1 min; an additional elongation step of 72 °C for 3 min and a holding cycle of 4 °C. Reaction products were analysed on a 1.5 % agarose gel (IX TAE) and PCR products of the correct size (501 bp, corresponding to Gateway- modified INSP062 ORF) were gel purified using a Qiaquick Gel Extraction Kit (Qiagen cat. no. 28704) and eluted in 50 μl of elution buffer (Qiagen). Gateway-modified INSP062 ORF was then transferred to pDONR201 using BP clonase as follows: 5 μl of Gateway- modified INSP062 ORF was incubated with 1.5 μl pDONR201 (0.1 μg/μl), 2 μl BP buffer and 1.5 μl of BP clonase enzyme mix (Invitrogen) at RT for 1 h. The reaction was stopped by addition of proteinase K (2 μg) and incubated at 37°C for a further 10 min. An aliquot of this reaction (5 μl) was incubated with 50 μl of E. coli TOP 10 cells (Invitrogen) for 30 min on ice, heat shocked at 42 °C for 30 s then diluted by addition of 250 μl of SOC medium. Cells were then incubated for 1 h at 37 °C with shaking. Transformants (50 ul) were plated on LB-kanamycin plates and incubated overnight at 37 °C. Plasmid mini prep DNA was isolated from 14 resultant colonies using a Qiaprep Turbo 9600 robotic system (Qiagen) and subjected to DNA sequencing with pΕNTR-Fl and pΕNTR-Rl sequencing primers using the BigDyeTerminator system (Applied Biosystems cat. no. 4390246) according to the manufacturer's instructions. Sequencing reactions were purified using Dye-Εx columns (Qiagen) or Montage SΕQ 96 cleanup plates (Millipore cat. no. LSKS09624) then analyzed on an Applied Biosystems 3700 sequencer. A map of the pΕNTR-INSP062-6HIS plasmid is shown in Figure 11.

Subcloning of the INSP062 ORF to expression vector pEAK12d

Plasmid eluate (1.5 μl) from a pDONR201 clone containing the correct sequence of the LNSP062 (pENTR-INSP062-6HIS, plasmid ID # 13409) was then used in a recombination reaction containing 1.5 μl pEAK12d vector (0.1 μg/μl), 2 μl LR buffer and 1.5 μl of LR clonase (Invitrogen) in a final volume of 10 μl. The mixture was incubated at RT for 1 h, stopped by addition of proteinase K (2 μg) and incubated at 37°C for a further 10 min. An aliquot of this reaction (1 μl) was used to transform 20 μl of E. coli DH10B cells (diluted 1/5 in sterile water) by electroporation using a Biorad Gene Pulser. Electroporated cells were diluted by addition of 1 ml of SOC medium and incubated for 1 h at 37 °C. Transformants (10 μl) were plated on LB-ampicillin plates and incubated overnight at 37 °C. Mini prep DNA was prepared from 5 colonies using a Qiaprep Turbo 9600 robotic system (Qiagen) and eluted in 50 μl of elution buffer. Plasmid mini prep DNA was then subjected to DNA sequencing with pEAK12-F and pEAK12-R sequencing primers using the BigDyeTerminator system (Applied Biosystems cat. no. 4390246) according to the manufacturer's instructions. Sequencing reactions were purified using Dye-Ex columns (Qiagen) or Montage SEQ 96 cleanup plates (Millipore cat. no. LSKS09624) then analyzed on an Applied Biosystems 3700 sequencer.

CsCl gradient purified maxi-prep DNA of plasmid pEAK12d-INSP062-6HIS (plasmid ID # 13408) was prepared from a 500 ml culture of a sequence verified clone (Sambrook J. et al, in Molecular Cloning, a Laboratory Manual, 2^nd edition, 1989, Cold Spring Harbor Laboratory Press), resuspended at a concentration of 1 μg/μl in sterile water and stored at - 20°C. A map of the ρEAK12d-INSP062-6HIS plasmid is shown in Figure 12.

Example 6: Cloning of INSP063 by exon assembly

As described above, the coding sequence of INSP063 spans 3 exons.

PCR amplification of exons encoding INSP063 from genomic DNA.

PCR primers were designed to amplify exons 1, 2 and 3 individually (Table 2 and Figure 13). The reverse primer for exon 1 (INSP063 - exonlR) has an overlap of 18 bases with exon 2 at its 5' end. The forward primer for exon 2 (INSP063 - exon2F) has an 18 bp overlap with exon 1 at its 5' end. The reverse primer for exon 2 (INSP063 - exon2R ) has an overlap of 18 bases with exon 3 at its 5' end. The forward primer for exon 3 (INSP063 - exon3F) contains an 18 bp overlap with exon 2 at its 5' end.

To generate INSP063 exon 1, the PCR reaction was performed in a final volume of 50 μl and contained 2 μl of 5 mM dNTPs (Amersham Pharmacia Biotech), 3 μl of INSP063- exonlF (10 μM), 3 μl of INSP063 -exonlR (10 μM) , 5 μl of 10X Amplitaq™ buffer and 0.5 μl of Amplitaq (5 U/μl) (Applied Biosystems cat. no. N808-0155). The PCR conditions were 94 °C for 2 min; 25 cycles of 94 °C for 30 s, 55 °C for 30 s and 72 °C for 30 s; an additional elongation cycle of 72 °C for 3 min; and a holding cycle of 4 °C. Reaction products were loaded onto a 1.5 % agarose gel (IX TAE) and PCR products of the correct size (55 bp) were gel- purified using a Qiaquick Gel Extraction Kit (Qiagen cat. no. 28704) and eluted in 50 μl of elution buffer (Qiagen).

For amplification of exon 2, the PCR reaction was performed in a final volume of 50 μl and contained 1 μl of human genomic DNA (0.1 μg/μl, Novagen cat. no. 69237), 2 μl of 5 mM dNTPs (Amersham Pharmacia Biotech), 3 μl of INSP063-exon2F (10 μM), 3 μl of INSP063-exon2R (10 μM), 5 μl of 10X Amplitaq™ buffer and 0.5 μl of Amplitaq™ (5 U/μl). The PCR conditions were 94 °C for 2 min; 30 cycles of 94 °C for 30 s, 66 °C for 30 s and 72 °C for 30 s; an additional elongation cycle of 72 °C for 3 min; and a holding cycle of 4 °C. Reaction products were loaded onto a 2 % agarose gel (IX TBE) and PCR products of the correct size (190 bp) were gel-purified using a Qiaquick Gel Extraction Kit (Qiagen cat. no. 28704) and eluted in 50 μl of elution buffer (Qiagen). For amplification of exon 3, the PCR reaction was performed in a final volume of 50 μl and contained 1 μl of human genomic DNA (0.1 μg/μl, Novagen cat. no. 69237), 2 μl of 5 mM dNTPs (Amersham Pharmacia Biotech), 3 μl of INSP063-exon3F (10 μM), 3 μl of INSP063-exon3R (10 μM), 5 μl of 10X Amplitaq™ buffer and 0.5 μl of Amplitaq™ (5 U/μl). The PCR conditions were 94 °C for 2 min; 30 cycles of 94 °C for 30 s, 66 °C for 30 s and 72 °C for 30 s; an additional elongation cycle of 72 °C for 3 min; and a holding cycle of 4 °C. Reaction products were loaded onto a 2 % agarose gel (IX TBE) and PCR products of the correct size (282 bp) were gel-purified using a Qiaquick Gel Extraction Kit (Qiagen cat. no. 28704) and eluted in 50 μl of elution buffer (Qiagen). Table 2

