EP1976550A2

EP1976550A2 - Identification of apoptosis inducing proteins

Info

Publication number: EP1976550A2
Application number: EP06831456A
Authority: EP
Inventors: Thomas Charles Freeman; Andrew David Miller; Ella Louise Palmer
Original assignee: Imperial Innovations Ltd
Current assignee: Ip2ipo Innovations Ltd
Priority date: 2005-12-22
Filing date: 2006-12-22
Publication date: 2008-10-08
Also published as: GB0526212D0; WO2007072042A3; WO2007072042A2

Abstract

The present invention relates to the treatment and/or prophylaxis of a disease through the regulation of one or more genes involved in apoptosis. The invention also relates to a vector for use in regulating expression of the one or more genes in the treatment and/or prophylaxis of a disease involving apoptosis.

Description

Methods

Apoptosis is a mechanism for regulating cell survival by which unwanted or damaged cells are induced to undergo a controlled cell death. During development, apoptosis is used to remove surplus cells and remodel tissues. After birth, apoptosis plays additional roles in tissue homeostasis, immune selection and in deleting cells that have become infected, irreparably damaged, or transformed (Meier et al. 2000) (Rich et al. 1999). The apoptotic cascade maybe triggered through two major pathways. Extracellular signals such as the tumour necrosis factor (TNF) family of proteins can activate the receptor-mediated extrinsic pathway. Alternatively, stress signals such as DNA damage or withdrawal of survival signals may trigger the mitochondrial intrinsic pathway. Regardless of the mechanism of activation, cells undergoing apoptosis show characteristic features which include chromatin aggregation, nuclear/cytoplasmic condensation and partitioning of the cytoplasm and nucleus into membrane bound-vesicles (apoptotic bodies) which contain ribosomes, morphologically intact mitochondria and nuclear material (Gallaher et al. 2001; Li and Yuan 1999). Numerous studies have focused on the identification of proteins and pathways that modulate apoptosis as alterations in their activity are a major contributing factor in many diseases, including cancer, autoimmune diseases and neurodegenerative disorders. Indeed, the regulation of apoptosis could lead to the treatment and/or prophylaxis of a number of these diseases.

hi a first aspect of the present invention there is provided an isolated or recombinant nucleic acid molecule comprising: (a) a nucleotide sequence comprising the sequence of Figures 1, 3, 5, 7, 9,

11, 13, 15, 17 or 19, or its RNA equivalent; (b) a nucleotide sequence which is complementary to the sequence of (a); (c) a nucleotide sequence which codes for the same polypeptide as the nucleotide sequence of (a) or (b); or

(d) a fragment of the nucleotide sequence of (a) for use in the treatment and/or prophylaxis of a disease through the regulation of apoptosis.

The present invention also provides the use of an isolated or recombinant nucleic acid molecule comprising: a) a nucleotide sequence comprising the sequence of Figures 1, 3, 5, 7, 9, 11, 13, 15, 17 or 19, or its RNA equivalent; b) a nucleotide sequence which is complementary to the sequence of (a); c) a nucleotide sequence which codes for the same polypeptide as the nucleotide sequence of a) orb); or d) a fragment of the nucleotide sequence of a) in the manufacture of a medicament for the treatment and/or prophylaxis of a disease through the regulation of apoptosis.

hi the present invention a number of genes have been identified that when expressed induce apoptosis. Further, it has been possible to identify common apoptotic pathways on which the different pro-apoptotic proteins appear to be acting thereby confirming their role in apoptosis.

The regulation of apoptosis may comprise increasing or decreasing expression of the identified genes, hi treating diseases such as cancer such that apoptosis of cancer cells is induced, the regulation of apoptosis may comprise over-expressing an identified gene. Alternatively, for diseases such as autoimmune diseases and neurodegenerative disorders, the regulation of apoptosis may comprise decreasing expression of the identified genes using nucleic acid sequences of the first aspect in conjunction with well-known gene "knock-out," ribozyme or triple helix methods to decrease expression of a polypeptide. Alternatively, endogenous polypeptide expression can also be reduced by inactivating or "knocking out" an identified gene as defined herein, or the promoter of such a gene, using targeted homologous recombination (e.g., see Smithies, et a!., 1985, Nature 317:230-234; Thomas & Capecchi, 1987, Cell 51:503- 512; Thompson et al, 1989, Cell 5:313-321; and Zijlstra et ah, 1989, Nature 342:435- 438). RNA interference (RNAi) or antisense techniques may also be used to suppress expression of the identified genes.

