AU772811B2 - A method of modulating cell survival and reagents useful for same - Google Patents

Description

WO 00/20578 PCT/AU99/00860 -1- A METHOD OF MODULATING CELL SURVIVAL AND REAGENTS USEFUL FOR SAME FIELD OF THE INVENTION The present invention relates generally to a method for modulating cell survival.

Modulation of cell survival includes inducing, enhancing or otherwise promoting cell survival such as the survival of neural cells as well as facilitating cell death such as the death of targeted cancer cells. The modulation of cell survival is mediated by a region identified on the p75 neurotrophin receptor (p75NTR) required for death signalling. The present invention further provides genetic molecules which encode the death signalling region of p 7 5 NTR which are useful in antagonising death signal function as well as promoting cell death when expressed in targeted cells. The present invention also contemplates recombinant peptides, polypeptides and proteins as well as chemical equivalents, derivatives and homologues thereof which comprise the death signalling portion of p75NT

R

Particularly useful molecules of the present invention comprise peptides corresponding to soluble forms of the death signalling portion of p75

R

These molecules antagonise p75NTR-mediated cell death.

BACKGROUND OF THE INVENTION Bibliographic details of the publications'numerically referred to in this specification are collected at the end of the description.

The subject specification contains nucleotide and amino acid sequence information prepared using the programme Patentln Version 2.0, presented herein after the bibliography. Each nucleotide or amino acid sequence is identified in the sequence listing by the numeric indicator <210> followed by the sequence identifier (e.g.

<210>1, <210>2, etc). The length, type of sequence (DNA, protein (PRT), etc) and I WO 00/20578 PCT/AU99/00860 -2source organism for each nucleotide or amino acid sequence are indicated by information provided in the numeric indicator fields <211>, <212> and <213>, respectively. Nucleotide and amino acid sequences referred to in the specification are defined by the information provided in numeric indicator field <400> followed by the sequence identifier (eg. <400>1, <400>2, etc).

The increasing sophistication of recombinant DNA technology is greatly facilitating research and development in the medical and allied health fields. This is particularly the case in the development of recombinant cytokines and growth factors for use in the treatment of diabetes, acquired immunodeficency syndrome (AIDS) and a number of cancers.

However, despite this developing knowledge of cytokine and growth factor effector molecules, their full exploitation requires an understanding of the corresponding cellular receptors and the complex biochemical and physiological signalling pathways initiated following interaction with ligands or following other stimulation such as disease, receptor aggregation or trauma.

A number of soluble trophic factors have been shown to exhibit an effect on neural survival in vivo. Many of these factors act directly on the developing neuron within, for example, the dorsal root ganglia (DRG). One factor of particular importance is nerve growth factor (NGF) The p75 neurotrophin receptor (hereinafter referred to as "p 7 5 which is capable of associating with trk growth factor receptors, facilitates high affinity NGF binding and survival signalling. Although NGF has been proposed as a potential therapeutic molecule to promote survival of neurons, NGF is a multifunctional molecule and its pleiotrophy may adversely effect a range of non-neural cells.

p 7 5 NTR is also multifunctional. It has now been shown that p 7 5 NTR is capable of acting as a death receptor. Elevated p 7 5 NTR expression results in increased cell death in vitro and in vivo Furthermore, down-regulation of p 7 5 NTR prevents WO 00/20578 PCT/AU99/00860 -3neural death after growth-factor withdrawal or axotomy Consistent with the dual functions of p 7 5 NTR, mice with deleted p 7 5 NTR genes have a dramatic reduction of NGF dependent neurons, such as dorsal root ganglia, but increased numbers of other neuron populations (sympathetic and basal forebrain neurons) suggesting lack of naturally occurring cell death p 7 5 NTR is also implicated in mediating death of neural, oligodendrocytes and Schwann cells 9].

p 7 5 NTR is a member of the tumor necrosis factor (TNF) receptor/Fas superfamily, showing homology not only to the extracellular ligand binding domain but also to a cytoplasmic motif known as the "death domain", so termed because of the cytotoxic actions of proteins containing the domain There is an accumulating body of evidence which suggests that p 7 5 NTR is involved in mediating cell death in a variety of degenerative diseases. During adulthood, p75 NTR expression is down-regulated in most brain areas but is rapidly induced in ischemia (stroke) and results in transient increased p75NT expression and apoptosis, as do both peripheral and motor nerve lesions [10-12]. p 7 5 NTR is also up regulated in patients with MND and in experimental allergic encephalomyelitis (a model of multiple sclerosis; Intriguingly, in the basal forebrain and hippocampus, areas involved in learning and memory, p 7 5 NTR is highly expressed in aged rodents and in Alzheimer's patients, where extensive neural death is occurring 16]. These data suggest that p 7 5 NTR is involved not only in normal developmental cell death, but may mediate the cell death occurring after injury or in neurodegenerative disease.

In work leading up to the present invention, the inventors sought to elucidate the region on p75 N T R which mediates death signalling. The inventors surprisingly determined that the death signal is not the cytoplasmic motif known as the death domain but is a region adjacent the membrane domain on p 7 5 NTR. The identification of this region provides for an opportunity to modulate cell survival by antagonising the death signalling region or promoting apoptosis by providing cells

I

WO 00/20578 PCT/AU99/00860 -4with the genetic material to express the death signalling region adjacent, proximal or otherwise juxtaposed or associated with the membrane or to express the death signalling region in multimeric form.

SUMMARY OF THE INVENTION Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

One aspect of the present invention provides an isolated nucleic acid molecule comprising a sequence of nucleotides or complementary sequence of nucleotides which encodes an amino acid sequence which is capable of signalling, inducing or otherwise facilitating the death of a cell in which said amino acid sequence is adjacent, proximal or otherwise juxtaposed to the membrane of said cell or when said amino acid sequence is in multimeric form.

Another aspect of the present invention is directed to a nucleic acid molecule comprising a sequence of nucleotides or complementary sequence of nucleotides which encodes a peptide, polypeptide or protein capable of signalling, inducing or otherwise facilitating death of a cell in which it is expressed wherein said peptide, polypeptide or protein comprises a membrane associating portion and/or a multimer-forming portion and a portion which corresponds to all or part of the cytoplasmic region of p 7 5 NTR or a functional equivalent, derivative or homologue thereof.

Yet another aspect of the present invention contemplates homologues, analogues and derivatives of a nucleic acid molecule which encodes a peptide, polypeptide or protein which is capable of signalling inducing or otherwise facilitating death of a WO 00/20578 PCT/AU99/00860 cell in which it is expressed wherein said peptide, polypeptide or protein comprises a membrane associating portion and/or a multimer-forming portion and a portion which corresponds to all or part of the cytoplasmic region of p 75 NTR or a functional equivalent, derivative or homologue thereof.

A further aspect of the present invention provides an isolated nucleic acid molecule comprising a sequence of nucleotides which encodes an amino acid sequence which inhibits or reduces p 7 5 NTR-mediated cell death wherein said amino acid sequence is a soluble form of the p 75 NTR receptor corresponding to an intracellular region adjacent, proximal or otherwise juxtaposed to the membrane of said cell.

Still another aspect of the present invention provides a nucleic acid molecule comprising a nucleotide sequence or complementary nucleotide sequence which is substantially as set forth in <400>3 or is a nucleotide sequence capable of hybridising to <400>3 or its complementary form under low stringency conditions or is a nucleotide sequence having at least 60% identity to <400>3.

Still yet another aspect of the present invention contemplates a nucleic acid molecule comprising a nucleotide sequence or a complementary form thereof, which nucleotide sequence encodes an amino acid sequence substantially as set forth in <400>4 or a derivative, homologue or chemical equivalent thereof or an amino acid sequence having at least 60% identity thereto.

Even yet another aspect of the present-invention provides a genetic construct comprising an isolated nucleic acid molecule which comprises a sequence of nucleotides which corresponds or is complementary to a death signal region from p 75 NTR or a homologue, analogue or derivative thereof.

Another aspect of the present invention contemplates an isolated peptide, polypeptide or protein comprising the cytoplasmic region of p 75 NTR which signals, induces or otherwise facilitates cell death when said peptide, polypeptide or protein WO 00/20578 PCT/AU99/00860 -6is adjacent, proximal or otherwise juxtaposed to a membrane-associating region such as from p 7 5 NTR or other membrane molecule and/or said peptide, polypeptide or protein is capable of forming multimers or a derivative, homologue, chemical equivalent or analogue of said peptide, polypeptide or protein. This aspect of the present invention does not extend to the full length p 7 5

NTR

Still another aspect of the present invention contemplates a method for inhibiting, reducing or otherwise antagonising a p75NTR-mediated death signal in a neural cell, said method comprising introducing a nucleic acid molecule capable of being expressed to an expression product which corresponds to a non-membrane associated form of the p 7 5 NTR death signal region or a derivative, functional equivalent or homologue thereof.

Yet another aspect of the invention contemplates a method for inhibiting, reducing or otherwise antagonising a p75NTR-mediated death signal in a neural cell, said method comprising contacting a cell carrying a p 7 5 NTR with a death signal-inhibiting effective amount of a molecule capable of antagonising the death signal of p 7 5

NTR

or a component of the death signalling pathway.

Even still another aspect of the present invention provides a biological composition comprising a genetic molecule capable of expressing a p 7 5 NTR death signal antagonist or a p75 N TR death signal.

Another aspect of the present invention is directed to a biological composition comprising a molecule capable of antagonising p75NTR-mediated death signalling of a cell.

Yet still another aspect of the present invention contemplates a method for modulating p75NTR-mediated death signal in a neural cell, said method comprising administering an agent which antagonises or agonises cleavage of the extracellular domain of p 7 5

NTR

WO 00/20578 PCT/AU99/00860 -7- Still another aspect of the present invention provides a method for inhibiting, reducing or otherwise antagonising p75NTR-mediated death signal in a neural cell, said method comprising administering a peptide, polypeptide or protein or analogues or mimetics thereof which correspond to a non-membrane associated form of the p 7 5 NTR death signal region or a derivative, functional equivalent or homologue thereof.

Another aspect of the present invention provides peptide antagonists of the p 7 5

NTR

death signal or functional analogues or mimetics thereof.

The terms "c35" and "35mer" are used interchangeably herein to refer to 35 amino acid domain juxtaposed to the membrane. When in soluble form, this peptide is referred to as soluble c35 or 35mer. The nucleotide and amino acid sequence of are shown in <400>7 and <400>8, respectively. The term "29mer" refers to a truncated form of the 35mer. Six amino acids have been deleted from the Cterminal end. The nucleotide and amino acid sequence of 29mer are shown in <400>11 and <400>12, respectively. The present invention extends to isolated forms of c35 and the 29 mer, to compositions comprising same and to genetic sequences encoding same.

WO 00/20578 PCT/AU99/00860 -8- BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a diagrammatic representation showing plasmid constructs with and without the death signalling region. The black region is the putative "death domain" but which is not directly involved in p 7 5 NT mediated cell death.

Figure 2 is a graphical representation showing survival of DRG neurons 17 hours after microinfection and cultured in LIF. The data show that the amino acid domain juxtaposed to the membrane is required for death signalling rather than the putative "death domain" Figure 3 is a graphical representation showing DRG survival 16 hours after microinjection and cultured in LIF. The data show that over 90% of cells die when expressing the death signal linked to the membrane.

Figure 4 is a graphical representation showing DRG survival 20 hours after microinjection and cultured in LIF. These data show that when the death signal is not associated with the membrane, that the ability to induce death is removed.

Figure 5 is a graphical representation showing that the c35 soluble protein (i.e.

p 7 5 NTR death signal region) inhibits death signalling mediated by p75

NTR

Figure 6 is a graphical representation showing that soluble c35 inhibits p 7 5

NTR-

mediated death signalling.

Figure 7 is a graphical representation showing protection of membrane-bound killing-domain by a soluble 35 amino acid peptide and a soluble 29 amino acid peptide. The cells were subjected to microinjection of sptc35 or GFP followed minutes later by either peptide c35 or the 29mer peptide.

Figure 8 is a graphical representation showing that peptide 29 which has a WO 00/20578 PCT/AU99/00860 -9palmitoyl group at the membrane (amino) end and which facilitates association with the membrane mediates to cell death. In contrast, the soluble 35 amino acid molecule tends to protect the cells. F, Fluoro tagged; pen, penetratin.

Figure 9 is a graphical representation showing the palmitoylated 29mer fused to penetratin mediates specific killing whereas non-palmitoylated 29mer blocks cell death. Cells were treated with 2 AM peptide for 1-2 hours then washed. pen, penetratin, F29, 29 mer; Palm, palmitoylation.

pen F29 pen F29 Palm pen gp130 Palm pen WO 00/20578 PCT/AU99/00860 DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT The present invention arose in part following an investigation of the neurotrophin receptor, p 75 NTR in its capacity as a death signalling protein. Although the p 75

NTR

molecule comprises a putative death domain in accordance with the present invention, this death domain is not directly associated with p75NTR-mediated cell death. Rather, a region adjacent, proximal or otherwise juxtaposed to the membrane domain of p 7 5 NTR is required for cell death. The nucleotide and corresponding amino acid sequence of the death domain is shown in <400>9 and <400>10, respectively.

Accordingly, one aspect of the present invention provides an isolated nucleic acid molecule comprising a sequence of nucleotides or complementary sequence of nucleotides which encode an amino acid sequence which is capable of signalling, inducing or otherwise facilitating the death of a cell in which said amino acid sequence is adjacent, proximal or otherwise juxtaposed to the membrane of said cell or when said amino acid sequence is in multimeric form.