Primer Sequence (5' -3')

GCP Forward G GGG ACA AGT TTG TAG AAA AAA GCA GGC TTC GCC ACC

GCP Reverse GGG GAC CAC TTT GTA CAA GAA AGC TGG GTT TCA ATG GTG ATG GTG ATG GTG

INSP063-exonlF ATG AAG GTC ACT AGC CCC ATG CTG CTG CTG GCT GAG

INSP063-exonlR AGC ACT CAA GGC CCT GGC CCT CAG CCA GCA GCA GCA T

INSP063- exon2F TGC TGC TGC TGG CTG AGG GCC AGG GCC TTG AGT GCT TC

INSP063- exon2R TGT AGC TGA GCT TCT GCC CTG CCT ACA GAC GCT GGG TC

INSP063- exon3F CCC AGC GTC TGT AGG CAG GGC AGA AGC TCA GCT ACA CT

INSP063- exon3R TCA CAG CAG AGC CCA GAG AAG GGG CTC AGG

INSP063-EX1 GCA GGC TTC GCC ACC ATG AAG GTC ACT AGC CCC ATG C

INSP063-EX2 GTG ATG GTG ATG GTG CAG CAG AGC CCA GAG GAA GG pEAK12-F GCC AGC TTG GCA CTT GAT GT pEAK12-R GAT GGA GGT GGA CGT GTC AG pENTR-Fl TCG CGT TAA CGC TAG CAT GGA TCT C pENTR-Rl GTA ACA TCA GAG ATT TTG AGA CAC underlined sequence = Kozak sequence

Bold = Stop codon

Italic sequence = His tag

Sequence in grey = overlap with adjacent exon

Assembly of exons 1-3 encoding the ORF of INSP063

Exons 1, 2 and 3 were assembled in a 100 μl PCR reaction containing 8 μl of purified exon 1, 3 μl of purified exon 2, 5 μl of purified exon 3, 4 μl of 5 mM dNTPs, 6 μl of INSP063- exonlF (10 μM), 6 μl of INSP063-exon3R (10 μM), 10 μl of 10X Amplitaq™ buffer, and 0.5 μl of Amplitaq™ (5 U/μl). The reaction conditions were: 94 °C, 4 min; 10 cycles of 94°C for 30 s, 48 °C for 30 s and 70 °C for 2 min; 25 cycles of 94 °C for 30 s, 52 °C, for 30 s and 70°C for 2 min; an additional elongation step of 70 °C for 10 min; and a holding cycle at 4 °C. Reaction products were analysed on a 1.5 % agarose gel (IX TAE). PCR products of the correct size (456 bp) were gel purified using a Qiaquick Gel Extraction Kit (Qiagen cat. no. 28704) and eluted in 30 μl of elution buffer (Qiagen). The resultant PCR product contains the ORF of LNSP063 (see Figure 14)

Subcloning of the INSP063 ORF into pDONR201

The INSP063 ORF was subcloned into ρDONR201 Gateway™ cloning system (Invitrogen). As described above, the first stage of the Gateway cloning process involves a two step PCR reaction which generates the ORF of INSP063 flanked at the 5' end by an attBl recombination site and Kozak sequence, and flanked at the 3' end by a sequence encoding an in-frame 6 histidine (6HIS) tag, a stop codon and the attB2 recombination site (Gateway compatible cDNA). The first PCR reaction contained 3 μl of gel purified INSP063 ORF, 2 μl of 5 mM dNTPs (Amersham Pharmacia Biotech), 3 μl of INSP063- EX1 (10 μM), 3 μl of INSP063-EX2 (10 μM) , 5 μl of Amplitaq™ buffer and 0.5 μl of Amplitaq™ (5 U/μl) in a final volume of 50 μl. The PCR conditions were 94 °C for 2 min; 30 cycles of 94 °C for 30 sec; 55 °C for 30 sec and 72 °C for 1 min; an additional elongation step of 72 °C for 3 min and a holding cycle of 4 °C. Reaction products were analysed on a 1.5 % agarose gel (IX TAE) and PCR products of the correct size (partial attBl -INSP063-5HIS - 486 bp) were gel purified using a Qiaquick Gel Extraction Kit (Qiagen cat. no. 28704) and eluted in 50 μl of elution buffer (Qiagen). The 2^nd PCR reaction contained 5 μl of gel purified partial attBl-INSP063-5HIS fragment, 2 μl of 5 mM dNTPs (Amersham Pharmacia Biotech), 3 μl of GCP-F (10 μM), 3 μl of GCP-R (10 μM), 5 μl of Amplitaq buffer and 0.5 μl of Amplitaq (5 U/μl) in a final volume of 50 μl. The PCR conditions were 94 °C for 2 min; 30 cycles of 94 °C for 30 sec; 55 °C for 30 sec and 72 °C for 1 min; an additional elongation step of 72 °C for 3 min and a holding cycle of 4 °C. Reaction products were analysed on a 1.5 % agarose gel (IX TAE) and PCR products of the correct size (541 bp, corresponding to Gateway-modified INSP063 ORF) were gel purified using a Qiaquick Gel Extraction Kit (Qiagen cat. no. 28704) and eluted in 50 μl of elution buffer (Qiagen). Gateway-modified INSP063 ORF was then transferred to pDONR201 using BP clonase as follows: 5 μl of Gateway-modified INSP063 ORF was incubated with 1.5 μl pDONR201 (0.1 μg/μl), 2 μl BP buffer and 1.5 μl of BP clonase enzyme mix (Invitrogen) at RT for 1 h. The reaction was stopped by addition of 1 μl proteinase K (2 μg) and incubated at 37°C for a further 10 min. An aliquot of this reaction (5 μl) was incubated with 50 μl of E. coli TOP 10 cells (Invitrogen) for 20 min on ice, heat shocked at 42 °C for 30 s then diluted by addition of 250 μl of SOC medium. Cells were then incubated for 1 h at 37 °C with shaking. Transformants (50 ul) were plated on LB- kanamycin plates and incubated overnight at 37 °C. Plasmid mini prep DNA was isolated from 8 resultant colonies using a Qiaprep Turbo 9600 robotic system (Qiagen) and subjected to DNA sequencing with pΕNTR-Fl and pΕNTR-Rl sequencing primers using the BigDyeTerminator system (Applied Biosystems cat. no. 4390246) according to the manufacturer's instructions. Sequencing reactions were purified using Dye-Εx columns (Qiagen) or Montage SΕQ 96 cleanup plates (Millipore cat. no. LSKS09624) then analyzed on an Applied Biosystems 3700 sequencer. A map of the pΕNTR-INSP063-6HIS plasmid is shown in Figure 15.