The term 'RNA equivalent' when used above indicates that a given RNA molecule has a sequence which is complementary to that of a given DNA molecule, allowing for the fact that in RNA 'U' replaces 'T' in the genetic code. The nucleic acid molecule of the present invention may be in isolated, recombinant or chemically synthetic form.

As used herein with respect to nucleic acid molecules, "isolated or "recombinant" means any of a) amplified in vitro by, for example, polymerase chain reaction (PCR), b) recombinantly produced by cloning, c) purified by, for example, gel separation, or d) synthesised, such as by chemical synthesis.

The nucleic acid molecules of the present invention, including DNA and RNA, may be synthesised using methods known in the art, such as using conventional chemical approaches or polymerase chain reaction (PCR) amplification. The nucleic acid molecules of the present invention also permit the identification and cloning of the identified genes, for instance by screening cDNA libraries, genomic libraries or expression libraries.

The fragment of the sequence of a) above may comprise at least 15 nucleotides, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, or at least 60 nucleotides. The fragment may comprise in the range of 18 to 20 nucleotides. A fragment of the sequence of a) may find use as a hybridisation probe to detect the nucleic acid molecule of a), b) or c) in a sample.

The present invention includes nucleic acid molecules comprising a sequence complementary to the sequence as defined in (a) above. Thus, for example, both strands of a double stranded nucleic acid molecule are included within the scope of the present invention (whether or not they are associated with one another). Also included are mRNA molecules and complementary DNA molecules (e.g. cDNA molecules).

The nucleic acid molecules of the present invention may be for use in the treatment and/or prophylaxis of cancer. The nucleic acid molecules of the present invention may be for use in the treatment and/or prophylaxis of cancers of all types including solid tumours and metastases. The nucleic acid molecule may be adapted to be over- expressed in cancer cells. The nucleic acid molecule may further comprise an inducible promoter.

The present invention also provides, in a second aspect, an expression vector comprising a nucleic acid molecule of the first aspect, for use in the treatment and/or prophylaxis of a disease through the regulation of apoptosis.

The vector may be for use in the treatment and/or prophylaxis of cancer.

The vectors for use in the present invention may be integrating or non-integrating vectors. The vectors may be selected from the group comprising retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, non- viral vectors and combinations of these vectors.

Retroviruses may be selected from murine leukaemia virus (MLV), mouse mammary tumour virus (MMTV), Rouse sarcoma virus (RSV), Moloney murine leukaemia virus (MoMLV), Fujinami sarcoma virus (FuSV), Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukaemia virus (A-MLV) and Avian erythroblastoma virus (AEV).

Lentiviruses may be selected from human immunodeficiency virus (HFV), simian immunodeficiency virus (SFV), feline immunodeficiency virus (FFV), equine infectious anaemia virus (EIAV), caprine arthritis encephalitis virus (CAEV), bovine immunodeficiency virus (BFV) and Jembrana disease virus (JDV) based vectors. Adenoviruses may be selected from adenovirus type 5 first and second generation and gutless vectors. Details of adenovirus can be found GenBank accession number M73260. Adeno-associated viruses may be selected from all adeno-associated serotypes.

Retroviruses integrate into host cell DNA and have the potential for lifelong expression. However, retroviruses can potentially cause insertional mutagenesis due to insertion into the host's chromosomes. Lentivirases can also integrate into the host's DNA. As with retroviruses, lentiviruses can potentially cause mutations when they are inserted into the host's chromosomes. Adeno-associated viruses can also be integrated into the host's DNA albeit to a lesser extent than retroviruses. Retroviruses, lentiviruses and adeno-associated viruses therefore have potential for long term expression in the host.

Adenoviruses can achieve transgene expression at high levels. However, they are usually non-integrating vectors and therefore do not insert themselves into the host's genome and accordingly have to be repeatedly administered in gene therapy applications.

Suitable retroviruses and lentiviruses for use in the present invention may be obtained from Coffin et al ("Retroviruses" 1997 Cold Spring Harbour Laboratory Press Eds: JM Coffin, SM Hughes, HE Varmus pp 758-763). Details on the structure of the genome of retroviruses are well known and may be found in the art. Details concerning lentivirases are well known and may also be found in the art. For example, details on HIV may be found from the NCBI Genbank (ie. Genome Accession No AF033819), details on EIAV maybe found from ICTVdB - The Universal Virus Database, version 3. http://www.ncbi.nlm.nih.gov/ICTVdb/ICTVdB/. (accession number 00.061.1.06.003), and details on FIV may be found from the ICTVdB (accession number 00.061.1.06.004). In addition, details concerning adenovirus type 5 may be found from the NCBI Genbank NCBI Genbank (ie. Genome Accession No. M73260). Details concerning adeno-associated virus type 2 may be found from the ICTVdB Virus accession number: 50103001. Viral vectors have a natural tropism for certain organs and are otherwise efficient mediators of nucleic acid molecule delivery. In the case of cancer, viral vectors have usually been administered by intratumoural injection. In the case of viral nucleic acid molecule delivery, the nucleic acid construct may be devised with the nucleic acid molecule of the first aspect included as appropriate for the virus type being used in the delivery process.