Reference herein to the signalling, inducing or otherwise facilitating the death of a cell or a death signal is meant to be construed in its broadest sense meaning that the amino acid sequence plays a role in a pathway leading to cell death. The death signal may also be regarded as an apoptopic signal. Although not wishing to limit the present invention to any one theory or mode of action, it is proposed herein that there is a pathway from p75TR activation to caspase activation and cellular degeneration, p75 TR-mediated cell death may also occur directly or indirectly via Bcl-2.

The present specification refers interchangeably to death signal, death signal region, signalling, inducing or otherwise facilitating the death of a cell and The nucleic acid molecule of the present invention may encode a non-full length WO 00/20578 PCT/AU99/00860 -11p 7 5 NTR molecule although to facilitate cell death, the nucleic acid molecule must encode all or part of the cytoplasmic portion of the p 7 5 NTR molecule and a sufficient amount of the membrane domain such that the region referred to herein as the death signal is membrane associated. A "part" of the cytoplasmic domain of p75

NT

includes all or a death-inducing functional part of a 35 amino acid region juxtaposed to the membrane domain. An example of a part of the 35 amino acid region is a truncated form. One such form is referred to herein as the "29 mer". Alternatively, the cytoplasmic domain of the p 7 5 NTR molecule is in multimeric form or capable of forming multimers. A multimer comprises two or more copies of the molecule such as a dimer, trimer or larger copy molecule.

The term "membrane associated" means that the death signal is adjacent, proximal or otherwise juxtaposed to the membrane of a cell expressing the nucleic acid molecule.

The "death signal region" and other related terms are used herein to describe functionally the region of the cytoplasmic portion of p 7 5 NTR which is adjacent, proximal or otherwise juxtaposed to a region of p 7 5 NT R which associates with the membrane or which cytoplasmic portion is in multimeric form. The death signal region is not the same portion of the molecules as the "death domain" although there may be functional similarities in death signalling.

Accordingly, another aspect of the present invention is directed to a nucleic acid molecule comprising a sequence of nucleotides or complementary sequence of nucleotides which encodes a peptide, polypeptide or protein capable of signalling, inducing or otherwise facilitating death of a cell in which it is expressed wherein said peptide, polypeptide or protein comprises a membrane associating portion and/or a multimer-forming portion and a portion which corresponds to all or part of the cytoplasmic region of p 7 5 N TR or a functional equivalent, derivative or homologue thereof.

WO 00/20578 PCT/AU99/00860 -12- In order to signal, induce or otherwise facilitate death of a cell, the death signal region is preferably adjacent, proximal or otherwise juxtaposed to the cell membrane. This may be facilitated by modifying a peptide such that it associates with the membrane. One example of this type of modification is palmi toylation.

This puts a palmitoyl group at the membrane (amino) end of the peptide.

Accordingly, another aspect of the present invention contemplates plamitoylated peptides, polypeptides or proteins comprising all or part of the death signal region of p 7 5 NTR. Such peptides are particularly useful in promoting cell death.

The present invention also extends to multimeric forms of death signal peptides, polypeptides and proteins and attachments which facilitate same. A multimer comprises two or more molecules. The present invention also extends to cleavage forms of the full length p 7 5 NTR molecule.

In one embodiment, the membrane portion is derived from p 7 5 NTR or a functional equivalent, derivative or homologue thereof. In another embodiment, the membrane domain is from another molecule such as a receptor or other ligandbinding molecule. Examples of receptors according to this aspect of the present invention include cytokine receptors the Leukaemia Inhibitory Factor (LIF) receptor, interleukin receptor, and colony-stimulating factor receptors). Examples of ligand-binding molecules include immunoglobulins and T cell receptors.

When in multimeric form, the molecule-is only optionally associated with the membrane to effect cell death.

The nucleic acid molecule may comprise cDNA or genomic DNA or may comprise ribonucleotides such as mRNA. The nucleic acid molecule may be derived from a cDNA or genomic molecule encoding p75NTR or a derivative or homologue thereof or may be prepared by the stepwise addition of nucleotides in a defined sequence.

WO 00/20578 PCT/AU99/00860 13- The nucleic acid molecule of the present invention may also be considered as corresponding to a "gene".

Reference herein to a "gene" is to be taken in its broadest context and includes: a classical genomic gene consisting of transcriptional and/or translational regulatory sequences and/or a coding region and/or nontranslated sequences introns, and untranslated sequences); (ii) mRNA or cDNA corresponding to the coding regions exons) optionally comprising or 3'-untranslated sequences of the gene; or (iii) an amplified DNA fragment or other recombinant nucleic acid molecule produced in vitro and comprising all or a part of the coding region and/or or 3'-untranslated sequences of the gene.

The term "gene" is also used to describe synthetic or fusion molecules encoding all or part of a functional product. A functional product is one which comprises a sequence of nucleotides or is complementary to a sequence of nucleotides which encodes a functional death signal from p 7 5 NTR or its derivative or homologue.

The nucleotide sequence of the present invention may correspond to the cDNA or genomic sequence of a gene encoding p75N R or a death signal region thereof or may be subjected to mutagenesis to produce single or multiple nucleotide substitutions, deletions and/or additions. Nucleotide insertional derivatives of the nucleic acid molecule of the present invention include 5' and 3' terminal fusions as well as intra-sequence insertions of single or multiple nucleotides. Insertional nucleotide sequence variants are those in which one or more nucleotides are introduced into a predetermined site in the nucleotide sequence although random insertion is also possible with suitable screening of the resulting product. Deletional variants are characterised by the removal of one or more nucleotides from the sequence. Substitutional nucleotide variants are those in which at least one nucleotide in the sequence has been removed and a different nucleotide inserted in its place. Such a substitution may be "silent" in that the substitution does not WO 00/20578 PCT/AU99/00860 -14change the amino acid defined by the codon. Alternatively, substituents are designed to alter one amino acid for another similar acting amino acid, or amino acid of like charge, polarity, or hydrophobicity.

Accordingly, another aspect of the present invention contemplates homologues, analogues and derivatives of a nucleic acid molecule which encodes a peptide, polypeptide or protein which is capable of signalling, inducing or otherwise facilitating death of a cell in which it is expressed wherein said peptide, polypeptide or protein comprises a membrane associating portion and/or multimer-forming portion and a portion which corresponds to all or part of the cytoplasmic region of p 7 5 NTR or a functional equivalent, derivative or homologue thereof.

For the present purpose, "homologues" of a nucleic acid molecule as herein defined or of a nucleotide sequence shall be taken to refer to an isolated nucleic acid molecule which is substantially the same as the nucleic acid molecule of the present invention or its complementary nucleotide sequence, notwithstanding the occurrence within said sequence, of one or more nucleotide substitutions, insertions, deletions, or rearrangements.

"Analogues" of a nucleic acid molecule as herein defined or of a nucleotide sequence set forth herein shall be taken to refer to an isolated nucleic acid molecule which is substantially the same as a nucleic acid molecule of the present invention or its complementary nucleotide sequence, notwithstanding the occurrence of any non-nucleotide constituents not normally present in said isolated nucleic acid molecule, for example carbohydrates, radiochemicals including radionucleotides, reporter molecules such as, but not limited to DIG, alkaline phosphatase or horseradish peroxidase, amongst others.

"Derivatives" of a nucleic acid molecule as herein defined or of a nucleotide sequence set forth herein shall be taken to refer to any isolated nucleic acid molecule which contains significant sequence similarity to said sequence or a part WO 00/20578 PCT/AU99/00860 thereof. Generally, the nucleotide sequence of the present invention may be subjected to mutagenesis to produce single or multiple nucleotide substitutions, deletions and/or insertions. Nucleotide insertional derivatives of the nucleotide sequence of the present invention include 5' and 3' terminal fusions as well as intra-sequence insertions of single or multiple nucleotides or nucleotide analogues.

Insertional nucleotide sequence variants are those in which one or more nucleotides or nucleotide analogues are introduced into a predetermined site in the nucleotide sequence of said sequence, although random insertion is also possible with suitable screening of the resulting product being performed. Deletional variants are characterised by the removal of one or more nucleotides from the nucleotide sequence. Substitutional nucleotide variants are those in which at least one nucleotide in the sequence has been removed and a different nucleotide or nucleotide analogue inserted in its place.

In one embodiment, the derivatives encode a peptide, polypeptide or protein which induces cell death. In another embodiment, the derivatives do not induce cell death but antagonise the death signal.

According to this latter embodiment, there is provided an isolated nucleic acid molecule comprising a sequence of nucleotides which encodes an amino acid sequence which inhibits or reduces p75NTR-mediated cell death wherein said amino acid sequence is a soluble form of the p 7 5 NTR receptor corresponding to an intracellular region adjacent, proximal or otherwise juxtaposed to the membrane of said cell.

The nucleic acid molecule of the present invention may be based on a nucleotide sequence of the gene or cDNA encoding p75

NT

R from any animal such as from mammals. Preferred mammals include humans, primates, livestock animals (e.g.

cows, sheep, horses, pigs, donkeys, goats), laboratory test animals rabbits, mice, rats, guinea pigs, hamsters), companion animals dogs, cats) and captive wild animals.

WO 00/20578 PCT/AU99/00860 -16- A particularly preferred sequence is from human or primate or murine p 7 5

NTR

Although not wishing to limit the present invention to any one theory or mode of action, it is proposed that the extracellular domain of p 7 5 NTR may be cleaved off resulting in active death signal (see Zupan et al Accordingly, by antagonising cleavage, cell death may be prevented or at least delayed or inhibited. Conversely, for targeted cancer cells, an agonist of p 7 5 NTR extracellular domain cleavage would promote cell death.

Accordingly, another aspect of the present invention contemplates a method for modulating p75NTR-mediated death signal in a neural cell, said method comprising administering an agent which antagonises or agonises cleavage of the extracellular domain of Preferably, to prevent neural cell death, extracellular p 7 5 NTR cleavage is antagonised.

The present invention is exemplified using a nucleotide sequence from rat p 7 5

NTR

cDNA. This is done, however, with the understanding that the nucleotide sequence may be from p 7 5 NTR genomic or cDNA from any animal.

Accordingly, another aspect of the present invention provides a nucleic acid molecule comprising a nucleotide sequence or a complementary form thereof wherein said nucleotide sequence is capable of hybridising to <400>1 or a complementary form thereof under low stringency conditions, such as at 42 °C.

The nucleotide sequence set forth in <400>1 is the cDNA sequence encoding p 7 5 NTR. The nucleic acid molecule according to this aspect of the present invention does not extend to the full length p 7 5 TR cDNA sequence but comprises a portion which encodes an amino acid sequence which signals, induces or otherwise facilitates cell death when associated with a membrane portion of p 7 5 NTR or other WO 00/20578 PCT/AU99/00860 -17molecules.

Accordingly, another aspect of the present invention provides a nucleic acid molecule comprising a nucleotide sequence or complementary nucleotide sequence which is substantially as set forth in <400>7 or is a nucleotide sequence capable of hybridising to <400>7 or a complementary form thereof under low stringency conditions such as at 42 °C or is a nucleotide sequence having at least identity to <400>7.

The nucleotide sequence set forth in <400>7 is the death signal defined herein associated with p 7 5 NT R This sequence encodes a 35 amino acid region also referred to herein as "c35". Truncated forms of c35 are also contemplated by the present invention such as a 25-30 amino acid molecules. One particular example is a 29mer which lacks carboxy terminal amino acids 30 to 35. As stated above, the present invention extends to palmitoylated c35 and its derivatives as well as molecules fused with molecules to facilitate membrane passage such as penetratin and the TAT protein from HIV.

Reference herein to a low stringency such as at 42 0 C includes and encompasses from at least about 0% v/v to at least about 15% v/v formamide and from at least about 1M to at least about 2M salt for hybridisation, and at least about 1M to at least about 2M salt for washing conditions. Alternative stringency conditions may be applied where necessary, such as medium stringency, which includes and encompasses from at least about 16%-v/v to at least about 30% v/v formamide and from at least about 0.5M to at least about 0.9M salt for hybridisation, and at least about 0.5M to at least about 0.9M salt for washing conditions, or high stringency, which includes and encompasses from at least about 31% v/v to at least about v/v formamide and from at least about 0.01M to at least about 0.15M salt for hybridisation, and at least about 0.01M to at least about 0.15M salt for washing conditions. Preferably, low stringency is determined at 42 0

C.

WO 00/20578 PCT/AU99/00860 -18- The present invention further contemplates a nucleic acid molecule comprising a nucleotide sequence or a complementary form thereof, which nucleotide sequence encodes an amino acid sequence substantially as set forth in <400>8 or a derivative, homologue or chemical equivalent thereof or an amino acid sequence having at least 60% identity thereto.

The amino acid sequence of <400>8 corresponds to the amino acid sequence of the p 75 NTR death signal.

The term "identity" as used herein includes exact identity between compared sequences at the nucleotide or amino acid level. Where there is non-identity at the nucleotide level, the term "similarity" may also be used and includes differences between sequences which result in different amino acids that are nevertheless related to each other at the structural, functional, biochemical and/or conformational levels. Where there is non-identity at the amino acid level, "similarity" includes amino acids that are nevertheless related to each other at the structural, functional, biochemical and/or conformational levels. In a particularly preferred embodiment, nucleotide and sequence comparisons are made at the level of identity rather than similarity. Any number of programs are available to compare nucleotide and amino acid sequences. Preferred programs have regard to an appropriate alignment.

One such program is Gap which considers all possible alignment and gap positions and creates an alignment with the largest number of matched bases and the fewest gaps. Gap uses the alignment method of Needleman and Wunsch Gap reads a scoring matrix that contains values for every possible GCG symbol match. GAP is available on ANGIS (Australian National Genomic Information Service) at website http://mel .angis.org.au..