Subcloning of the INSP063 ORF to expression vector pEAK12d

Plasmid eluate (1.5 μl) from a pDONR201 clone containing the correct sequence of the INSP063 (pENTR-INSP063-6HIS, plasmid ID # 13371) was then used in a recombination reaction containing 1.5 μl pEAK12d vector (0.1 μg/μl), 2 μl LR buffer and 1.5 μl of LR clonase (Invitrogen) in a final volume of 10 μl. The mixture was incubated at RT for 1 h, stopped by addition of 1 μl proteinase K (2 μg) and incubated at 37°C for a further 10 min.

An aliquot of this reaction (2 μl) was incubated with 50 μl of E. coli TOP 10 cells (Invitrogen) for 20 min on ice, heat shocked at 42 °C for 30 s then diluted by addition of

250 μl of SOC medium. Cells were then incubated for 1 h at 37 °C with shaking.

Transformants (50 μl) were plated on LB-ampicillin plates and incubated overnight at

37°C.

Twelve of the resultant colonies were picked into 50 ul of sterile water and 25 ul was then subjected PCR in a total reaction volume of 50 μl containing 2 μl of 5mM dNTPs, 2.5 μl of 10 μM INSP063-exonlF, 2.5 μl of 10 μM INSP063-exon3R, 5 μl of 10X AmpliTaqTM buffer and 0.5 μl AmpliTaqTM (Applied Biosystems cat. no. N808-0155). The cycling conditions were as follows: 96 °C for 2 min; 30 cycles of 94 °C for 30 sec, 55°C for 30 sec, and 72 °C for 2 min; 1 cycle, 72 °C for 3 min. Reaction products were analysed on a 1.5 % agarose gel (IX TAE). CsCl gradient purified maxi-prep DNA was prepared from a 500 ml culture of 1 of the colonies which gave the correct size PCR product (781 bp) according to the published method (Sambrook J. et al., in Molecular Cloning, a Laboratory Manual, 2nd edition, 1989, Cold Spring Harbor Laboratory Press), and resuspended at a concentration of 1 μg/μl in sterile water. Two hundred ng of purified plasmid DNA (pEAK12d- INSP063-6HIS (plasmid ID # 13369) was then subjected to DNA sequencing with pEAK12-F and pEAK12-R sequencing primers using the BigDyeTerminator system (Applied Biosystems cat. no. 4390246) according to the manufacturer's instructions. Sequencing reactions were purified using Dye-Ex columns (Qiagen) or Montage SEQ 96 cleanup plates (Millipore cat. no. LSKS09624) then analyzed on an Applied Biosystems 3700 sequencer. A map of the pEAK12d-INSP063-6HIS plasmid is shown in Figure 16.

Further experiments may now be performed to determine the tissue distribution and expression levels of the INSP062 and INSP063 polypeptides in vivo, on the basis of the nucleotide and amino acid sequence disclosed herein.

The presence of the transcripts for LNSP062 and INSP063 may be investigated by PCR of cDNA from different human tissues. The INSP062 and INSP063 transcripts may be present at very low levels in the samples tested. Therefore, extreme care is needed in the design of experiments to establish the presence of a transcript in various human tissues as a small amount of genomic contamination in the RNA preparation will provide a false positive result. Thus, all RNA should be treated with DNAse prior to use for reverse transcription. In addition, for each tissue a control reaction may be set up in which reverse transcription was not undertaken (a - RT control).

For example, 1 μg of total RNA from each tissue may be used to generate cDNA using Multiscript reverse transcriptase (ABI) and random hexamer primers. For each tissue, a control reaction is set up in which all the constituents are added except the reverse transcriptase (- RT control). PCR reactions are set up for each tissue on the reverse transcribed RNA samples and the minus RT controls. INSP062-specific and INSP063- specific primers may readily be designed on the basis of the sequence information provided herein. The presence of a product of the correct molecular weight in the reverse transcribed sample together with the absence of a product in the minus RT control may be taken as evidence for the presence of a transcript in that tissue. Any suitable cDNA libraries may be used to screen for the LNSP062 and INSP063 transcripts, not only those generated as described above.

The tissue distribution pattern of the INSP062 and INSP063 polypeptides will provide further useful information in relation to the function of those polypeptides.

In addition, further experiments may now be performed using the pEAK12d-INSP062- 6HIS and pEAK12d-INSP063-6HIS expression vectors. Transfection of mammalian cell lines with these vectors may enable the high level expression of the INSP062 and INSP063 proteins and thus enable the continued investigation of the functional characteristics of the INSP062 and INSP9063 polypeptides. The following material and methods are an example of those suitable in such experiments:

Cell Culture

Human Embryonic Kidney 293 cells expressing the Epstein-Barr virus Nuclear Antigen (HEK293-EBNA, Invitrogen) are maintained in suspension in Ex-cell VPRO serum-free medium (seed stock, maintenance medium, JRH). Sixteen to 20 hours prior to transfection (Day-1), cells are seeded in 2x T225 flasks (50 ml per flask in DMEM / F12 (1:1) containing 2% FBS seeding medium (JRH) at a density of 2x10⁵ cells/ ml). The next day (transfection day 0) transfection takes place using the JetPEI™ reagent (2μl/μg of plasmid DNA, PolyPlus-transfection). For each flask, plasmid DNA is co-transfected with GFP (fluorescent reporter gene) DNA. The transfection mix is then added to the 2xT225 flasks and incubated at 37°C (5%CO ) for 6 days. Confirmation of positive transfection may be carried out by qualitative fluorescence examination at day 1 and day 6 (Axiovert 10 Zeiss).

On day 6 (harvest day), supernatants from the two flasks are pooled and centrifuged (e.g. 4°C, 400g) and placed into a pot bearing a unique identifier. One aliquot (500 μl) is kept for QC of the 6His-tagged protein (internal bioprocessing QC).

Scale-up batches may be produced by following the protocol called "PEI transfection of suspension cells", referenced BP/PEI/HH/02/04, with PolyEthylenelmine from Polysciences as transfection agent. Purification process

The culture medium sample containing the recombinant protein with a C-terminal 6His tag is diluted with cold buffer A (50 mM NaH₂PO₄; 600 mM NaCI; 8.7 % (w/v) glycerol, pH 7.5). The sample is filtered then through a sterile filter (Millipore) and kept at 4°C in a sterile square media bottle (Nalgene).