Non-viral vectors may be selected from all vectors that do not integrate into host chromosomes. Non- viral vectors can either be physical in character (e.g. hydrodynamics, electroporation, biolistics, injection etc.) or synthetic (e.g. cationic liposome/micelle-based or cationic polymer-based). Physical vectors may be designed essentially for local/regional delivery only, and intratumoural delivery is normal. Synthetic vectors may be used for local/regional delivery without specific targeting ligands or may be equipped with ligands for longer range targeting. Typical ligands are integrin-targeting peptides, but for cancer cells there has been a tendency to use transferrin, anti-transferrin receptor antibodies, or else folate ligands with some degree of success.

In the case of non- viral nucleic acid delivery, nucleic acid constructs may be plasmid DNA (integrating or non-integrating). Alternatively, plasmid mini-circles, cosmids and artificial chromosomes may be used to express the nucleic acid molecule of the first aspect.

In a third aspect, the present invention provides a method for the prophylaxis and/or treatment of a disease through the regulation of apoptosis comprising administering a nucleic acid molecule of the first aspect to a subject. In a fourth aspect, the present invention provides a method for the prophylaxis and/or treatment of a disease through the regulation of apoptosis comprising administering a vector of the second aspect to a subject.

In a fifth aspect, the present invention provides a nucleic acid molecule that is antisense to a portion of a nucleic acid molecule of the first aspect for use in the manufacture of a medicament for the treatment and/or prophylaxis of a disease through the regulation of apoptosis.

The nucleic acid molecule may be of sufficient length to enable expression of a nucleic acid molecule of the first aspect to be blocked. The nucleic acid may comprise at least 10 nucleotides, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, or at least 60 nucleotides. The nucleic acid may comprise in the range of 18 to 20 nucleotides.

In a sixth aspect, the present invention provides a method for the prophylaxis and/or treatment of a disease through the regulation of apoptosis comprising administering a nucleic acid molecule comprising at least six nucleotides that is antisense to a portion of a nucleic acid molecule of the first aspect to a subject.

As used herein, an "antisense" nucleic acid molecule refers to a nucleic acid capable of hybridising by virtue of some sequence complementarity to a portion of RNA (preferably rnRNA) derived from a nucleic acid molecule of the first aspect. The antisense nucleic acid may be complementary to a coding and/or non-coding region of rnRNA derived from a nucleic acid molecule of the first aspect. Such antisense nucleic acids have utility as compounds that inhibit expression, and can be used in the treatment and/or prevention of diseases through the regulation of apoptosis.

A hybridising nucleic acid molecule of the present invention may have a high degree of sequence identity along its length with a nucleic acid molecule within the scope of (a)- (d) in the first aspect and (a) -(c) of the second aspect above (e.g. at least 50%, at least 75% or at least 90% or 95% sequence identity). As will be appreciated by the skilled person, the higher the sequence identity a given single stranded nucleic acid molecule has with another nucleic acid molecule, the greater the likelihood that it will hybridise to a nucleic acid molecule which is complementary to that other nucleic acid molecule under appropriate conditions.

The "percent identity" of two nucleic acid sequences can be or is generally determined by aligning the sequences for optimal comparison purposes (e.g., gaps can be introduced in either sequences for best alignment with the other sequence) and comparing the nucleotides at corresponding positions. The "best alignment" is an alignment of two sequences that results in the highest percent identity. The percent identity is determined by the number of identical nucleotides in the sequences being compared (i.e., % identity = # of identical positions/total # of positions x 100).

The determination of percent identity between two sequences can be accomplished using a mathematical algorithm known to those of skill in the art. An example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. The NBLAST and XBLAST programs of Altschul, et al. (1990) J. MoI. Biol. 215:403-410 have incorporated such an algorithm. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to a nucleic acid molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-Blast can be used to perform an iterated search which detects distant relationships between molecules (Id.). When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.

Another example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). The ALIGN program (version 2.0) which is part of the GCG sequence alignment software package has incorporated such an algorithm. Other algorithms for sequence analysis known in the art include ADVANCE and ADAM as described in Torellis and Robotti (1994) Comput. Appl. Biosci., 10 :3-5; and FASTA described in Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85:2444-8. Within FASTA, ktup is a control option that sets the sensitivity and speed of the search.