The present invention further comprises a nucleic acid molecule comprising the nucleotide sequence: nX l b a -n'y}c a nd wherein WO 00/20578 PCT/AU99/00860 -19nj is a sequence of x nucleotides encoding an extracellular portion of a receptor or ligand-binding molecule; n'y} is a sequence of y nucleotides encoding a transmembrane peptide, polypeptide or protein or a molecule capable of inducing multimerisation; n" is a sequence of z nucleotides comprising a nucleotide sequence substantially as set forth in <400>7 or a nucleotide sequence encoding an amino acid sequence substantially as set forth in <400>8 or a nucleotide sequence capable of hybridising to <400>7 or a complementary form thereof under low stringency conditions such as at 42 oC or a nucleotide sequence having at least 60% identity to <400>7; b, c and d may be the same or difference and each is 0, 1 or >1; x, y and z may be the same or different and each is 0, 1 or >1; a is a nucleotide bond; wherein when c is 1 or >1 and d is 1 or >1 and wherein when the molecule is expressed in a neural cell, the expression product signals, induces or otherwise facilitates cell death.

Preferably, nx} comprises the nucleotide sequence substantially as set forth in <400>3 or is a nucleotide sequence having at least about 60% identity thereto or is capable of hybridising to <400>3 or its complementary form under low stringency conditions such as at 42 °C.

Preferably, n'y} comprises the nucleotide sequence substantially as set forth in <400>5 or is a nucleotide sequence having at least about 60% identity thereto or WO 00/20578 PCT/AU99/00860 is capable of hybridising to <400>5 or its complementary form under low stringency conditions such as at 42 oC.

The nucleotide sequences nj, n'y} and n"z may be in any order and in any combination.

For the production of a recombinant peptide, polypeptide or protein comprising the death signal, the nucleic acid molecule of the present invention is placed, in the sense orientation, in operable connection with a suitable promoter sequence and introduced into a suitable expression system, for example a bacterial, yeast, baculovirus, plant, animal or other expression system.

Accordingly, a further aspect of the present invention provides a genetic construct comprising an isolated nucleic acid molecule which comprises a sequence of nucleotides which corresponds or is complementary to a death signal region from p 7 5 NTR or a homologue, analogue or derivative thereof.

According to this embodiment, the coding region of the death signal from p 7 5

NTR

may be placed in operable connection with a promoter sequence such that a gene product is capable of being expressed under the control of said promoter sequence.

Optionally, said genetic construct further comprises a terminator sequence.

In the present context, the term "in operable connection with" is used to indicate that expression of the isolated nucleotide sequence is under the control of the promoter sequence with which it is connected.

The term "terminator" refers to a DNA sequence at the end of a transcriptional unit which signals termination of transcription. Terminators are 3'-non-translated DNA sequences containing a polyadenylation signal, which facilitates the addition of polyadenylate sequences to the 3'-end of a primary transcript. Terminators active WO 00/20578 PCT/AU99/00860 -21in plant cells are known and described in the literature. They may be isolated from bacteria, fungi, viruses, animals and/or plants.

Examples of terminators particularly suitable for use in the genetic constructs of the present invention include the SV40 polyadenylation signal, amongst others.

Reference herein to a "promoter" is to be taken in its broadest context and includes the transcriptional regulatory sequences of a classical genomic gene, including the TATA box which is required for accurate transcription initiation in eukaryotic cells, with or without a CCAAT box sequence and additional regulatory elements (i.e.

upstream activating sequences, enhancers and silencers). For expression in prokaryotic cells, such as bacteria, the promoter should at least contain the -35 box and -10 box sequences.

A promoter is usually, but not necessarily, positioned upstream or of the nucleotide sequence encoding the death signal of p 75 NTR, the expression of which it regulates. Furthermore, the regulatory elements comprising a promoter are usually positioned within 2 kb of the start site of transcription of the gene.

In the present context, the term "promoter" is also used to describe a synthetic or fusion molecule, or derivative which confers, activates or enhances expression of an isolated nucleic acid molecule, in a cell, such as a plant, animal, insect, fungal, yeast or bacterial cell. Preferred promoters may contain additional copies of one or more specific regulatory elements, to further enhance expression of a nucleic acid molecule which expression it regulates and/or to alter the spatial expression and/or temporal expression of same. For example, regulatory elements which confer copper inducibility may be placed adjacent to a heterologous promoter sequence driving expression of a nucleic acid molecule, thereby conferring copper inducibility on the expression of said molecule.

WO 00/20578 PCT/AU99/00860 -22- Placing an isolated nucleic acid molecule under the regulatory control of a promoter sequence means positioning said molecule such that expression is controlled by the promoter sequence. Promoters are generally positioned 5' (upstream) to the genes that they control. In the construction of heterologous promoter/structural gene combinations it is generally preferred to position the promoter at a distance from the gene transcription start site that is approximately the same as the distance between that promoter and the gene it controls in its natural setting, the gene from which the promoter is derived. As is known in the art, some variation in this distance can be accommodated without loss of promoter function. Similarly, the preferred positioning of a regulatory sequence element with respect to a heterologous gene to be placed under its control is defined by the positioning of the element in its natural setting, the genes from which it is derived. Again, as is known in the art, some variation in this distance can also occur.

Examples of promoters suitable for use in genetic constructs of the present invention include viral, fungal, bacterial, animal and plant derived promoters capable of functioning in plant, animal, insect, fungal, yeast or bacterial cells. The promoter may regulate the expression of the nucleic acid molecule constitutively, or differentially with respect to the tissue in which expression occurs or, with respect to the developmental stage at which expression occurs, or in response to external stimuli such as physiological stresses, or plant pathogens, or metal ions, amongst others.

Preferably, the promoter is capable of regulating expression of a nucleic acid molecule in a yeast or bacterial cell.

Examples of preferred promoters include the bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac promoter, tac promoter, SV40 early promoter, and the like.

The genetic construct contemplated herein is introduced into a suitable expression WO 00/20578 PCT/AU99/00860 -23system for a time and under conditions sufficient for expression of said death signal or inhibitor portion from p 7 5 NTR to occur.

The genetic construct may also comprise a nucleotide sequence corresponding to all or part of the membrane domain of p 7 5 or other membrane molecules.

Accordingly, a further aspect of the invention contemplates a recombinant peptide, polypeptide or protein produced by expressing the isolated nucleic acid molecule herein described in a suitable host cell. The present invention extends also to a synthetic peptide fragment of said recombinant gene product.

The present invention further contemplates an isolated peptide, polypeptide or protein comprising the cytoplasmic region of p 7 5 NTR which signals, induces or otherwise facilitates cell death when said peptide, polypeptide or protein is adjacent, proximal or otherwise juxtaposed to a membrane-associating region such as from p 7 5 NR or other membrane molecule and/or is in multimeric form or a derivative, homologue, chemical equivalent or analogue of said peptide, polypeptide or protein. This aspect of the present invention does not extend to the full length p75

R

Suitable molecules according to this aspect of the present invention include a peptide, polypeptide or protein corresponding to a soluble form of the death signalling region of p 7 5 NTR or a molecule capable of antagonising that region or a component of the death signalling pathway. An example of a possible component of the death signalling pathway is Bcl-2.

The peptide, polypeptide or protein of this aspect of the present invention is useful inter alia as a therapeutic molecule to antagonise p75 -mediated death signalling. For example, the peptide, polypeptide or protein may themselves be administered to directly antagonise p75NTR-mediated death signalling or the peptide, polypeptide or protein may need to be chemically modified to facilitate penetration WO 00/20578 PCT/AU99/00860 -24into the cell. One such chemical modification is fusion to or co-expression with penetratin or the TAT protein from HIV. Alternatively, the death signalling region of p 7 5 NTR may be used to screen for antagonists of this region. Such antagonists may, for example, be identified following natural product screening or the screening of chemical libraries. For natural product screening suitable environments include, but are not limited to, plants, bacteria and other microorganisms, river and sea beds, coral and arctic or antarctic regions. The present invention also contemplates antagonists directed to other components of the death signalling pathway. Such components to be targeted include but are not limited to Bcl-2 or related or homologous molecules. Preferably, for peptides, polypeptides and proteins designed to induce cell death, the molecules are palmitoylated.

Preferably, the peptide, polypeptide or protein comprises an amino acid sequence substantially as set forth in <400>8 or an amino acid sequence having at least identity thereto or a chemical equivalent, derivative, homologue or analogue of said peptide, polypeptide or protein.

The term "isolated" means that the peptide, polypeptide or protein of the present invention is provided in a form which is distinct from that which occurs in nature, preferably wherein one or more contaminants have been removed. Accordingly, the isolated peptide, polypeptide or protein of the invention may be partially-purified or substantially pure, in which a substantial amount of the contaminants have been removed or in sequencably pure or substantially homogeneous form.

The term "sequencably pure" means that the isolated peptide, polypeptide or protein is provided in a form which is sufficiently purified to facilitate amino acid sequence determination using procedures known to those skilled in the art.

The term "substantially homogeneous" means that the isolated peptide, polypeptide or protein of the present invention is at least about 95% free of WO 00/20578 PCT/AU99/00860 contaminants, more preferably at least about 99% free of contaminants, including 100% purity.

The present invention extends to a range of derivatives and chemical analogues of the peptide, polypeptide or protein.

Furthermore, the amino acids of a homologous polypeptide may be replaced by other amino acids having similar properties, for example hydrophobicity, hydrophilicity, hydrophobic moment, charge or antigenicity, and so on.

"Analogues" encompass death signal containing peptides, polypeptides or proteins which are at least about 60% identical to the p75Nm death signal sequence notwithstanding the occurrence of any non-naturally occurring amino acid analogues therein. "Analogues" also encompass polypeptide mimotypes.

The term "derivative" in relation to a peptide, polypeptide or protein shall be taken to refer hereinafter to mutants, parts or fragments derived from the functional p 7 5 "R molecule or death signal region thereof or derivatives thereof which may or may not possess the death signal activity of the functional p 7 5 NT". Derivatives include modified peptides in which ligands are attached to one or more of the amino acid residues contained therein, such as carbohydrates, enzymes, proteins, polypeptides or reporter molecules such as radionuclides or fluorescent compounds. Glycosylated, fluorescent, acylated or alkylated forms of the subject peptides are particularly contemplated by the present invention. Additionally, derivatives of the peptide, polypeptide or protein described herein comprise fragments or parts of an amino acid sequence disclosed herein are within the scope of the invention, as are homopolymers or heteropolymers comprising two or more copies of the subject polypeptides. Procedures for derivatizing peptides are wellknown in the art.

A homologue, analogue or derivative of <400>2 or <400>8 may comprise an amino

-I

WO 00/20578 PCT/AU99/00860 -26acid substitution or said <400> 2 or 8 may encompass amino acid alterations in which an amino acid is replaced with a different naturally-occurring or a nonconventional amino acid residue. Such substitutions may be classified as "conservative", in which case an amino acid residue contained in a phospholipase inhibitory protein is replaced with another naturally-occurring amino acid of similar character, for example Gly*-Ala, Val*-lle*-+Leu, Asp*-Glu, Lys*-Arg, Asn+-GIn or Phe-Trp++Tyr.

Substitutions encompassed by the present invention may also be "nonconservative", in which an amino acid residue which is present in a phospholipase inhibitory protein is substituted with an amino acid having different properties, such as a naturally-occurring amino acid from a different group (eg. substituted a charged or hydrophobic amino acid with alanine), or alternatively, in which a naturally-occurring amino acid is substituted with a non-conventional amino acid.

Amino acid substitutions are typically of single residues, but may be of multiple residues, either clustered or dispersed.

Naturally-occurring amino acids include those listed in Table 1. Non-conventional amino acids encompassed by the invention include, but are not limited to those listed in Table 2.

Amino acid deletions will usually be of the order of about 1-10 amino acid residues, while insertions may be of any length. Deletions and insertions may be made to the N-terminus, the C-terminus or be internal deletions or insertions. Generally, insertions within the amino acid sequence will be smaller than amino-or carboxylterminal fusions and of the order of 1-4 amino acid residues.