The purification is performed at 4°C on the VISION workstation (Applied Biosystems) connected to an automatic sample loader (Labomatic). The purification procedure is composed of two sequential steps, metal affinity chromatography on a Poros 20 MC (Applied Biosystems) column charged with Ni ions (4.6 x 50 mm, 0.83 ml), followed by gel filtration on a Sephadex G-25 medium (Amersham Pharmacia) column (1,0 x 10 cm).

For the first chromatography step the metal affinity column is regenerated with 30 column volumes of EDTA solution (100 mM EDTA; 1 M NaCI; pH 8.0), recharged with Ni ions tlirough washing with 15 column volumes of a 100 mM NiSO solution, washed with 10 column volumes of buffer A, followed by 7 column volumes of buffer B (50 mM NaH₂PO₄; 600 mM NaCI; 8.7 % (w/v) glycerol, 400 mM; imidazole, pH 7.5), and finally equilibrated with 15 column volumes of buffer A containing 15 mM imidazole. The sample is transferred, by the Labomatic sample loader, into a 200 ml sample loop and subsequently charged onto the Ni metal affinity column at a flow rate of 10 ml/min. The column is washed with 12 column volumes of buffer A, followed by 28 column volumes of buffer A containing 20 mM imidazole. During the 20 mM imidazole wash loosely attached contaminating proteins are eluted from the column. The recombinant His-tagged protein is finally eluted with 10 column volumes of buffer B at a flow rate of 2 ml/min, and the eluted protein is collected.

For the second chromatography step, the Sephadex G-25 gel-filtration column is regenerated with 2 ml of buffer D (1.137 M NaCI; 2.7 mM KCI; 1.5 mM KH₂PO₄; 8 mM Na₂HPO₄; pH 7.2), and subsequently equilibrated with 4 column volumes of buffer C (137 mM NaCI; 2.7 mM KCI; 1.5 mM KH₂PO₄; 8 mM Na₂HPO₄; 20 % (w/v) glycerol; pH 7.4). The peak fraction eluted from the Ni-column is automatically loaded onto the Sephadex G- 25 column through the integrated sample loader on the VISION and the protein is eluted with buffer C at a flow rate of 2 ml/min. The fraction was filtered through a sterile centrifugation filter (Millipore), frozen and stored at -80°C. An aliquot of the sample is analyzed on SDS-PAGE (4-12% NuPAGE gel; Novex) Western blot with anti-His antibodies. The NuPAGE gel may be stained in a 0.1 % Coomassie blue R250 staining solution (30 % methanol, 10 % acetic acid) at room temperature for 1 h and subsequently destained in 20 % methanol, 7.5 % acetic acid until the background is clear and the protein bands clearly visible.

Following the electrophoresis the proteins are electrotransferred from the gel to a nitrocellulose membrane. The membrane is blocked with 5 % milk powder in buffer E (137 mM NaCI; 2.7 mM KCI; 1.5 mM KH₂PO₄; 8 mM Na₂HPO₄; 0.1 % Tween 20, pH 7.4) for 1 h at room temperature, and subsequently incubated with a mixture of 2 rabbit polyclonal anti-His antibodies (G-18 and H-15, 0.2ug/ml each; Santa Cruz) in 2.5 % milk powder in buffer E overnight at 4°C. After a further 1 hour incubation at room temperature, the membrane is washed with buffer E (3 x 10 min), and then incubated with a secondary HRP-conjugated anti-rabbit antibody (DAKO, HRP 0399) diluted 1/3000 in buffer E containing 2.5 % milk powder for 2 hours at room temperature. After washing with buffer E (3 x 10 minutes), the membrane is developed with the ECL kit (Amersham Pharmacia) for 1 min. The membrane is subsequently exposed to a Hyperfilm (Amersham Pharmacia), the film developed and the western blot image visually analysed.

For samples that showed detectable protein bands by Coomassie staining, the protein concentration may be determined using the BCA protein assay kit (Pierce) with bovine serum albumin as standard.

Furthermore, overexpression or knock-down of the expression of INSP062 and INSP063 polypeptides in cell lines may be used to determine the effect on transcriptional activation of the host cell genome. Dimerisation partners, co-activators and co-repressors of the INSP062 and INSP063 polypeptides may be identified by immunoprecipitation combined with Western blotting and immunoprecipitation combined with mass spectroscopy. Sequences:

SEQ ID NO:l (AAX19982 Nucleotide sequence exon 1)

1 ATGGAGAGGC TCGTCCTAAC CCTGTGCACC CTCCCGCTGG CTGTGGCGTC TGCTGGCTGC 61 GCCACGACGC CAG

SEQ ID NO:2 (AAX19982 Protein sequence exon 1)

1 MERLVLTLCT LPLAVASAGC ATTPA

SEQ ID NO:3 (AAX19982 Nucleotide sequence exon 2) 1 CTCGCAACCT GAGCTGCTAC CAGTGCTTCA AGGTCAGCAG CTGGACGGAG TGCCCGCCCA 61 CCTGGTGCAG CCCGCTGGAC CAAGTCTGCA TCTCCAACGA GGTGGTCGTC TCTTTTA

SEQ ID NO:4 (AAX19982 Protein sequence exon 2)

1 RNLSCYQCFK VSS TECPPT WCSP DQVCI SNEWVSFK

SEQ ID NO:5 (AAX19982 Nucleotide sequence exon 3)

1 AATGGAGTGT ACGCGTCCTG CTCAGCAAAC GCTGTGCTCC CAGATGTCCC AACGACAACA

61 TGAAGTTCGA ATGGTCGCCG GCCCCCATGG TGCAAGGCGT GATCACCAGG CGCTGCTGTT

121 CCTGGGCTCT CTGCAACAGG GCACTGACCC CACAGGAGGG GCGCTGGGCC CTGCGAGGGG 181 GGCTCCTGCT CCAG

SEQ ID NO:6 (AAX19982 Protein sequence exon 3)

1 SVRVLLSKR CAPRCPNDNM KFEWSPAPMV QGVITRRCCS WALCNRALTP QEGRALRGG 61 LLLQ

SEQ ID NO:7 (AAX19982 Nucleotide sequence exon 4)

1 GACCCTTCGA GGGGCAGAAG AACCTGGGTG CGGCCACAGC TGGGGCTCCC ACTCTGCCTT 61 CCCATTTCCA GCCCCCTCTG CCCAAGGGAA ACCCAGGAAG GATAA

SEQ ID NO:8 (AAX19982 Protein sequence exon 4)

1 DPSRGRRT V RPQLGLPLCL PISSPLCPRE TQEG*

SEQ ID NO:9 (AAX19982 Nucleotide sequence)

1 ATGGAGAGGC TCGTCCTAAC CCTGTGCACC CTCCCGCTGG CTGTGGCGTC TGCTGGCTGC 61 GCCACGACGC CAGCTCGCAA CCTGAGCTGC TACCAGTGCT TCAAGGTCAG CAGCTGGACG