In view of the foregoing description, the skilled person will appreciate that a large number of nucleic acids are within the scope of the present invention. Unless the context indicates otherwise, nucleic acid molecules of the present invention may have one or more of the following characteristics:

1) they may be DNA or RNA;

2) they may be single or double stranded;

3) they may be provided in recombinant form, e.g. covalently linked to a 5' and/or a 3' flanking sequence to provide a molecule which does not occur in nature;

4) they may be provided without 5' and/or 3' flanking sequences which normally occur in nature;

5) they may be provided in substantially pure form. Thus they may be provided in a form which is substantially free from contaminating proteins and/or from other nucleic acids; and

6) they may be provided with introns or without introns (e.g. as cDNA).

The DNA or RNA molecules may be in the form of aptamers

Diseases can be treated and/or prevented through the regulation of apoptosis using RNA interference (RNAi) to suppress expression of the identified genes. RNA interference is a process by which double stranded RNA can induce sequence specific post-transcriptional gene silencing or inhibition (WO01/75164).

Thus, in a seventh aspect, the present invention provides a RNA molecule comprising a double stranded structure which has a nucleotide sequence which is identical to a portion of the sequence of Figures 1, 3, 5, 7, 9, 11, 13, 15, 17 or 19. In an eighth aspect, there is provided a nucleic acid molecule that when transcribed provides the RNA molecule of the seventh aspect.

In a ninth aspect, the present invention provides the use of a RNA molecule of the seventh aspect, or nucleic acid molecule of the eighth aspect, in the manufacture of a medicament for the prophylaxis and/or treatment of a disease through the regulation of apoptosis.

In a tenth aspect, the present invention provides a method for the prophylaxis and/or treatment of a disease through the regulation of apoptosis, comprising administering to a subject a RNA molecule of the seventh aspect, or nucleic acid molecule of the eighth aspect.

The RNA molecule may have a length of from 19 to 25 nucleotides or 19 to 23 nucleotides, or 21 nucleotides. At least one strand may have a 3' overlap from 1 to 5 nucleotides, 1 to 3 nucleotides or 2 nucleotides. At least one of the RNA strands may be blunt ended.

One strand of the RNA molecule may have a 3' overhang and the other strand can be blunt-ended or have an overhang. If both strands comprise an overhang, the length of the overhangs may be the same or different for each strand. The RNA may comprise 21 nucleotide strands which are paired and which have overhangs of from about 1 to 3, particularly 2, nucleotides on both 3' ends of the RNA. Li order to further enhance the stability of the RNA, the 3' overhangs can be stabilised against degradation through the inclusion of purine nucleotides, such as adenosine or guanosine nucleotides. Alternatively, pyrimidine nucleotides may be substituted by modified analogues, e.g., substitution of uridine 2 nucleotides 3' overhangs by T- deoxythymidine is tolerated and does not affect the efficiency of RNAi. The RNA molecules of this aspect of the present invention can be obtained using a number of techniques known to those of skill in the art. For example, the RNA can be chemically synthesised or recombinantly produced using methods known in the art.

In the present invention, the RNA is useful as a sequence-specific mediator of RNA degradation and thus, for inhibiting mRNA of the identified genes associated with or causative of apoptosis. 21-23 nt RNAs can be produced and tested for their ability to mediate RNAi in a cell, such as a human or other primate cell. Those 21-23 nt human RNA molecules shown to mediate RNAi can be tested, if desired, in an appropriate animal model to further assess their in vivo effectiveness. Additional copies of 21-23 nt RNAs shown to mediate RNAi can then be produced.

Any dsRNA can be used in the methods of the present invention, provided that it has sufficient homology to the targeted portion to which it is identical in sequence to mediate RNAi. The dsRNA for use in the present invention corresponds to a nucleic acid molecule as defined above.

The RNA can be introduced into human cells or a human in order to mediate RNA interference in the cells or in cells in the individual, such as to prevent or treat a disease through the regulation of apoptosis. hi this method, the identified gene is targeted, and the corresponding mRNA is degraded by RNAi. In this embodiment, an RNA of about 21 to about 23 nucleotides that targets the corresponding mRNA (the mRNA of the targeted gene) for degradation is introduced into the cell or organism. The cell or organism is maintained under conditions under which degradation of the corresponding mRNA occurs, thereby mediating RNA interference of the mRNA of the gene in the cell or organism. In the event that the RNA is introduced into a cell in which RNAi does not normally occur, the factors needed to mediate RNAi are introduced into such a cell or the expression of the factors is induced in such a cell. Alternatively, an ex vivo method may be used to treat cells from an individual to degrade the identified gene that causes or is associated with apoptosis. In this embodiment, cells to be treated are obtained from the individual using known methods (e.g., phlebotomy or collection of bone marrow) and RNAs that mediate degradation of the corresponding mRNA(s) are introduced into the cells, which are then re-introduced into the individual. If necessary, biochemical components needed for RNAi to occur can also be introduced into the cells.