WO 00/20578 WO 0020578PCT/AU99/00860 27 TABLE 1 Amino Acid Three-letter One-letter Abbreviation Symbol Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys C Glutamine Gin Q Glutamic acid Glu E Glycine Gly G Histidine His H Isoleucine lieI Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Praline Pro P Senine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V Any amino acid as above Xaa X WO 00/20578 WO 0020578PCT/AU99/00860 28 TABLE 2 Non-conventional Code Non-conventional Code amino acid amino acid a-aminobutyric acid a-amino-a-methylbutyrate amninocyclopropanecarboxylate amninoisobutyric acid aminonorbomylcarboxylate cyclohexylala nine cyclopentylalanine D-alanine D-arginine D-aspartic acid D-cysteine D-g lutamine D-glutamic acid D-histidine D-isoleucine D-Ieucine D-lysine D-methionine D-ornithine D-phenylalanine D-proline D-serine D-threonine D-tryptophan Abu Mgabu Cpro Aib Norb Chexa Cpen Dal Darg Dasp Dcys Dgln Dglu Ohis Dile Dieu Dlys Dmet Dom Dphe Dpro Dser Dthr Dtrp L- N-methylala nine L-N-methylarginine L-N-methylasparagine L-N-methylaspartic acid L-N-methylcysteine L-N-methylglutamine L-N-methylglutamic acid L-N-methylhistidine L-N-methyl isolleucine L-N-methylleucine L-N-methyllysine L-N-methyl methionine L-N-methylnorleucine L-N-methylnorvaline L-N-methylornithine L-N-methyl phenyl ala nine L-N-methylproline L-N-methylserine L-N-methylthreonine L-N-methyltryptophan L-N-methyltyrosine L-N-methylvaline L-N-methylethylglycine L-N-methyl-t-butylgtycine L-norleucine L-norvaline Nmala Nmarg Nmasn Nmasp Nmcys Nmgln Nmglu Nmnhis Nmile Nmleu Nmlys Nmmet Nmnle Nmnva Nmorn Nmphe Nmpro Nmser Nmthr Nmtrp Nmtyr Nmval Nmetg Nmtbug NMe Nva WO 00/20578 WO 0020578PCT/AU99/00860 -29 D-tyrosine D-valine D-a-methylalanine D-a-methylarginine D-a-methylasparagine D-a-methylaspartate D-a-methylcysteine D-a-methylglutamine D-a-methylhistidine D-a-methylisoleucine D-a-methylleucine D-a-methyllysine D-a-methylmethionine D-a-methylornithine D-a-methylphenylalanine D-a-methylproline D-a-methylserine D-ct-methylthreonime D-ct-methyltryptophan D-a-methyltyrosine D-a-methylvaline D-N-methylalanine D-N-methylarginine D-N-methylasparagine D-N-methylaspartate D-N-methylcysteine Dtyr a-methyl-aminoisobutyrate DvaI cx-methyl-y-aminobutyrate Dmala cx-methyl cyclohexylalIan ine Dmarg a-methylcylcopentylalanine Dmasn a-methyl-a-napthylalanine Dmasp cx-methylpenicillamine Dmcys N-(4-aminobutyl)glycine Dmgln N-(2-aminoethyl)glycine Dmnhis N-(3-aminopropyl)glycine Dmile N-amino-cx-methylbutyrate Dmleu a-napthylalanine Dmnlys N-benzylglycine Omnmet N-(2-carbamylethyl)glycine Dmom N-(carbamylmethyl)glycine Dmnphe N-(2-carboxyethyl)glycine Dmnpro N-(carboxymethyl)glycine Dmnser N-cyclobutylglycine Dmthr N-cycloheptylglycine Dmtrp N-cyctohexylglycine Dmnty N-cyclodecylglycine Dmval N-cylcododecylglycine Dnmala N-cyclooctylglycine Dnmarg N-cyclopropylglycine Dnmasn N-cycloundecylglycine Dnmasp N-(2,2-diphenylethyl) glycine Dnmcys N-(3,3-diphenylpropyl) glycine Maib Mgabu Mchexa Mcpen Manap M pen Nglu Naeg Norn Nmaabu Anap Nphe NgIn Nasn Nglu Nasp Ncbut Nchep Nchex Ncdec Ncdod Ncoct Ncpro Nound Nbhm Nbhe WO 00/20578 WO 0020578PCT/AU99/00860 D-N-methylglutamine D-N-methylg luta mate D-N-methylhistidine D-N-methylisoleucine D-N-methylleucine D-N-methyllysine N-methylcyclohexylalanine D-N-methylornithine N-methylglycine, N-methylaminoisobutyrate N-(1 -methylpropyl)glycine N-(2-methylpropyl)glycine D-N-methyltryptophan D-N-methyltyrosine D-N-methylvaline y-aminobutyric acid L-t-butylglycine L-ethylglycine L-homnophenylalanine L-a-methylarginine L-a-methylaspartate L-a-methylcysteine L-a-methylglutamine L-a-methylhistidine L-a-methylisoleucine Dnmgln N-(3-guanidinopropyl) glycine Dn mglu N-(1I -hyd roxyethyl)glycine Dnmhis N-(hydroxyethyl))glycine Dnmile N-(imidazolylethyl)) glycine Dnmleu N-(3-indolylyethyl) glycine Dn mlys N-methyl-y-aminobutyrate Nmchexa D-N-methylmethionine Dnmorn N-methylcyclopentylalanine Nala D-N-methylphenylalanine Nmaib D-N-methylproline Nile D-N-methylserine Nleu D-N-methylthreonine Dnmtrp N-(1 -methylethyl)glycine Dnmtyr N-methyla-napthylalanine Dnmval N-methylpenicillamnine Gabu N-(p-hydroxyphenyl)glycine Tbug N-(thiomethyl)glycine Etg penicillamnine Hphe L-a-methylalanine Marg L-a-methylasparagine Masp L-a-methyl-t-butylglycine Mcys L-methylethylglycine Mgln L-a-methylglutamate Mhis L-a-methylhomo phenylalanine Mile N-(2-methylthioethyl) glycine Mleu L-a-methyllysine Narg Nthr Nser Nhis Nhtrp Nmgabu Dn mmet Nmcpen Dnmphe Dn mpro Dnmser Dnmthr NvaI Nmanap Nmpen N htyr Ncys Pen Mala Masn Mtbug Metg Mglu Mhphe Nmet Mlys L-c-methylleucine WO 00/20578 PCT/AU99/00860 -31 L-a-methylmethionine L-a-methylnorvaline L-a-methylphenylalanine L-a-methylserine L-a-methyltryptophan L-a-methylvaline N-(N-(2,2-diphenylethyl) carbamylmethyl)glycine 1-carboxy-1-(2,2-diphenylethylamino)cyclopropane Mmet L-a-methylnorleucine Mnva L-a-methylornithine Mphe L-a-methylproline Mser L-a-methylthreonine Mtrp L-a-methyltyrosine Mval L-N-methylhomo phenylalanine N-(N-(3,3-diphenylpropyl) Nnbhm carbamylmethyl)glycine Mnle Morn Mpro Mthr Mtyr Nmhphe Nnbhe Nmbc The present invention provides therefore, peptides, polypeptides and proteins which inhibit p 7 5 NTR death signalling and/or cleavage of extracellular domain of p 7 5

TR.

Accordingly, another aspect of the present invention contemplates a method for inhibiting, reducing or otherwise antagonising p75NTR-mediated death signal in a neural cell, said method comprising administering a peptide, polypeptide or protein or analogues or mimetics thereof which correspond to a non-membrane associated form of the p75 N T R death signal region or a derivative, functional equivalent or homologue thereof.

Yet another aspect of the present invention is directed to peptide antagonists of the p 7 5 NTR death signal or functional analogues or mimetics thereof.

The present invention provides for a method of treatment or prophylaxis of disease conditions associated with neural death or where cell death is to be promoted such as in treating or preventing cancer growth and/or development.

WO 00/20578 PCT/AU99/00860 -32- In one embodiment, it has been determined in accordance with the present invention that expression of a nucleic acid molecule encoding only death signal and not adjacent, proximal or juxtaposed to a membrane-associating sequence results in antagonising of the death signal.

According to this embodiment, the present invention contemplates a method for inhibiting, reducing or otherwise antagonising a p75NTR-mediated death signal in a neural cell, said method comprising introducing a nucleic acid molecule capable of being expressed to an expression product which corresponds to a non-membrane associated form of the p 7 5 NTR death signal region or a derivative, functional equivalent or homologue thereof.

In a related embodiment there is provided a method for inhibiting, reducing or otherwise antagonising a p75NTR-mediated death signal in a neural cell, said method comprising contacting a cell carrying a p 75 NTR with a death signal-inhibiting effective amount of a molecule capable of antagonising the death signal of p 75

NTR

or a component of the death signalling pathway.

This aspect of the present invention is useful for the treatment of a range of neurodegenerative diseases such as cerebral palsy, trauma induced paralysis, vascular ischaemia associated with stroke, neural tumours, motoneurone disease, Parkinson's disease, Huntington's disease, Alzheimer's disease, multiple sclerosis and peripheral neuropathies associated with diabetes, heavy metal or alcohol toxicity, renal failure and/or infectious diseases such as herpes, rubella, measles, chicken pox, HIV and/or HTLV-1. This aspect is also useful for treating neurons or glia damaged by trauma or disease.

Alternatively, the method is aimed at targeting certain cells such as cancer cells wherein expression is required of a death signal from p 7 5 NTR or a derivative, functional equivalent or homologue thereof adjacent, proximal or otherwise juxtaposed to a membrane-associating portion of p 75 NTR or other membrane WO 00/20578 PCT/AU99/00860 -33molecules or is in multimeric form. The nucleic acid molecule may require modification to ensure appropriate targeting to the cell or the nucleic acid molecule may be injected directly into cancerous tissue.

Another aspect of the present invention provides a biological composition comprising a genetic molecule capable of being expressed into a p 75 NTR death signal antagonist or a p 75 NTR death signal. The biological composition further comprises one or more pharmaceutically acceptable carriers and/or diluents. The nucleic acid molecules according to this aspect of the present invention may be naked nucleic acid molecules or contained or associated with a viral vector or other suitable delivery mechanism.

Another aspect of the present invention is directed to a biological composition comprising a molecule capable of antagonising p75NTR-mediated death signalling of a cell.

Suitable molecules according to this aspect of the present invention are as contemplated above and include a peptide, polypeptide or protein comprising a soluble form of the p 75 NTR death signalling region or an antagonist of a component of the p 75 NTR death signalling pathway.

The present invention is also useful as a culture agent such as preventing or reducing the death of cells in vitro. The present invention is particularly useful in vitro when used in combination with LIP. Even more particularly, the present invention is useful for culturing recombinant cell lines.

The present invention also provides for the use of the death signal of p 75 NTR in the manufacture of a medicament for the treatment of neurodegenerative diseases in animals. Preferred animals include humans, primates, livestock animals, laboratory test animals, companion animals and captive wild animals.

WO 00/20578 PCT/AU99/00860 34 The present invention is further described by the following non-limiting Examples.

WO 00/20578 PCT/AU99/00860 EXAMPLE 1 The aim of this example was to determine the protein domains on p75 N T R responsible for death signalling.

In order to investigate how p 7 5 NTR signals neural death, the inventors devised a robust in vitro assay for p75 T R induced death. Plasmid expression constructs were microinjected into individual neurons in the presence of the growth factor LIF, and the survival of the neurons expressing the different plasmids was determined. A series of plasmid constructs which encode incomplete p 7 5 NTR proteins were made (see Figure 1) and the ability of each protein to signal death when over expressed was assessed.

The p75 NT protein is a transmembrane protein comprised of a large extracellular domain with four cysteine rich motifs responsible for interacting with soluble growth factors, and a short cytoplasmic, intracellular tail. The cytoplasmic domain does not contain a kinase domain but contains a domain with significant homology to a motif known as a "death domain" [<400>9, <400>10], found in apoptosis-inducing Tumour Necrosis Factor Receptors (TNFR) and TNFR-associating death-effector proteins Using expression plasmids of p 7 5 NTR proteins deleted for either the entire cytoplasmic domain (p75nc) or a significant portion of the cytoplasmic domain including the entire death domain (p75tm), the inventors found that the cytoplasmic domain is responsible for death signalling. Surprisingly, the intracellular 35 amino acid domain juxtaposed to the membrane, and not the death domain, is responsible for death signalling (Figure This region of the p 7 5 NTR protein shows no homology to other death inducing proteins or to known functional motifs.

To further investigate the domain required for death signalling the inventors made WO 00/20578 PCT/AU99/00860 -36constructs expressing p 7 5 NTR proteins deleted for the extracellular domain or the extracellular and transmembrane domains. Proteins without extracellular domains retain the signal peptide which is responsible for correctly transporting the protein into the cell membrane. Proteins without transmembrane domains are expressed free in the cytoplasm of the cell and are epitope tagged with a FLAG motif for detection.

The inventors found that the extracellular domain of p 7 5 NTR had a significant inhibitory effect of the ability of the cytoplasmic domain to signal cell death.

Furthermore, the membrane linked 35 amino acid cytoplasmic domain (c35) was a potent stimulant of neural death with over 90% of cells injected with the plasmid dead after 16 hours (Figure However, if the cytoplasmic 35 amino acid domain is not associated with the membrane, the ability of the domain to induce death is removed (Figure 4).

These results indicate that the domain responsible for death induction is within the first 35 amino acids of the cytoplasmic tail but that the transmembrane domain, or at least association with the membrane, is required for death-signal activation. This may be related to the ability of the transmembrane protein to more efficiently form death-signal inducing multimers, or that the position of the p75 NTR protein in relation to other membrane-bound accessory molecules might be important in initiating death signalling.

The inventors hypothesised that the free cytoplasmic expressed 35 amino acid domain might be able to interfere with death signalling from full length p 7 5 N TR proteins by a dominant-negative mechanism, and attempted to inhibit the death by co-expressing the proteins. Given the results presented below regarding the ability of overexpression of Bcl-2 to enhance p 7 5 N TR killing, this paradigm was used to test the ability of the c35 protein to inhibit death signalling. The inventors found that indeed the expression of the c35 protein was able to inhibit this killing (Figure This further indicates that p 7 5 N TR signals killing via interaction of an accessory WO 00/20578 WO 0020578PCT/AU99/00860 -37molecule to a motif within the first 35 amino acids of the cytoplasmic domain.

EXAMPLE 2 The aim of this example is to determine the minimum number of amino acid residues on c35 require to mediate death signalling.

A series of deletion and truncation mutants in the c35 region are produced and tested for the ability to induce death signalling.