121 GAGTGCCCGC CCACCTGGTG CAGCCCGCTG GACCAAGTCT GCATCTCCAA CGAGGTGGTC

181 GTCTCTTTTA AATGGAGTGT ACGCGTCCTG CTCAGCAAAC GCTGTGCTCC CAGATGTCCC

241 AACGACAACA TGAAGTTCGA ATGGTCGCCG GCCCCCATGG TGCAAGGCGT GATCACCAGG

301 CGCTGCTGTT CCTGGGCTCT CTGCAACAGG GCACTGACCC CACAGGAGGG GCGCTGGGCC 361 CTGCGAGGGG GGCTCCTGCT CCAGGACCCT TCGAGGGGCA GAAGAACCTG GGTGCGGCCA 421 CAGCTGGGGC TCCCACTCTG CCTTCCCATT TCCAGCCCCC TCTGCCCAAG GGAAACCCAG 481 GAAGGATAA

SEQ ID NO:10 (AAX19982 Protein sequence) 1 MERLVLTLCT LPLAVASAGC ATTPARNLSC YQCFKVSS T ECPPTWCSPL DQVCISNEVV 61 VSFK SVRVL LSKRCAPRCP NDN KFE SP APMVQGVITR RCCS ALCNR ALTPQEGRA 121 LRGGLLLQDP SRGRRT VRP QLGLPLCLPI SSPLCPRETQ EG*

SEQ ID NO:ll (INSP062 Nucleotide sequence exon 1) 1 ATGGAGAGGC TCGTCCTAAC CCTGTGCACC CTCCCGCTGG CTGTGGCGTC TGCTGGCTGC 61 GCCACGACGC CAG

SEQ ID NO:12 (INSP062 Protein sequence exon 1)

1 MERLVLTLCT LPLAVASAGC ATTPA

SEQ ID NO: 13 (INSP062 Nucleotide sequence exon 2)

1 CTCGCAACCT GAGCTGCTAC CAGTGCTTCA AGGTCAGCAG CTGGACGGAG TGCCCGCCCA 61 CCTGGTGCAG CCCGCTGGAC CAAGTCTGCA TCTCCAACGA GGTGGTCGTC TCTTTTA

SEQ ID NO:14 (INSP062 Protein sequence exon 2)

1 RNLSCYQCFK VSS TECPPT CSPLDQVCI SNEVWSFK

SEQ ID NO:15 (INSP062 Nucleotide sequence exon 3)

1 AATGGAGTGT ACGCGTCCTG CTCAGCAAAC GCTGTGCTCC CAGATGTCCC AACGACAACA 61 TGAAGTTCGA ATGGTCGCCG GCCCCCATGG TGCAAGGCGT GATCACCAGG CGCTGCTGTT

121 CCTGGGCTCT CTGCAACAGG GCACTGACCC CACAGGAGGG GCGCTGGGCC CTGCGAGGGG

181 GGCTCCTGCT CCAGGTGGGC CTCAGCCTCC TCAGGGCCCT GTTGTGA

SEQ ID NO: 16 (INSP062 Protein sequence exon 3) 1 SVRVLLSKR CAPRCPNDNM KFE SPAPMV QGVITRRCCS WALCNRALTP QEGRALRGG 61 LLLQVGLSLL RALL*

SEQ ID NO:17 (INSP062 Nucleotide sequence)

121 GAGTGCCCGC CCACCTGGTG CAGCCCGCTG GACCAAGTCT GCATCTCCAA CGAGGTGGTC

181 GTCTCTTTTA AATGGAGTGT ACGCGTCCTG CTCAGCAAAC GCTGTGCTCC CAGATGTCCC

241 AACGACAACA TGAAGTTCGA ATGGTCGCCG GCCCCCATGG TGCAAGGCGT GATCACCAGG

301 CGCTGCTGTT CCTGGGCTCT CTGCAACAGG GCACTGACCC CACAGGAGGG GCGCTGGGCC 361 CTGCGAGGGG GGCTCCTGCT CCAGGTGGGC CTCAGCCTCC TCAGGGCCCT GTTGTGA SEQ ID NO:18 (INSP062 Protein sequence)

1 MERLVLTLCT LPLAVASAGC ATTPARNLSC YQCFKVSSWT ECPPTWCSPL DQVCISNEW 61 VSFK SVRVL LSKRCAPRCP NDNMKFEWSP APMVQGVITR RCCSWALCNR ALTPQEGRWA 121 LRGGLLLQVG LSLLRALL*

SEQ ID NO: 19 (INSP063 Nucleotide sequence exon 1)

1 ATGAAGGTCA CTAGCCCCAT GCTGCTGCTG GCTGAGG

SEQ ID NO:20 (INSP063 Protein sequence exon 1)

1 MKVTSPMLLL AEG

SEQ ID NO:21 (INSP063 Nucleotide sequence exon 2)

1 GCCAGGGCCT TGAGTGCTTC CAGTGCTACG GTGTCCTGGA CCCCAGCCTG TGTCACCCCG 61 TCTCCTATCC CATGCAGGCT CAAAGCTGCC CCTCCTCTGT GGTCACTGGC ACTATCGATG 121 GTGAGTCCTG GGTGGGACCC AGCGTCTGTA GGCAG

SEQ ID NO:22 (INSP063 Protein sequence exon 2)

1 QGLECFQCYG VLDPSLCHPV SYPMQAQSCP SSVVTGTIDG ES VGPSVCR Q

SEQ ID NO:23 (TNSP063 Nucleotide sequence exon 3)

1 GGCAGAAGCT CAGCTACACT AGCAAGGGCT GTGGCCCCAC TCTGTGCCCA GATTATGAAC

61 CTCACCCATC CTGTGGTCCC TGGAGGGTCT TACCCCACAG AAATTGAGGA TAGACTGATT

121 GACTCGAAGA TTGAGAAGCT GGACATGACC TGCTGTGAAA ATAGCCTCCG TAACAAGGCG 181 GCCACAGTGC GGCGTGGCCT CTGGTGCCAG GCTGTCAGGG AGCTCCTGCT CAGCCTGAGC

241 CCCTTCCTCT GGGCTCTGCT GTGA

SEQ ID NO:24 (INSP063 Protein sequence exon 3)

1 GRSSATLARA VAPLCAQIMN LTHPVVPGGS YPTEIEDRLI DSKIEKLDMT CCENSLRNKA 61 ATVRRGL CQ AVRELLLSLS PFL ALL

SEQ ID NO:25 (INSP063 Nucleotide sequence)

1 ATGAAGGTCA CTAGCCCCAT GCTGCTGCTG GCTGAGGGCC AGGGCCTTGA GTGCTTCCAG

61 TGCTACGGTG TCCTGGACCC CAGCCTGTGT CACCCCGTCT CCTATCCCAT GCAGGCTCAA 121 AGCTGCCCCT CCTCTGTGGT CACTGGCACT ATCGATGGTG AGTCCTGGGT GGGACCCAGC

181 GTCTGTAGGC AGGGCAGAAG CTCAGCTACA CTAGCAAGGG CTGTGGCCCC ACTCTGTGCC

241 CAGATTATGA ACCTCACCCA TCCTGTGGTC CCTGGAGGGT CTTACCCCAC AGAAATTGAG

301 GATAGACTGA TTGACTCGAA GATTGAGAAG CTGGACATGA CCTGCTGTGA AAATAGCCTC

361 CGTAACAAGG CGGCCACAGT GCGGCGTGGC CTCTGGTGCC AGGCTGTCAG GGAGCTCCTG 421 CTCAGCCTGA GCCCCTTCCT CTGGGCTCTG CTGTGA

SEQ ID NO:26 (INSP063 Protein sequence)

1 MKVTSPMLLL AEGQGLECFQ CYGVLDPSLC HPVSYPMQAQ SCPSSVVTGT IDGES VGPS 61 VCRQGRSSAT LARAVAPLCA QIMNLTHPW PGGSYPTEIE DRLIDSKIEK LDMTCCENSL 121 RNKAATVRRG L CQAVRELL LSLSPFL AL L*

SEQ ID NO:27 (INSP062 Mature Protein sequence exon 1 A)

1 S GC ATT PA

SEQ ID NO:28 (INSP062 Mature Protein sequence A)

1 SAGCATTPAR NLSCYQCFKV SSWTECPPT CSPLDQVCIS NEVWSFK S VRVLLSKRCA 61 PRCPNDNMKF EWSPAPMVQG VITRRCCSWA LCNRALTPQE GRWALRGGLL LQVGLSLLRA 121 LL*

SEQ ID NO:29 (INSP062 Mature Protein sequence exon 1 B)

1 TTPA

SEQ ID NO:30 (INSP062 Mature Protein sequence B) 1 TTPARNLSCY QCFKVSSWTE CPPTWCSPLD QVCISNEVVV SFKWSVRVLL SKRCAPRCPN 61 DNMKFEWSPA PMVQGVITRR CCSWALCNRA LTPQEGRWAL RGGLLLQVGL SLLRALL*

SEQ ID NO:31 (INSP063 Mature Protein sequence exon 2)

1 ECFQCYGVLD PSLCHPVSYP MQAQSCPSSV VTGTIDGESW VGPSVCRQ

SEQ ID NO:32 (INSP063 Mature Protein sequence)

1 ECFQCYGVLD PSLCHPVSYP MQAQSCPSSV VTGTIDGESW VGPSVCRQGR SSATLARAVA 61 PLCAQIMNLT HPWPGGSYP TEIEDRLIDS KIEKLDMTCC ENSLRNKAAT VRRGLWCQAV 121 RELLLSLSPF LWALL*

SEQ ID NO:33 (Oligonucleotide primer - GCP Forward)

GGGGACAAGTTTGTACAAAAAAGCAGGCTTCGCCACC

SEQ ID NO:34 (Oligonucleotide primer - GCP Reverse) GGGGACCACTTTGTACAAGAAAGCTGGGTTTCAATGGTGATGGTGATGGTG

SEQ ID NO:35 (Oligonucleotide primer - INSP062-exonlF) ATGGAGAGGCTCGTCCTAACCCTGTGCACCCTCCCGCTGGCTGTGGCGTCTGCTG

SEQ ID NO:36 (Oligonucleotide primer - INSP062-exonlF2)

ATGGAGAGGCTCGTCCTAACCCTGTG

SEQ ID NO:37 (Oligonucleotide primer - INSP062-exonlR)

AGCAGCTCAGGTTGCGAGCTGGCGTCGTGGCGCAGCCAGCAGACGCCACAGCCA

SEQ ID NO:38 (Oligonucleotide primer - INSP062- exon2F) GCTGCGCCACGACGCCAGCTCGCAACCTGAGCTGCTAC

SEQ ID NO:39 (Oligonucleotide primer - INSP062- exon2R)

GGACGCGTACACTCCATTTAAAAGAGACGACCACCTCG

SEQ ID NO:40 (Oligonucleotide primer - INSP062- exon3F)

AGGTGGTCGTCTCTTTTAAATGGAGTGTACGCGTCCTG

SEQ ID NO:41 (Oligonucleotide primer - INSP062- exon3R)

TCACAACAGGGCCCTGAGGAGGCTGA

SEQ ID NO:42 (Oligonucleotide primer- INSP062-EX1)

GCAGGCTTCGCCACCATGGAGAGGCTCGTCCTAAC

SEQ ID NO:43 (Oligonucleotide primer - INSP062-EX2) GTGATGGTGATGGTGCAACAGGGCCCTGAGGAGGCTGA

SEQ ID NO:44 (Oligonucleotide primer - pEAK12-F)

GCCAGCTTGGCACTTGATGT

SEQ ID NO:45 (Oligonucleotide primer - pEAK12-R)

GATGGAGGTGGACGTGTCAG

SEQ ID NO:46 (Oligonucleotide primer - pENTR-Fl)

TCGCGTTAACGCTAGCATGGATCTC

SEQ ID NO:47 (Oligonucleotide primer - pENTR-Rl)

GTAACATCAGAGATTTTGAGACAC SEQ ID NO:48 (Oligonucleotide primer - INSP063-exonlF)

ATGAAGGTCACTAGCCCCATGCTGCTGCTGGCTGAG

SEQ ID NO:49 (Oligonucleotide primer - INSP063-exonlR)

AGCACTCAAGGCCCTGGCCCTCAGCCAGCAGCAGCAT

SEQ ID NO:50 (Oligonucleotide primer - INSP063- exon2F)

TGCTGCTGCTGGCTGAGGGCCAGGGCCTTGAGTGCTTC

SEQ ID NO:51 (Oligonucleotide primer - INSP063- exon2R)

TGTAGCTGAGCTTCTGCCCTGCCTACAGACGCTGGGTC

SEQ ID NO:52 (Oligonucleotide primer- INSP063- exon3F) CCCAGCGTCTGTAGGCAGGGCAGAAGCTCAGCTACACT

SEQ ID NO:53 (Oligonucleotide primer - INSP063- exon3R)

TCACAGCAGAGCCCAGAGAAGGGGCTCAGG

SEQ ID NO:54 (Oligonucleotide primer- INSP063-EX1)

GCAGGCTTCGCCACCATGAAGGTCACTAGCCCCATGC

SEQ ID NO:55 (Oligonucleotide primer - INSP063-EX2)

GTGATGGTGATGGTGCAGCAGAGCCCAGAGGAAGG

SEQ ID NO:56 (Cloned INSP062 nucleotide sequence)

1 ACAAGTTTGT ACAAAAAAGC AGGCTTCGCC ACCATGGAGA GGCTCGTCCT AACCCTGTGC 61 ACCCTCCCGC TGGCTGTGGC GTCTGCTGGC TGCGCCACGA CGCCAGCTCG CAACCTGAGC