The deletion mutants from the membrane distal end are as follows:

KRWNSCKQNKQGANSRPVNQTPPPEGEKLHSDSG;

KRWNSCKQNKQGANSRPVNQTPPPEGEKLHSDS;

KRWNSCKQNKQGANSRPVNQTPPPEGEKLHSD;

KRWNSCKQNKQGANSRPVNQTPPPEGEKLHS;

KRWNSCKQNKQGANSRPVNQTPPPEGEKLH;

KRWNSCKQNKQGANSRPVNQTPPPEGEKL;

KRWNSCKQNKQGANSRPVNQTPPPEGEK;

KRWNSCKQNKQGANSRPVNQTPPPEGE;

KRWNSCKQNKQGANSRPVNQTPPPEG;

KRWNSCKQNKQGANSRPVNQTPPPE;

KRWNSCKQNKQGANSRPVNQTPPP;

KRWNSCKQNKQGANSRPVNQTPP;

KRWNSCKQNKQGANSRPVNQTP;

KRWNSCKQNKQGANSRPVNQT;

KRWNSCKQNKQGANSRPVNQ;

KRWNSCKQNKQGANSRPVN;

KRWNSCKQNKQGANSRPV;

KRWNSCKQNKQGANSRP;

KRWNSCKQNKQGANSR;

KRWNSCKQNKQGANS;

WO 00/20578 WO 0020578PCT/AU99/00860 38

KRWNSCKQNKQGAN;

KRWNSCKQNKQGA;

KRWNSCKQNKQG;

KRWNSCKQNKQ;

KRWNSCKQNK;

KRWNSCKQN;

KRWNSCKQ;

KRWNSCK;

KRWNSC;

KRWNS;

KRWN;

KRW;

KR; and

K.

The deletion mutants from the membrane proximal end are as follows:

RWNSCKQNKQGANSRPVNQTPPPEGEKLHSDSGI;

WNSCKQNKQGANSRPVNQTPPPEGEKLHSDSGI;

NSCKQNKQGANSRPVNQTPPPEGEKLHSDSGI;

SCKQNKQGANSRPVNQTPPPEGEKLHSDSGI;

CKQNKQGANSRPVNQTPPPEGEKLHSDSGI;

KQNKQGANSRPVNQTPPPEGEKLHSDSGI;

QNKQGANSRPVNQTPPPEGEKLHSDSGI;

NKQGANSRPVNQTPPPEGEKLHSDSGI;

KQGANSRPVNQTPPPEGEKLHSDSGI;

QGANSRPVNQTPPPEGEKLHSDSGI;

GANSRPVNQTPPPEGEKLHSDSGI;

ANSRPVNQTPPPEGEKLHSDSGI;

NSRPVNQTPPPEGEKLHSDSGI;

SRPVNQTPPPEGEKLHSDSGI;

WO 00/20578 WO 0020578PCT/AU99/00860 39

RPVNQTPPPEGEKLHSDSGI;

PVNQTPPPEGEKLHSDSGI;

VNQTPPPEGEKLHSDSGI;

NQTPPPEGEKLHSDSGI;

QTPPPEGEKLHSDSGI;

TPPPEGEKLHSDSGI;

PPPEGEKLHSDSGI;

PPEGEKLHSDSGI;

PEGEKLHSDSGI;

EGEKLHSDSGI;

GEKLHSDSGI;

EKLHSDSGI;

KLHSDSGI;

LHSDSGI;

HSDSGI;

SDSGI;

DSGI;

SGI;

GI; and 1.

WO 00/20578 PCT/AU99/00860 EXAMPLE 3 ROLE OF BCL-2 IN PROMOTING p75NT R MEDIATED DEATH SIGNALLING As the inventors had shown that the death of dorsal root ganglia (DRG) sensory neurons in vitro and in vivo, was, at least in part, mediated by p 7 5 NTR, p 7 5 NTR was over-expressed in these cells by microinjecting rat p 7 5 NTR cDNA expressing plasmid into the nucleus of mouse sensory neurons. These were cultured in the presence of the LIF to prevent neural death not linked to p 7 5 NTR mechanisms. It was found that the expression of the rat p 7 5 NTR could be detected by surface immunofluorescence within 24 hours of injection. The injected neurons were observed over a 48 hour period and the viability was assessed. It was found that within the first 16 hours, a significantly higher number of p 7 5

N

R plasmid injected neurons had died compared to neurons injected with control plasmids 3galactosidase, or a truncated p 7 5 NTR protein lacking the entire cytoplasmic domain (p75NTRnc). It was found that p 7 5 NTR-mediated neural death occurred later in the experiment similar to Fas/TNF-induced rapid cell death. Since both full-length p 7 5 NTR and p75NRnc protein showed a similar level of expression after injection, this indicates that the cytoplasmic domain of p 7 5 NTR is required for death signalling.

This was expected since the cytoplasmic tail contains a sequence with homology to the Fas/TNFR "death domain" The inventors next examined whether deletion of the "death domain" also abolished the ability of p 7 5 N TR to kill. It was found that the neural death observed after expression of p 7 5 NTR with a truncated cytoplasmic tail (p75NRtr) was equivalent to the full-length p 7 5 NTR protein. This demonstrated that the "death domain" was not required for p 7 5 NTR killing and, since the p 7 5 NTR death domain has recently been shown to have a different tertiary structure to TNFR family death domain and does not self-associate in vitro, it suggests that the p 7 5 NTR "death domain" may not normally function to induce death. Together, these results predict that an alternative pathway involving proteins other than "death domain" adapter proteins, such as TRADD and FADD, is responsible for p75NTR-mediated killing.

WO 00/20578 PCT/AU99/00860 -41- The Bcl-2 family of proteins is involved in mediating apoptotic signalling pathways, and can homodimerise or heterodimerise with other family members. Bcl-2 and Bcl-xL are well characterised inhibitors of stress-induced apoptosis, JNK activation and neural death due to growth-factor limitation. However, both are poor inhibitors of Fas and TNFR mediated opoptosis. As it had been shown previously that high levels of Bcl-2 or Bcl-xL blocked neural cell death in a variety of models, the inventors examined whether over-expression of these proteins could block the death induced by p75T

R

The inventors found that over-expression of Bcl-xL protected neurons against death, supporting the hypothesis that p 7 5 NTR signals through an alternative pathway to TNFR-induced apoptosis. In contrast, while Bcl-2 overexpression alone had no effect on cell survival in the presence of LIF, Bcl-2 in combination with p75 R over-expression, surprisingly, induced a significant increase in neural death above that seen with p 7 5 NTR over-expression alone. Bcl-2 in combination with p75 Rnc did not cause significant cell death and furthermore, the cell death observed with p75 R and Bcl-2 over-expression was totally ablated if the cells were cultured in NGF. Bcl-2 was able to protect against neural death induced by NGF withdrawal, but not withdrawal of LIF. Thus, at the same expression levels in the same neural population, Bcl-2 was able to prevent or enhance neural cell death depending on the nature of the death signal.

These results are surprising since Bcl-2 has previously been shown to have similar actions to Bcl-xL in almost all cell-death systems.

To determine whether the paradoxical effect of Bcl-2 on p75NTR-induced killing was related to its known anti-apoptotic activity, Bcl-2 proteins with inactivating point mutation, G145E, in the "Bcl-2 Homology" BH1 domain and W188A in the BH2 domain were utilised. Like wildtype Bcl-2, expression of either Bcl-2 mutant alone did not effect neural survival. In combination with p75 N T R expression, the enhanced killing effect seen with Bcl-2 co-expression was abrogated by the G145E mutation, WO 00/20578 PCT/AU99/00860 -42even though the proteins were expressed to comparable levels. Thus, an intact BH1 homology region is required for the death promoting activity of Bcl-2.

Mutation of the equivalent G138 residue in Bcl-xL results in a conformational change between a-helices 4 and 5, disrupting access to the hydrophobic cleft formed by BH1, BH2 and BH3 domains. Therefore, the molecular mechanism by which Bcl-2 participates in the p75NTR killing pathway may be dependent on interactions either directly with the BH domains or with the hydrophobic cleft, as indicated with experiments using the W188A mutation. Co-expression of p75

TR

with the Bcl-2 W188A protein not only abrogated the increased p 75 NTR killing but, more importantly, protected neurons from any p75NTR-induced death, reminiscent of that seen with Bcl-xL. These experiments suggest that the conformation of the Bcl- 2 protein is integral to the opposing functions observed herein.

The inventors had observed that DRG neurons isolated from newborn mice depleted for p 7 5 NTR were less susceptible to NGF withdrawal, as is the case with sympathetic neurons, when compared to neurons from wildtype mice. This is indicative of absent or delayed naturally occurring cell death observed in these mice. The inventors attempted to induce cell death in p 7 5 NTR "knock out" DRG neurons by re-introducing p 7 5 NTR expression. Surprisingly, apoptosis was not induced by re-expression into "knock out" DRG neurons, the inventors found that neural death was significantly increased under these conditions. This implicated an absolute requirement for Bcl-2 in mediating p75NTR killing.

The inventors tested, therefore, whether high endogenous Bcl-2 levels might be necessary for successful p75NTR-mediated killing in normal neurons by assaying p 7 5 NTR killing in Bcl-2 depleted cells. Endogenous Bcl-2 was down regulated by antisense as previously described. When the Bcl-2 antisense plasmid was injected at the same time as p 7 5 NTR plasmids no diminishment in the death signal was seen.

If, however, the Bcl-2 antisense was microinjected first (to give time to reduced Bcl- 2 production and deplete endogenous Bcl-2; and then a day later the p 7 5 NTR or WO 00/20578 PCT/AU99/00860 -43p 7 5NTRnc constructs were microinjected, there was no difference in survival between p 7 5 NTR and p75NTRnc expressing neurons, strongly suggesting that endogenous Bcl-2 is required for p 7 5 NTR killing effects. To confirm this observation, the inventors isolated neurons from newborn Bcl-2 "knock out" mice (an heterozygous line of mice containing a disrupted Bcl-2 gene) and their wild-type litter mates and compared the effect of p 7 5 NT over-expression with control plasmid p 7 5

NT

R

It was found that the neurons isolated from Bcl-2 deficient mice were significantly protected from p 7 5 NTR killing, showing a 56.9% reduction in death compared wildtype neurons, supporting the hypothesis that endogenous Bcl-2 is required for p7 5 NTR killing.

Bcl-2 has previously been observed to increase cell death when highly expressed both in vitro and in vivo when expressed at high levels as a transgene, causing increased apoptosis in the brain under a neuron specific promoter, or in photoreceptor cells when expressed specifically under a rhodopsin promoter.

Thus, it is possible that the high level of Bcl-2 is able to "prime" the death pathway such that an apoptotic stimulus via p 7 5 N TR results in rapid cell death.

Bcl-2 and Bcl-xL when cleaved by caspases have also been shown to be capable of promoting apoptosis in vitro, with cells expressing non-cleavable mutant Bcl-2 and Bcl-xL proteins showing increased viability compared to cells expressing wildtype proteins. Cleavage of Bcl-2 is possible in this system, however, the Bcl-2 mutations which results in loss of death promoting activity, would not prevent cleavage of Bcl-2, indicating that cleavage of Bcl-2 would only be part of the mechanism by which Bcl-2 promotes killing. In addition, if cleavage was the dominant mechanism, Bcl-xL might be expected to act as a death signalling protein in this system.

To investigate whether the p75NTR-Bcl-2 death-signalling cascade was dependent on caspase activation, inhibitors of caspases were employed. In the presence of z- VAD, a nonspecific caspase peptide inhibitor, or after co-expression of modified WO 00/20578 PCT/AU99/00860 -44crmA plasmids, designed to inhibit Group II caspases such as caspases 2 and 3, death was significantly reduced. Similarly, the modified crmA was able to block the killing induced by co-expression of p 7 5 NTR and Bcl-2. This indicates that p 7 5 NTR induced apoptosis is a caspase dependent pathway and that the mechanism by which Bcl-2 assists killing is through the same pathway.

EXAMPLE 4 ANTAGONSIM OF p 7 5 NTR MEDIATED DEATH SIGNALLING Figure 6 shows that soluble c35 (35mer) [<400>7 and <400>8] protects cells from death signalling in a dose-dependent manner against membrane bound Furthermore, the 35mer when expressed from a genetic construct, protected Schwann cells against NGF-induced death. c35 when expressed in soluble form can also protect cells against membrane bound c35. The inventors show in Figure 7 that soluble c35 can also protect against membrane-linked, expressed c35. A truncated form of c35, a 29mer [<400>11 and <400>12], also protected against membrane-bound c35, when in soluble form.

Figure 8 shows that the 29mer with a palmitoyl group at the membrane (amino) end resulted in cell death. The palmitoylation links the peptide to the plasma membrane. This membrane-linked 29mer leads to cell death whereas its soluble form protects cells against p75NTR-mediated death signalling.

EXAMPLE EFFECTS OF PALMITOYLATION Figure 9 shows the effects of palmitoylated 29 mer (fused to penetratin) on mediating cell death. Cells were washed with peptide (2 AM) for approximately 110 minutes and the cells were then washed. Controls included penetratin-fused 29 mer, palmitoylated penetratin-fused 29 mer palmitoylated penetratin-fused gp130 and penetratin alone. Palmitoylated, penetratin fused 29 mer mediated significant WO 00/20578 PCT/AU99/00860 cell death.

EXAMPLE 6 PASSAGE ACROSS BLOOD BRAIN BARRIER The ability for peptides to cross the blood brain barrier is tested using fluorescence-linked peptides injected intraperitonealy into mice. The peptides may be fused to penetratin or fused or associated with the TAT protein from HIV (18).

EXAMPLE 17 ANIMAL MODELS Peptides are delivered with penetratin or TAT (18) to various animal models for neurodegenerative diseases. The animal models used include: Animal Model Disease Axotomy of newborn rat sensory Peripheral neuropathies neurons Axotomy of newborn rat motor neurons Motor neuron disease (SOD1 mice: B6SJL-TgN [SOD1- G93A] 1 Gurdl) Ischemia of adult rats Stroke Experimental allergic encephamylitis Multiple sclerosis and optic nerve axotomy Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described.

It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or WO 00/20578 PCT/AU99/00860 -46collectively, and any and all combinations of any two or more of said steps or features.