121 TGCTACCAGT GCTTCAAGGT CAGCAGCTGG ACGGAGTGCC CGCCCACCTG GTGCAGCCCG

181 CTGGACCAAG TCTGCATCTC CAACGAGGTG GTCGTCTCTT TTAAATGGAG TGTACGCGTC 241 CTGCTCAGCA AACGCTGTGC TCCCAGATGT CCCAACGACA ACATGAAGTT CGAATGGTCG

301 CCGGCCCCCA TGGTGCAAGG CGTGATCACC AGGCGCTGCT GTTCCTGGGC TCTCTGCAAC

361 AGGGCACTGA CCCCACAGGA GGGGCGCTGG GCCCTGCGAG GGGGGCTCCT GCTCCAGGTG

421 GGCCTCAGCC TCCTCAGGGC CCTGTTGCAC CATCACCATC ACCATTGAAA CCCAGCTTTC 481 TTGTACAAAG TGGT SEQ ID NO:57 (Cloned INSP062 amino acid sequence)

MERLVLTLCTLPLAVASAGCATTPARNLSCYQCFKVSSWTECPPTWCSPLDQVCISNEWVSFKWSVRVLLSK RCAPRCPNDNMKFEWSPAPMVQGVITRRCCSWALCNRALTPQEGRWALRGGLLLQVGLSLLRALLHHHHHH

SEQ ID NO:58 (Cloned INSP063 nucleotide sequence)

1 ACAAGTTTGT ACAAAAAAGC AGGCTTCGCC ACCATGAAGG TCACTAGCCC

51 CATGCTGCTG CTGGCTGAGG GCCAGGGCCT TGAGTGCTTC CAGTGCTACG

101 GTGTCCTGGA CCCCAGCCTG TGTCACCCCG TCTCCTATCC CATGCAGGCT

151 CAAAGCTGCC CCTCCTCTGT GGTCACTGGC ACTATCGATG GTGAGTCCTG 201 GGTGGGACCC AGCGTCTGTA GGCAGGGCAG AAGCTCAGCT ACACTAGCAA

251 GGGCTGTGGC CCCACTCTGT GCCCAGATTA TGAACCTCAC CCATCCTGTG

301 GTCCCTGGAG GGTCTTACCC CACAGAAATT GAGGATAGAC TGATTGACTC

351 GAAGATTGAG AAGCTGGACA TGACCTGCTG TGAAAATAGC CTCCGTAACA

401 AGGCGGCCAC AGTGCGGCGT GGCCTCTGGT GCCAGGCTGT CAGGGAGCTC 451 CTGCTCAGCC TGAGCCCCTT CCTCTGGGCT CTGCTGCACC ATCACCATCA

501 CCATTGAAAC CCAGCTTTCT TGTACAAAGT GGT

SEQ ID NO:59 (Cloned INSP063 amino acid sequence) MKVTSPMLLLAEGQGLECFQCYGVLDPSLCHPVSYPMQAQSCPSSWTGTIDGESWVGPSVCRQGRSSATLAR AVAPLCAQIMNLTHPVVPGGSYPTEIEDRLIDSKIEKLDMTCCENSLRNKAATVRRGLWCQAVRELLLSLSPF LWALLHHHHHH

Claims

1. A polypeptide, which polypeptide:

(i) comprises the amino acid sequence as recited in SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:28 and/or SEQ ID NO:30; (ii) is a fragment thereof which is a member of the three finger toxin fold family, or having an antigenic determinant in common with the polypeptide of (i); or

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

2. A polypeptide according to claim 1 wliich:

(i) comprises the amino acid sequence as recited in SEQ ID NO: 18, SEQ ID NO:28 or SEQ ID NO:30;

(ii) is a fragment thereof which is a member of the three finger toxin fold family, or having an antigenic determinant in common with the polypeptide of (i); or

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

3. A polypeptide according to claim 1 or 2 which: (i) consists of the amino acid sequence as recited in SEQ ID NO: 16, SEQ ID

NO: 18, SEQ ID NO:28 and or SEQ ID NO:30;

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

4. A polypeptide, wliich polypeptide:

(i) comprises the amino acid sequence as recited in SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:31 and/or SEQ ID NO:32;

(ii) is a fragment thereof which is a member of the three finger toxin fold family, or having an antigenic determinant in common with the polypeptide of (i); or (iii) is a functional equivalent of (i) or (ii).

5. A polypeptide according to claim 4 which:

(i) comprises the amino acid sequence as recited in SEQ ID NO:26 or SEQ ID NO:32; (ii) is a fragment thereof which is a member of the three finger toxin fold family, or having an antigenic determinant in common with the polypeptide of (i); or

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

6. A polypeptide according to claim 4 or 5 which: (i) consists of the amino acid sequence as recited in SEQ ID NO:20, SEQ ID

NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:31 and/or SEQ ID NO:32;

(ii) is a fragment tliereof which is a member of the three finger toxin fold family, or having an antigenic determinant in common with the polypeptide of (i); or

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

7. A polypeptide which is a functional equivalent according to part (iii) of any of the above claims, characterised in that it is homologous to the amino acid sequence as recited in SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:31 or SEQ ID NO:32, and is a member of the three finger toxin fold family.

8. A polypeptide which is a fragment or a functional equivalent as recited in any one of claims 1 to 7, which has greater than 80% sequence identity with the amino acid sequence recited in SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:31 or SEQ ID NO:32, or with an active fragment thereof, preferably greater than 85%, 90%, 95%, 98% or 99% sequence identity.

9. A polypeptide which is a functional equivalent as recited in any one of claims 1 to 8, which exhibits significant structural homology with a polypeptide having the amino acid sequence recited in SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:31 or SEQ ID NO:32.

10. A polypeptide which is a fragment as recited in claims 1-6 and claim 8 having an antigenic determinant in common with the polypeptide of part (i) of any one of claim 1 to claim 6 which consists of 7 or more amino acid residues from the amino acid sequence recited in SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO.22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:31 or SEQ ID NO:32.

11. A purified nucleic acid molecule which encodes a polypeptide according to any one of the preceding claims.

12. A purified nucleic acid molecule according to claim 11, which comprises the nucleic acid sequence as recited in SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23 and or SEQ ID NO:25, or is a redundant equivalent or fragment thereof.

13. A purified nucleic acid molecule according to claim 11 which consists of the nucleic acid sequence as recited in SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23 and/or SEQ ID NO:25, or is a redundant equivalent or fragment thereof.

14. A purified nucleic acid molecule which hybridizes under high stringency conditions with a nucleic acid molecule according to any one of claims 11 to 13.

15. A vector comprising a nucleic acid molecule as recited in any one of claims 11 to 14.

16. A host cell transformed with a vector according to claim 15.

17. A ligand which binds specifically to the three finger toxin fold family polypeptide according to any one of claims 1 to 10.

18. A ligand according to claim 17, which is an antibody.

19. A compound that either increases or decreases the level of expression or activity of a polypeptide according to any one of claims 1 to 10.