WO 00/20578 PCT/AU99/00860 -47-

BIBLIOGRAPHY

1. Levi-Montalcini, 1982. Ann. Rev. Neurosci. 5: 341-362.

2. Rabizadeh,et al., Science, 1993. 261(5119): p. 345-8.

3. Majdan, et al., Journal of Neuroscience, 1997. 17(18): p. 6988-98.

4. Barrett, G.L. and A. Georgiou, Journal of Neuroscience Research, 1996.

45(2): p. 117-28.

Barrett, G.L. and P.F. Bartlett, Proceedings of the National Academy of Sciences of the United States of America, 1994. 91(14): p. 6501-5.

6. Cheema, G.L. Barrett, and P.F. Bartlett, Journal of Neuroscience Research, 1996. 46(2): p. 239-45.

7. Bamji, et al., Journal of Cell Biology, 1998. 140(4): p. 911-23.

8. Van der Zee, et al., Science, 1996. 274(5293): p. 1729-32.

9. Feinstein, et al.,Trends in Biochemical Sciences, 1995. 20(9): p. 342-4.

Moix, et al., Brain Research, 1991. 564(1): p. 176-80.

11. Lee, et al., Journal of Neuroscience Research, 1995.41(5): p. 684-95.

12. Rende, et al., Journal of Comparative Neurology, 1995. 363(2): p. 249-63.

13. Seeburger, et al., Brain Research, 1993. 621(1): p. 111-5.

14. De Simone, et al., Neuropathology Applied Neurobiology, 1996. 22(1): p.

54-9.

Conner, et al., Journal of Comparative Neurology, 1992. 319(3): p. 454-62.

16. Wiley, et al., Journal of the Neurological Sciences, 1995. 128(2): p. 157-66.

17. Needleman and Wunsch, 1970. J. Mol. Biol. 48: 443-453.

18. Schwarze et al, 1999. Science -285: 1569-1572.

19. Nataf et al, 1998. J. Neurosci. Res. 52 83-92.

Zupan et al, 1999. J. Cell Biol. 264: 11714-11720.

EDITORIAL NOTE APPLICATION NUMBER 10194/00 The following Sequence Listing pages 1 to 14 are part of the description. The claims pages follow on pages "48" to "53".

WO 00/20578 WO 0020578PCT/AU99/00860 SEQUENCE LISTING <110> THE WALTER AND ELIZA HALL INSTITUTE OF MEDICAL RESEARCH (US ONLY: Perry Francis BARTLETT, Elizabeth Jane COU.LSON, Kate REID, Katrina FIELDEW, Manuel BACCA, Surindar CHEEMA, Trevor KILPATRICK, Kylie SHIPHAM) <120> A METHOD OF MODULATING CELL SURVIVAL AND REAGENTS USEFUL FOR SAME <130> EJE/AF <140> <14 1> <150> PP6353 <151> 1998-10-06 <150> PP6351 <151> 1998-10-07 <160> 12 <170> Patentln Ver. <210> 1 <211> 3260 <212> DNA <213> Human <220> <223> <220> <221> <222> Description of Artificial Sequence: Synthetic

CDS

(115) (1389) <400> 1 acagctccgg cgggcagcag gcgctggagc gcatcgcagt tcagctcagc gcagcaccat cggtctgcgg agcggactga gctagaagcg gagcgctgac gccggaggcg tgca atg Met agg agg gca ggt gct gcc tgc agc gcc atg gac Arg Arg Ala Gly Ala Ala Cys Ser Ala Met Asp 10 ctg ctg ctg att cta ggg gtg tcc tct gga ggt Leu Leu Leu Ile Leu Gly Val Ser Ser Gly Gly 25 cgg ctg cgc ctg ctg Arg Leu Arg Leu Leu gcc aag gag aca tgt Ala Lys Glu Thr Cys 165 213 WO 00/20578 WO 0020578PCT/AU99/00860 -2tcc aca Ser Thr ggc ctg tac acc cac agc gga. gag tgc tgc aaa gcc tgc aac Gly Leu Tyr Thr His Ser Gly Glu Cys Cys Lys Ala Cys Asn ttg Leu so ggc gaa ggc gtg Gly Giu Gly Val cag ccc tgc gga Gin Pro Cys Gly aac cag acc gtg Asn Gin Thr Val tgt Cys gaa ccc tgc ctg Glu Pro Cys Leu aat gtt aca ttc Asn Val Thr Phe tcc Ser gat gtg gtg agc Asp Val Val Ser gcc act Ala Thr gag ccg tgc Gin Pro Cys gct ccc tgt Ala Pro Cys 100 aag Lys ccg tgc acc gag Pro Cys Thr Gin ctg ggc ctg cag Leu Gly Len Gin agc atg tcc Ser Met Ser gtg gag gca gac Val Giu Ala Asp gat Asp 105 gca gtg tgc aga tgt gcc tat ggc Ala Val Cys Arg Cys Ala Tyr Gly 110 tac tac Tyr Tyr 115 cag gac gag gag Gin Asp Gin Gin act Thr 120 ggc cac tgt gag Gly His Cys Gin gct Ala 125 tgc agc gtg tgc Cys Ser Val Cys gag Gin 130 gtg ggc tcg gga Val Gly Ser Gly ctc Len 135 gtg ttc tcc tgc Val Phe Ser Cys cag Gin 140 gac aaa cag aac Asp Lys Gin Asn aca Thr 145 gtg tgt gaa gag Val Cys Glu Gin tgc Cys 150 cca gag ggc aca.

Pro Gin Gly Thr tac Tyr 155 tca gac gaa gcc Ser Asp Gin Ala aac cac Asn His 160 gtg gac ccg Val Asp Pro tta. cgc gag Len Arg Gin 180 tgc Cys 165 cta. ccc tgc acg Len Pro Cys Thr gtg Val 170 tgc gag gac act Cys Gin Asp Thr gag cgc cag Gin Arg Gin 175 gag atc cct Gin Ile Pro tgc acg ccc tgg Cys Thr Pro Trp gat gct gaa tgc Asp Ala Gin Cys ggt cga Gly Arg 195 tgg atc cca agg Trp Ile Pro Arg acg ccc ccg gag Thr Pro Pro Giu ggc Gly 205 tcc gac agc aca Ser Asp Ser Thr gcg Ala 210 ccc agc acc cag Pro Ser Thr Gin cct gag gtt cct Pro Gin Val Pro gag caa gac ctt Gin Gin Asp Len gta Val 225 741 789 837 ccc agt aca gtg Pro Ser Thr Val gcg Ala 230 gat atg gtg acc Asp Met Val Thr act Thr 235 gtg atg ggc agc Val Met Gly Ser tcc cag Ser Gin 240 WO 00/20578 WO 0020578PCT/AU99/00860 -3cct gta gtg Pro Val Val tcc atc ttg Ser Ile Leu 260 ac c Thr 245 cgc ggc acc acc Arg Gly Thr Thr aac ctc att cct Asn Leu Ile Pro gtc tat tgc Val Tyr Cys 255 att gct ttc Ile Ala Phe 885 933 gct gc-t gtg gtc Ala Ala Val Val gtg Val 265 ggc ctt gtg gcc Gly Leu Val Ala tat Tyr 270 aag agg Lys Arg 275 tgg aac agc tgc Trp Asn Ser Cys aaa Lys 280 caa aat aaa caa Gin Asn Lys Gin ggc Gly 285 gcc aac agc cgc Ala Asn Ser Arg ccc Pro 290 gtg aac cag acg Val Asn Gin Thr cca ccg gag gga Pro Pro Giu Gly gag Glu 300 aaa ctg cac agc Lys Leu His Ser gac Asp 305 981 1029 1077 agt ggc atc tct Ser Gly Ile Ser gtg Val 310 gac agc cag agc Asp Ser Gin Ser ctg Leu 315 cac gac cag cag His Asp Gin Gin acc cat Thr His 320 acg cag act Thr Gin Thr agt agc ctg Ser Ser Leu 340 gcc Ala 325 tca ggc cag gcc Ser Gly Gin Ala aag ggt gat ggc Lys Gly Asp Giy aac ctc tac Asn Leu Tyr 335 ctg ctc aac Leu Leu Asn 1125 1173 ccc ctg acc aag Pro Leu Thr Lys cgt Arg 345 gag gag gta gag Giu Glu Val Giu aaa Lys 350 ggg gat Gly Asp 355 acc tgg cga cat Thr Trp Arg His ctg Leu 360 gca ggc gag ctg Ala Gly Glu Leu ggt Gly 365 tac cag cct gaa Tyr Gin Pro Giu cat His 370 gcc Ala ata gac tcc ttt Ile Asp Ser Phe agc tgg ggt gcc Ser Trp Gly Ala 390 acc Thr 375 cac gag gcc tgc His Giu Ala Cys gtg cga gcc ctg Val Arg Ala Leu 1221 1269 1317 cag gac agt gca Gin Asp Ser Ala ctt gat gcc ctt Leu Asp Ala Leu tta gcc Leu Ala 400 gcc ctg cga Ala Leu Arg gag tcc act Glu Ser Thr 420 cgc Arg 405 atc cag aga gct Ile Gin Arg Ala att gtg gag agt Ile Vai Giu Ser cta tgc agc Leu Cys Ser 415 1365 gcc aca tcc cca gtg tgaactcaca gactgggagc ccctgtcctg Ala Thr Ser Pro Val 425 1419 tcccacattc cgacgactga tgttctagcc agcccccaca gagctgcccc ctctccctcg 1479 gggatggccc aacggtcaga acggagcatc tctgtgcagg gcctctgtgt tcccactcct 1539 WO 00/20578 WO 0020578PCT/AU99/00860 -4gactccgttg gagagagagg tctacacatt ccctaagact aacaaggggc ggccaaagaa acttctctct cccaaccaca gtgtgcttgt ccgccctcaa gggctggtcc gcatttgtgg acggccctta actatactac gatatcttgc actgccagtg cccgggtctg gtgcgtggag tgcaaaagag tggcagcttt ggaaggggca tttgcttgct gggtatgcac gaccaggagc cagacacctg gtggtggggg ctgctcccga accacccgag agctgtgtca caggaggtac ggggcattgt gaggattacg cacacacagg ggaagaacaa tgacagggat ccacttccag attggtctat agagatgaat atgtcagccc attacacaca ttttctcccc agagatccgg gcaggccaag gaaatgctcc c agactggtg ctC ct Cagc t agagggttcg gttggaatga agactgacca attgtacgca ggaagtggga gctgcaggta gggggCCctt cctgacttgc gatctggggg tgaagaacca ggtaggctag gacctatctg atggtaaaac atgaaggctg ggcgtgact t gcccctaccc tctgatggag ctgttagtgc tcggggtcag cacacacaca atggctcttt aggggacaga aatgactgca cactcctccc tgagaacaca tcaaggcccc gtt cagtggc gtgggcaccc ccatccctcc t acgcgggtg cattctttgt cggtttagc a gcttctgacc tccatttcca tttgacacta gagccatgga accttcctta agctgaaagc ccagagaaag atacactccg tcagggaaat aacccttgtg tcaagctaag gctcattctt gaaccaagga cacacacaca tggggctgag gctgagcttc tctgagctgg cttcttgaag agaaaaagca tgcaaaggac gctttctccc cctctattta ccacccaggg gtatttttat gtatttattt tgtgtttggt accctctcct tctcaggcct ggagaaggga ctccacactg gcccctccct aggt ttggaa gcagggactg tttctgaatg atctggaagc cagatgaact ggctcaggct ggcataagcc ctcccacccc cacacacaca actagatcct atggggctgt tgtctgtctt ctgccccag gatgctggcc ggatttcctg ggctccttgg gcatgaagga tccacccaac ggacc ccaat tcctccccag tctgggggtc cagcaagaga 1599 ttccttcctt 1659 gcgggggcac 1719 tgaaccggag 1779 tctcccctct 1839 cccagcccac 1899 acctaggcca 1959 agggcgtcaa 2019 catgtctgcc 2079 gtttgttcaa 2139 tatccataag 2199 tgaagccaac 2259 acaatccaac 2319 cacacacaca 2379 gctgggagtc 2439 cttcctcgcc 2499 ccaatggcct 2559 aagactacag 2619 ctgcagtctg 2679 agcacggcca 2739 cctgttctgt 2799 gccccaggca 2859 ccggtgaaga 291 9 ctgcaattcc 2979 gagctgggga 3039 tctccagcct 3099 WO 00/20578 WO 0020578PCT/AU99/00860 tgttttgggc caagttggaa cctctggccc tccagctggt gactatgaac tccagacccc 3159 ttcgtgctcc ccgacgcctt ccccttgcat cctgtgtaac catttcgttg ggccctccca 3219 aaacctacac ataaaacata caggaggacc attaaattgg c 3260 <210> 2 <211> 425 <212> PRT <213> Human <400> 2 Met Arg Arg 1 Leu Leu Len Cys Ser Thr Asn Leu Gly Cys Giu Pro Thr Giu Pro Ser Ala Pro Gly Tyr Tyr 115 Cys Gin Val 130 Thr Val Cys 145 His Val Asp Gin Len Arg Ala Len Gly Glu Cys Cys Cys 100 Gin Gly Giu Pro Gin 180 Gly Ala Ala Cys Ser Ala Met Asp Arg Leu Arg Leu 5 10 Ile Leu Gly Leu Lys Val Asp Ser Glu Cys 165 Cys Leu Tyr Val Asp 70 Pro Gin Glu Gly Cys I50 Leu Thr Gly rhr Ala 55 Asn Cys Ala Gin Len 135 Pro Pro Pro Val Ser Ser 25 His Ser Gly 40 Gin Pro Cys Val Thr Phe Thr Gin Cys 90 Asp Asp Ala 105 Thr Gly His 120 Val Phe Ser Giu Gly Thr Cys Thr Val 170 Trp Ala Asp 185 Gly Gin Gly Ser 75 Leu Val Cys Cys Tyr 155 Cys Gly Cys Ala Asp Gly Cys Gin Gin 140 Ser Gin Ala Cys Asn Val Leu Arg Ala 125 Asp Asp Asp Cys Ljys Lys Gin Val Gin Cys 110 Cys Lys Glu Thr Gin Gin Ala Thr Ser Ser Ala Ser Gin Ala Gin 175 Gin Thr Cys Val Ala Met Tyr Val Asn Asn 160 Arg Ile Ala Glu 190 Pro Gly Arg Trp Ile Pro Arg Ser Thr Pro Pro Gin Gly Ser Asp Ser WO 00/20578 WO 0020578PCT/AU99/00860 -6- Thr Val 225 Gin cys Phe Arg Asp 305 His Tyr Asn Giu Leu 385 Ala Ala 210 Pro Pro Ser Lys Pro 290 Ser Thr Ser Gly His 370 Ala Ala 195 Pro Ser Val1 Ile Arg 275 Val Gly Gin Ser Asp 355 Ile Ser Leu Ser Thr Gin Thr Vai Leu 260 Trp, Asn Ile Thr Leu 340 Thr Asp Trp Arg Vai Thr 245 Ala Asn Gin Ser Ala 325 Pro Trp Ser Gly Arg 405 Ala 230 Arg Ala Ser Thr Val 310 Ser Leu Arg Phe Ala 390 Ile Giu Pro 215 Asp Met Giy Thr Vai Val Cys Lys 280 Pro Pro 295 Asp Ser Gly Gin Thr Lys His Leu.

360 Thr His 375 Gin Asp Gin Arg 205 Giu Val Pro Pro Giu Gin Asp Leu 220 Val Thr Thr Val Met Giy Ser Ser 235 240 Thr Asp Asn Leu Ile Pro Val Tyr 250 255 Val Gly Leu Val Ala Tyr Ile Ala 265 270 Gin Asn Lys Gin Gly Ala Asn Ser 285 Pro Giu Gly Giu Lys Leu His Ser 300 Gin Ser Leu His Asp Gin Gin Thr 315 320 Ala Leu Lys Gly Asp Gly Asn Leu 330 335 Arg Glu Glu Vai Glu. Lys Leu Leu 345 350 Ala Gly Glu. Leu Gly Tyr Gin Pro 365 Giu Ala Cys Pro Val Arg Ala Leu 380 Ser Ala Thr Leu. Asp Ala Leu Leu 395 400 Ala Asp Ile Val Giu Ser Leu. Cys 410 415 Ser Giu Ser Thr Ala Thr Ser Pro Val 420 425 <210> 3 <211> 867 <212 DNA <213> Rat <220> <223> Description of Artificial Sequence: Synthetic WO 00/20578 WO 0020578PCT/AU99/00860 <220> <221> CDS <222> (115)..(867) <400> 3 acagctccgg cgggcagcag gcgctggagc gcatcgcagt tcagctcagc gcagcaccat cggtctgcgg agcggactga gctagaagcg gagcgctgac gccggaggcg tgca atg 11' Met 1 agg agg gca ggt gct gcc tgc agc Arg Arg Ala Gly Ala Ala Cys Ser gcc Ala 10 atg gac cgg ctg Met Asp Arg Leu cgc ctg ctg Arg Leu Leu gag aca tgt Glu Thr Cys 165 ctg ctg ctg Leu Leu Leu att cta ggg gtg Ile Leu Gly Val tct gga ggt gcc Ser Gly Gly Ala tcc aca Ser Thr ggc ctg tac acc cac agc gga gag tgc Gly Leu Tyr Thr His Ser Gly Glu Cys 40 tgc Cys aaa gcc tgc aac Lys Ala Cys Asn ttg Leu gaa Glu ggc gaa ggc gtg Gly Glu Gly Val ccc tgc ctg gac Pro Cys Leu Asp cag ccc tgc gga Gin Pro Cys Gly aac cag acc gtg Asn Gln Thr Val tgt Cys 309 357 aat gtt aca ttc Asn Val Thr Phe gat gtg gtg agc Asp Val Val Ser gcc act Ala Thr gag ccg tgc Glu Pro Cys gct ccc tgt Ala Pro Cys 100 aag Lys ccg tgc acc gag Pro Cys Thr Glu tgc Cys 90 ctg ggc ctg cag Leu Gly Leu Gln agc atg tcc Ser Met Ser gcc tat ggc Ala Tyr Gly 405 453 gtg gag gca gac Val Glu Ala Asp gat Asp 105 gca, gtg tgc aga Ala Val Cys Arg tac tac Tyr Tyr .115 cag gac gag gag Gln Asp Glu Glu ggc cac tgt gag Gly His Cys Glu tgc agc gtg tgc Cys Ser Val Cys 501 549 gag Glu 130 gtg ggc tcg gga.

Val Gly Ser Gly ctc Leu 135 gtg ttc tcc tgc Val Phe Ser Cys cag Gln 140 gac aaa cag aac Asp Lys Gln Asn aca Thr 145 gtg tgt gaa gag Val Cys Glu Glu tgc Cys 150 cca gag ggc aca tac tca gac gaa gcc Pro Glu Gly Thr Tyr Ser Asp Glu Ala aac cac Asn His 160 597 WO 00/20578 WO 0020578PCT/AU99/00860 -8gtg gac ccg Vai Asp Pro tta cgc gag Leu Arg Giu 180 cta ccc tgc acg Leu Pro Cys Thr gtg Val 170 tgc gag gac act Cys Giu Asp Thr gag cgc cag Glu Arg Gin 175 gag atc cct Glu Ile Pro 645 693 tgc acg ccc tgg Cys Thr Pro Trp, gct Ala 185 gat gct gaa tgc Asp Ala Glu Cys gaa Giu 190 ggt cga Gly Arg 195 tgg atc cca agg Trp Ile Pro Arg tct Ser 200 acg ccc ccg gag Thr Pro Pro Glu ggc Gly 205 tcc gac agc aca Ser Asp Ser Thr 741 789 gcg Al a 210 ccc agc acc cag Pro Ser Thr Gin gag Glu 215 cct gag gtt cct Pro Glu Val Pro cca Pro 220 gag caa gac ctt Giu Gin Asp Leu gta Val 225 ccc agt aca gtg gcg gat atg gtg acc Pro Ser Thr Vai Ala Asp Met Val Thr 230 gtg atg ggc agc Val Met Gly Ser tcc cag Ser Gin 240 cct gta gtg Pro Val Val acc Thr 245 cgc ggc acc acc Arg Gly Thr Thr gac aac Asp Asn 250 867 <210> 4 <211> 251 <212> PRT <213> Rat <400> 4 Met Arg Arg 1 Leu Leu Leu Cys Ser Thr Ala Gly 5 Ala Ala Cys Ser Met Asp Arg Leu Arg Leu Ile Leu Gly Val Ser Ser Gly Gly Ala Lys Glu Thr Lys Ala Cys Gly Leu Tyr Thr His 40 Ser Gly Giu Cys Cys Asn Leu Gly Glu Gly Val Ala 55 Gin Pro Cys Gly Ala Asn Gin Thr Val Cys Glu Pro Cys Leu Asp Asn Val Thr Phe Ser Asp Val Val Ser Ala 70 75 Thr Glu Pro Cys Lys Pro Cys Thr Glu Cys Leu Gly Leu Gin Ser Met 90 Ser Ala Pro Cys Val Giu Ala Asp Asp Ala Val Cys Arg Cys Ala Tyr 100 105 110 WO 00/20578 WO 0020578PCT/AU99/00860 -9- Gly Tyr Tyr Gin Asp Glu Giu Thr Gly His Cys Giu Ala Cys Ser Val 115 120 125 Cys Thr 145 His Gin Pro Thr Val 225 Gin Giu 130 Val Val Leu Gly Ala 210 Pro Pro Val Cys Asp Arg Arg 195 Pro Ser Val Ser Glu Cys 165 Cys Ile Thr Val Thr 245 Gly Cys 150 Leu Thr Pro Gin Ala 230 Arg Leu 135 Pro Pro Pro Arg Glu 215 Asp Gly Phe Gly Thr Ala 185 Thr Giu Val Thr Ser Thr Val 170 Asp Pro Val Thr Asp 250 Gin 140 Ser Glu Glu Glu Pro 220 Val Asp Asp Asp Cys Gly 205 Glu Met Asn Asn 160 Arg Ile Ser Leu Ser 240 <210> <211> 66 <212> DNA <213> Artificial Sequence <220> <221> CDS <222> <220> <223> Description of Artificial Sequence:Synthetic <400> ctc att cct gtc tat tgc tcc atc ttg gct gct gtg gtc gtg ggc ctt 48 Leu Ile Pro Val Tyr Cys Ser Ile Leu Ala Ala Val Val Val Gly Leu 1 5 10 gtg gcc tat att gct ttc 66 Val Ala Tyr Ile Ala Phe WO 00/20578 WO 0020578PCT/AU99/00860 <210> 6 <211> 22 <212> PRT <213> Artificial Sequence <400> 6 Leu Ile Pro Val Tyr Cys Ser Ile Leu Ala Ala Val Val Val Gly Leu 1 5 10 Val Ala Tyr Ile Ala Phe <210> 7 <211> 105 <212> DNA <213> Artificial Sequence <220> <221> CDS <222> (105) <220> <223> Description of Artificial Sequence:Synthetic <400> 7 aag agg tgg aac agc tgc aaa Lys Arg Trp Asn Ser Cys Lys 1 5 caa aat aaa Gin Asn Lys 10 ccg gag gga Pro Glu Gly 25 caa ggc gcc aac Gin Gly Ala Asn agc cgc Ser Arg ccc gtg aac Pro Val Asn cag acg ccc cca Gin Thr Pro Pro gag aaa ctg Glu Lys Leu cac agc gac His Ser Asp agt ggc atc 105 Ser Gly Ile <210> 8 <211> <212> PRT <213> Artificial Sequence <400> 8 Lys Arg Trp Asn Ser Cys Lys Gin Asn Lys Gin Gly Ala Asn Ser Arg Pro Val Asn Gin Thr Pro Pro Pro Giu 25 Gly Giu Lys Leu His Ser ASP Ser Gly Ile WO 00/20578 WO 0020578PCT/AU99/00860 -11 <210> 9 <211> 2222 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:Synthetic <220> <221> CDS <222> <400> 9 tct gtg Ser Val 1 gac agc cag agc ctg cac gac Asp Ser Gin Ser Leu His Asp 5 cag Gin 10 cag acc cat acg cag act Gin Thr His Thr Gin Thr gcc tca ggc Ala Ser Gly ccc ctg acc Pro Leu Thr cag Gin gcc ctc aag ggt Ala Leu Lys Gly gat Asp ggc aac ctc tao agt agc ctg Gly Asn Leu Tyr Ser Ser Leu aag cgt gag gag Lys Arg Glu Glu gt a Val 40 gag aaa ctg ctc aac ggg gat acc Glu Lys Leu Leu Asn Gly Asp Thr tgg cga Trp Arg cat ctg gca ggc His Leu Ala Giy gag Glu ctg ggt tac cag Leu Gly Tyr Gin gaa cat ata gac Glu His Ile Asp tc Ser ttt acc cac gag Phe Thr His Glu gcc Ala 70 tgc cca gtg cga Cys Pro Val Arg ctg ctg gcc agc Leu Leu Ala Ser tgg Trp s0 192 240 288 ggt gcc cag gac Gly Ala Gin Asp agt Ser gca acg ctt Ala Thr Leu gat gc Asp Ala 90 gag agt Giu Ser 105 ott tta goc gc Leu Leu Ala Ala otg cga Leu Arg cgc atc cag Arg Ile Gin aga Arg 100 got gao att gtg Ala Asp Ile Val cta tgc agc Leu Cys Ser gag too act Glu Ser Thr 110 336 goc aca too cca gtg tgaactoaca gactgggagO cootgtcctg tccoacattc 391 Ala Thr Ser Pro Val 115 ogaogactga tgttotagco agccccaca gagctgcocc ototooctog gggatggcco 451 aacggtcaga acggagcatc totgtgcagg goototgtgt tcccactcct gaotoogttg 511 WO 00/20578 WO 0020578PCT/AU99/00860 -12ctgctcccga accacccgag agctgtgtca caggaggtac ggggcattgt gaggattacg cacacacagg ggaagaacaa tgacagggat ccacttccag attggtctat agagatgaat atgtcagccc attacacaca ttttctcccc agagatccgg gcaggccaag gaaatgctcc cagactggtg ctcctcagct agagggtt cg gttggaatga agactgacca attgtacgca ggaagtggga gctgcaggta gggggccctt cctgacttgc gatctggggg tgaagaacca ggtaggctag gacctatctg atggtaaaac atgaaggctg ggcgtgactt gcccctaccc t ctgatggag ctgttagtgc tcggggtcag cacacacaca atggctcttt aggggacaga aatgactgca cactcctccc tgagaacaca tcaaggcccc gttcagtggc gtgggcaccc ccatccctcc tacgcgggtg cattctttgt cggtttagca gcttctgacc tccatttcca tttgacacta gagccatgga accttcctta agctgaaagc ccagagaaag atacactccg tcagggaaat aacccttgtg tcaagctaag gctcattctt gaaccaagga cacacacaca tggggctgag gctgagcttc tctgagctgg cttcttgaag agaaaaagca tgcaaaggac gctttctccc cctctattta ccacccaggg gtatttttat gtatttattt tgtgtttggt accctctcct cagcaagaga gagagagagg 571 tctcaggcct ggagaaggga ctccacactg gcccctccct aggt ttggaa gcagggactg tttctgaatg atctggaagc cagatgaact ggctcaggct ggcataagcc ctcccacccc cacacacaca actagatcct atggggctgt tgtctgtctt ctgcccccag gatgctggcc ggatttcctg ggctccttgg gcatgaagga tccacccaac ggaccccaat tcctccccag tctgggggtc ttccttcctt gcgggggcac tgaaccggag tctcccctct cccagcccac acctaggcca agggcgtcaa catgtctgcc gtttgttcaa tatccataag tgaagccaac acaatccaac cacacacaca gctgggagtc ct tcc tcgc c ccaatggcct aagactacag ctgcagtctg agcacggcca cctgttctgt gccccaggca ccggtgaaga ctgcaattcc gagctgggga tctccagcct tctacacatt 631 ccctaagact 691 aacaaggggc 751 ggccaaagaa 811 acttctctct 871 cccaaccaca 931 gtgtgcttgt 991 ccgccctcaa 1051 gggctggtcc 1111 gcatttgtgg 1171 acggccctta 1231 actatactac 1291 gatatcttgc 1351 actgccagtg 1411 cccgggtctg 1471 gtgcgtggag 1531 tgcaaaagag 1591 tggcagcttt 1651 ggaaggggca 1711 tttgcttgct 1771 gggtatgcac 1831 gaccaggagc 1891 cagacacctg 1951 gtggtggggg 2011 tgttttgggc 2071 WO 00/20578 WO 0020578PCT/AU99/00860 13 caagttggaa cctctggccc tccagctggt gactatgaac tccagacccc ttcgtgctcc 2131 ccgacgcctt ccccttgcat cctgtgtaac catttcgttg ggccctccca aaacctacac 2191 ataaaacata caggaggacc attaaattgg c 2222 <210> <211> 117 <212> PRT <213> Artificial Sequence <400> Ser Val Asp Ser Gin Ser Leu His Asp Gin Gin Thr His Thr Gin Thr 1 5 10 Ala Ser Gly Gin Ala Leu Lys Gly Asp Gly Asn Leu Tyr Ser Ser Leu 25 Pro Leu Thr Lys Arg Glu Glu Val Glu Lys Leu Leu Asn Gly Asp Thr 40 Trp Arg His Leu Ala Gly Glu Leu Gly Tyr Gin Pro Giu His Ile Asp 55 Ser Phe Thr His Glu Ala Cys Pro Val Arg Ala Leu Leu Ala Ser Trp 70 75 Gly Ala Gin Asp Ser Ala Thr Leu Asp Ala Leu Leu Ala Ala Leu Arg 90 Arg Ile Gin Arg Ala Asp Ile Val Glu Ser Leu Cys Ser Glu Ser Thr 100 105 110 Ala Thr Ser Pro Val 115 <210> 11 <211> 87 <212> DNA <213> Artificial Sequence <220> <221> CDS <222> <220> <223> Description of Artificial Sequence:Synthetic WO 00/20578 WO 0020578PCT/AU99/00860 -14- <400> 11 aag agg tgg aac agc Lys Arg Trp Asn Ser 1 5 tgc aaa caa aat Cys Lys Gin Asn caa ggc gcc Gin Gly Ala aac agc cgc Asn Ser Arg ccc gtg aac cag acg ccc cca ccg Pro Val Asn Gin Thr Pro Pro Pro gga gag aaa ctg Gly Glu Lys Leu <210> 12 <211> 29 <212> PRT <213> Artificial Sequence <400> 12 Lys Arg Trp Asn Ser Cys Lys Gin Asn Lys Gin Gly Ala Asn Ser Arg 1 5 10 Pro Val Asn Gin Thr Pro Pro Pro Glu Gly Glu Lys Leu