20. A compound according to claim 19 that binds to a polypeptide according to any one of claims 1 to 10 without inducing any of the biological effects of the polypeptide.

21. A compound according to claim 20, which is a natural or modified substrate, ligand, enzyme, receptor or structural or functional mimetic.

22. A polypeptide according to any one of claims 1 to 10, a nucleic acid molecule according to any one of claims 11 to 14, a vector according to claim 15, a host cell according to claim 16, a ligand according to claim 17 or claim 18, or a compound according to any one of claims 19 to 21, for use in therapy or diagnosis of disease.

23. A method of diagnosing a disease in a patient, comprising assessing the level of expression of a natural gene encoding a polypeptide according to any one of claims 1 to 10, or assessing the activity of a polypeptide according to any one of claims 1 to 10, 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.

24. A method according to claim 23 that is carried out in vitro.

25. A method according to claim 23 or claim 24, which comprises the steps of: (a) contacting a ligand according to claim 17 or claim 18 with a biological sample under conditions suitable for the formation of a ligand-polypeptide complex; and (b) detecting said complex.

26. A method according to claim 23 or claim 24, 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 11 to 14 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.

27. A method according to claim 23 or claim 24, 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 11 to 14 and the primer; b) contacting a control sample witii 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.

28. A method according to claim 23 or claim 24 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 11 to 14 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.

29. The method of claim 28, 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.

30. The method of claim 28 or claim 29, 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.

31. A method according to any one of claims 23 to 30, wherein said disease includes, but is not limited to, cell proliferative disorders, including neoplasm, melanoma, lung, colorectal, breast, pancreas, head and neck and other solid tumours; myeloproliferative disorders, such as leukemia, non-Hodgkin lymphoma, leukopenia, thrombocytopenia, angiogenesis disorder, Kaposis' sarcoma; autoinimune/inflammatory disorders, including allergy, inflammatory bowel disease, arthritis, psoriasis and respiratory tract inflammation, asthma, and organ transplant rejection; cardiovascular disorders, including hypertension, oedema, angina, atherosclerosis, thrombosis, sepsis, shock, reperfusion injury, and ischemia; neurological disorders including central nervous system disease, Alzheimer's disease, brain injury, amyotrophic lateral sclerosis, and pain; developmental disorders; metabolic disorders including diabetes mellitus, osteoporosis, and obesity, AIDS and renal disease; infections including viral infection, bacterial infection, fungal infection and parasitic infection.

32. A method according to any one of claims 23 to 30, wherein said disease is a disease in which lymphocyte antigens are implicated.

33. Use of a polypeptide according to any one of claims 1 to 10 as a three finger toxin fold protein.

34. A pharmaceutical composition comprising a polypeptide according to any one of claims 1 to 10, a nucleic acid molecule according to any one of claims 11 to 14, a vector according to claim 15, a host cell according to claim 16, a ligand according to claim 17 or claim 18, or a compound according to any one of claims 19 to 21.

35. A vaccine composition comprising a polypeptide according to any one of claims 1 to 10 or a nucleic acid molecule according to any one of claims 11 to 14.

36. A polypeptide according to any one of claims 1 to 10, a nucleic acid molecule according to any one of claims 11 to 14, a vector according to claim 15, a host cell according to claim 16, a ligand according to claim 17 or claim 18, a compound according to any one of claims 19 to 21, or a pharmaceutical composition according to claim 34, for use in the manufacture of a medicament for the treatment of cell proliferative disorders, including neoplasm, melanoma, lung, colorectal, breast, pancreas, head and neck and other solid tumours; myeloproliferative disorders, such as leukemia, non-Hodgkin lymphoma, leukopenia, thrombocytopenia, angiogenesis disorder, Kaposis' sarcoma; autoimmune/inflammatory disorders, including allergy, inflammatory bowel disease, arthritis, psoriasis and respiratory tract inflammation, asthma, and organ transplant rejection; cardiovascular disorders, including hypertension, oedema, angina, atherosclerosis, thrombosis, sepsis, shock, reperfusion injury, and ischemia; neurological disorders including central nervous system disease, Alzheimer's disease, brain injury, amyotrophic lateral sclerosis, and pain; developmental disorders; metabolic disorders including diabetes mellitus, osteoporosis, and obesity, AIDS and renal disease; infections including viral infection, bacterial infection, fungal infection and parasitic infection and other pathological conditions.

37. A polypeptide according to any one of claims 1 to 10, a nucleic acid molecule according to any one of claims 11 to 14, a vector according to claim 15, a host cell according to claim 16, a ligand according to claim 17 or claim 18, a compound according to any one of claims 19 to 21, or a pharmaceutical composition according to claim 34, for use in the manufacture of a medicament for the treatment of a disease in which lymphocyte antigens are implicated.

38. A method of treating a disease in a patient, comprising administering to the patient a polypeptide according to any one of claims 1 to 10, a nucleic acid molecule according to any one of claims 11 to 14, a vector according to claim 15, a host cell according to claim 16, a ligand according to claim 17 or claim 18, a compound according to any one of claims 19 to 21, or a pharmaceutical composition according to claim 34.

39. A method according to claim 38, wherein, for diseases in which the expression of the natural gene or the activity of the polypeptide is lower in a diseased patient when compared to the level of expression or activity in a healthy patient, the polypeptide, nucleic acid molecule, vector, ligand, compound or composition administered to the patient is an agonist.

40. A method according to claim 38, wherein, for diseases in which the expression of the natural gene or activity of the polypeptide is higher in a diseased patient when compared to the level of expression or activity in a healthy patient, the polypeptide, nucleic acid molecule, vector, ligand, compound or composition administered to the patient is an antagonist.

41. 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 a polypeptide according to any one of claims 1 to 10, or the level of expression of a nucleic acid molecule according to any one of claims 11 to 14 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.

42. A method for the identification of a compound that is effective in the treatment and/or diagnosis of disease, comprising contacting a polypeptide according to any one of claims 1 to 10, or a nucleic acid molecule according to any one of claims 11 to 14 with one or more compounds suspected of possessing binding affinity for said polypeptide or nucleic acid molecule, and selecting a compound that binds specifically to said nucleic acid molecule or polypeptide.

43. 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 11 to 14; 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.

44. The kit of claim 43, further comprising a third container holding an agent for digesting unhybridised RNA.

45. 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 11 to 14.

5 46. A kit comprising one or more antibodies that bind to a polypeptide as recited in any one of claims 1 to 10; and a reagent useful for the detection of a binding reaction between said antibody and said polypeptide.

47. 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 1 to 10.

10 48. A method for screening for a compound effective to treat disease, by contacting a non- human transgenic animal according to claim 47 with a candidate compound and determining the effect of the compound on me disease of the animal.

49. A method according to any one of claims 37-46 or claim 48, wherein said disease is one of the diseases set for the in claim 31 or 36.