Claims (8)

19- 3-04:12:21PM:OAVIES COLLISON CAVE 5/ 16 C:r VDanitnud Sc tim CVlwOl 4.i.wllnln.chopp teu.edlClaiiav2.4dotLgsO4 -48- 1. A method for inhibiting, reducing or otherwise antagonising p 7 5 NTR mediated death signal in a neural cell, said method comprising administering a peptide, polypeptide or protein or analogues or mimetics thereof which correspond to a non-membrane associated form-of p75 NTR death signal region or a derivative, functional equivalent or homologue thereof. 2. A method for inhibiting, reducing or otherwise antagonising a p 7 5 NTR- mediated death signal in a neural cell, said method comprising Introducing a nucleic acid molecule capable of being expressed to an expression product which corresponds to a non-membrane associated form of the p7 5 N R death signal region or a derivative, functional equivalent or homologue thereof. 3. A method for inhibiting, reducing or otherwise antagonising a p 7 5 NTR mediated death signal in a neural cell, said method comprising contacting a cell carrying a p 75 NTR with a death signal-inhibiting effective amount of a molecule capable of antagonising the death signal of p75 N R or a component of the death signalling pathway. 4. An isolated nucleic acid molecule comprising a sequence of nucleotides or complementary sequence of nucleotides which encodes an amino acid sequence which comprises a membrane-associating portion and/or multimer- forming portion and a portion which corresponds to all or part of the cytoplasmic region of p75NTR or a functional equivalent, derivative or homologue thereof which is capable of signalling, Inducing or otherwise facilitating the death of a cell in which said amino acid sequence is adjacent, proximal or otherwise juxtaposed to the membrane of said cell or when said amino acid is in multimeric form, when used according to the method of any one of claims 1 to 3. The isolated nucleic acid molecule of claim 4 wherein the membrane- associated portion is derived from p 7 5 N "R or a functional equivalent, derivative or homologue thereof. COMS ID No: SMBI-00672060 Received by IP Australia: Time 12:31 Date 2004-03-19 19- 3-04;12:21PM:DAVIES COLLISON CAVE 6/ 16 Chcunn ipiind UKRC)ctop0aiOI4t.hlll.dewarJnmatal.rvL4.i2Mlat -49- 6. An isolated nucleic acid molecule comprising a sequence of nucleotides which encodes an amino acid sequence which inhibits or reduces p 7 5 NTR mediated cell death wherein said amino acid sequence is a soluble form of the receptor corresponding to an intracellular region adjacent, proximal or otherwise juxtaposed to the membrane of said cell when used according to the method of any one of claims 1 to 3. 7. The isolated nucleic acid molecule of claim 4 or 6 wherein the p 7 5 N is of human, primate or murine origin. 9* 8. The isolated nucleic acid molecule of Claim 4 or 6 comprising a nucleotide sequence substantially capable of hybridizing to <400>1 or its complementary form under low stringency conditions. 9. The isolated nucleic acid molecule of claim 8 comprising a nucleotide sequence substantially as set forth in <400>7 or a nucleotide sequence capable of hybridizing to <400>7 or its complementary form under low stringency conditions or a nucleotide sequence having at least 60% identity to <400>7. 9* 10. The isolated nucleic acid molecule of claim 9 comprising a nucleotide sequence encoding an amino acid sequence set forth in <400>8 or an amino acid sequence having at least 60% identity thereto. 11. A nucleic acid molecule when used according to the method of any one of claims 1 to 3 comprising the nucleotide sequence: (ni nxb a n'y} a n"}d wherein nx is a sequence of x nucleotides encoding an extracellular portion of a receptor or ligand-binding molecule; COMS ID No: SMBI-00672060 Received by IP Australia: Time 12:31 Date 2004-03-19 19- 3-04:12:21PM;DAVIES COLLISON CAVE 7/ 16 C1Dcusema n1senin t o alJ l$101U>4t.LdCh L ioparama d.brlqaitd1W OtH n'y} is a sequence of y nucleotides encoding a transmembrane peptide, polypeptide or protein or a molecule capable of inducing multimerisation; {n"i n"z} is a sequence of z nucleotides comprising a nucleotide sequence substantially as set forth in <400>7 or a nucleotide sequence encoding an amino acid sequence substantially as set forth in <400>8 or a nucleotide sequence capable of hybridising to <400>7 or a complementary form thereof under low stringency conditions such as at 42 0 C or a nucleotide sequence having at least 60% Identity to <400>7; b, c and d may be the same or different and each is 0, 1 or >1; x, y and z may be the same or different and each is 0, 1 or >1; a is a nucleotide bond; wherein when c is 1 or >1 and d is 1 or >1 and wherein when the molecule is expressed in a neural cell, the expression product signals, induces or otherwise facilitates cell death cell. 12. The nucleic acid molecule of claim 11 wherein {ni nx~ comprises the nucleotide sequence substantially as set forth in <400>3 or is a nucleotide sequence having at least about 60% identity thereto or is capable of hybridising thereto under low stringency conditions at 42°C. 13. The nucleic acid molecule of claim 11 wherein {n'l ny} comprises the nucleotide sequence substantially as set forth in <400>5 or is a nucleotide sequence having at least about 60% identity thereto or is capable of hybridising thereto under low stringency conditions at 42 0 C. COMS ID No: SMBI-00672060 Received by IP Australia: Time 12:31 Date 2004-03-19 19- 3-04:12:21PM:DAVIES COLLISON CAVE 8/ 16 CtO«Mci....J idlh.;A.Vl 4Cap*l l I *wipwa.Htalcir m flt sioc.WO2M -51 14. A genetic construct comprising an isolated nucleic acid molecule which comprises a sequence of nucleotides which corresponds or is complementary to a death signal region from p 7 5 N "R or a homologue, analogue or derivative thereof. The genetic construct of claim 14 wherein the coding region of the death signal from p75 TR is placed in operable connection with a promoter sequence such that a gene product is capable of being expressed under the control of said promoter sequence. 16. The genetic construct of claim 14 or 15 wherein said genetic construct further comprises a terminator sequence. 17. A peptide antagonist of the p75 N T death signal or functional analogues or mimetics thereof. 18. An isolated peptide, polypeptide or protein produced by expressing the nucleic acid molecule according to any one of claims 4 to 13 in a suitable host cell or a derivative, homologue or analogue of said peptide, polypeptide or protein. eooo 0. 19. The isolated peptide, polypeptide or protein according to claim 18 comprising the cytoplasmic region of p 7 5 NTR which signals, induces or otherwise facilitates cell death when said peptide, polypeptide or protein is adjacent, proximal or otherwise juxtaposed to a membrane-associating region such as from p 7 5 NTR or other membrane molecule and/or is in multimeric form or a derivative, homologue, chemical equivalent or analogue of said peptide, polypeptide or protein. The peptide, polypeptide or protein of claim 19 comprising an amino acid sequence substantially as set forth in <400>8 or an amino acid sequence having at least 60% identity thereto or a chemical equivalent, derivative, homologue or analogue of said peptide, polypeptide or protein. COMS ID No: SMBI-00672060 Received by IP Australia: Time 12:31 Date 2004-03-19 19- 3-04:12:21PM;DAVIES COLLISON CAVE 9/ 16 C.&iDMCfml 9ltiV^Ots>vp~IlI9 14 nii dpwpvn.oaaia t.vda calMGW -52-

21. A method for inhibiting, reducing or otherwise antagonising a p75 NT R mediated death signal in a neural cell, said method comprising contacting a cell carrying a p75 NT R with a death signal-inhibiting effective amount of a molecule capable of antagonising the death signal of p 7 5 NTR or a component of the death signalling pathway.

22. The method of any one of claims 1 to 3 for the treatment of a range of neurodegenerative diseases such as cerebral palsy, trauma induced paralysis, S. vascular ischaemia associated with stroke, neural tumours, motoneurone disease, Parkinson's disease, Huntington's disease, Alzheimer's disease, multiple sclerosis and peripheral neuropathies associated with diabetes, heavy metal or alcohol toxicity, renal failure and/or infectious diseases such as herpes, rubella, measles, *chicken pox, HIV and/or HTLV-1.

23. The method of any one of claims 1 to 3 for treating neurons and neural cells damaged by trauma or disease.

24. A biological composition comprising a genetic molecule capable of being expressed into a p 75 NT R death signal antagonist or a p 7 5 N TR death signal said composition further comprising one or more pharmaceutically acceptable carriers and/or diluents. A method for modulating p75NTR-mediated death signal in a neural cell, said method comprising administering an agent which antagonises or agonises cleavage of the extracellular domain of p 7 5 NT

26. Use of an antagonist of the death signal of p75 NT R in the manufacture of a medicament for the treatment of neurodegenerative diseases in animals. COMS ID No: SMBI-00672060 Received by IP Australia: Time 12:31 Date 2004-03-19 19- 3-04;12:21PM;DAVIES COLLISON CAVE 10/ 16 C(a cizc1 ti&t;1ir|Acntto&0«la«« dIa~w'nCMdUbii nofl.ntaoc1K1 -53-

27. Use of claim 26 wherein the animal is a human, primate, livestock animal, laboratory test animal, companion animal and/or captive wild animal.

28. A method according to any one of claims 1 to 3 and 21 to 23 and 25 or an isolated nucleic acid molecule according to any one of claims 4 to 10 or a nucleic acid according to any one of claims 11 to 13 or a genetic construct according to any one of claims 15 and 16 or a peptide according to any one of claims 17 to 20 or a biological composition according to claim 24 or a use according to any one of claims 26 and 27 substantially as hereinbefore defined 1'.with reference to the Figures and/or Examples. A@* S' 0@ 0 o* oeeo a 0 0.00 a a weo. ewe. C C C C C* S S C* S CC @9 C COMS ID No: SMBI-00672060 Received by IP Australia: Time 12:31 Date 2004-03-19