In an eleventh aspect, the present invention provides an isolated or recombinant polypeptide comprising: a) the amino acid sequence shown in Figures 2, 4, 6, 8, 10, 12, 14, 16, 18 or 20; or b) a fragment of a polypeptide as defined in a), for use in the treatment and/or prophylaxis of a disease through the regulation of apoptosis.

The polypeptides of the present invention can be coded for by a large variety of nucleic acid molecules, taking into account the well known degeneracy of the genetic code. All of these molecules are within the scope of the present invention. They can be inserted into vectors and cloned to provide large amounts of DNA or RNA for further study. Suitable vectors may be introduced into host cells to enable the expression of polypeptides used in the present invention using techniques known to the person skilled in the art.

The polypeptides or fragments thereof of the present invention maybe provided in isolated or recombinant form, and may be fused to other moieties. The polypeptides or fragments thereof may be provided in substantially pure form, that is to say free, to a substantial extent, from other proteins. Thus, a polypeptide may be provided in a composition in which it is the predominant component present (i.e. it is present at a level of at least 50%; preferably at least 75%, at least 90%, or at least 95%; when determined on a weight/weight basis excluding solvents or carriers).

In order to more fully appreciate the present invention, polypeptides within the scope of a)-b) above will now be discussed in greater detail.

Polypeptides within the scope of a) A polypeptide within the scope of a), may consist of the particular amino acid sequence given in Figure 2, 4, 6, 8, 10, 12, 14, 16, 18 or 20 or may have an additional N-terminal and/or an additional C-terminal amino acid sequence.

Additional N-terminal or C-terminal sequences may be provided for various reasons. Techniques for providing such additional sequences are well known in the art.

Additional sequences may be provided in order to alter the characteristics of a particular polypeptide. This can be useful in improving expression or regulation of expression in particular expression systems. For example, an additional sequence may provide some protection against proteolytic cleavage. This has been done for the hormone Somatostatin by fusing it at its N-terminus to part of the β galactosidase enzyme (Itakwa et al, Science 198: 105-63 (1977)).

Additional sequences can also be useful in altering the properties of a polypeptide to aid in identification or purification. For example, a fusion protein may be provided in which a polypeptide is linked to a moiety capable of being isolated by affinity chromatography. The moiety may be an antigen or an epitope and the affinity column may comprise immobilised antibodies or immobilised antibody fragments which bind to said antigen or epitope (desirably with a high degree of specificity). The fusion protein can usually be eluted from the column by addition of an appropriate buffer.

Additional N-terminal or C-terminal sequences may, however, be present simply as a result of a particular technique used to obtain a polypeptide and need not provide any particular advantageous characteristic to the polypeptide. Such polypeptides are within the scope of the present invention.

Whatever additional N-terminal or C-terminal sequence is present, it is preferred that the resultant polypeptide should exhibit the immunological or biological activity of the polypeptide having the amino acid sequence shown in Figure 2, 4, 6, 8, 10, 12, 14, 16, 18 or 20. Polypeptides within the scope of b)

As discussed supra, it is often advantageous to reduce the length of a polypeptide, provided that the resultant reduced length polypeptide still has a desired activity or can give rise to useful antibodies. Feature b) of the eleventh aspect of the present invention therefore covers fragments of polypeptides a) above.

The skilled person can determine whether or not a particular fragment has activity using the techniques disclosed above.

"Fragment" refers to a peptide or polypeptide comprising an amino acid sequence of at least 5 amino acid residues (preferably, at least 10 amino acid residues, at least 15 amino acid residues, at least 20 amino acid residues, at least 25 amino acid residues, at least 40 amino acid residues, at least 50 amino acid residues, at least 60 amino residues, at least 70 amino acid residues, at least 80 amino acid residues, at least 90 amino acid residues, at least 100 amino acid residues, at least 125 amino acid residues, at least 150 amino acid residues, at least 175 amino acid residues, at least 200 amino acid residues, or at least 250 amino acid residues) of the amino acid sequence of a). The fragment may or may not possess a functional activity of the polypeptide defined in a).

A polypeptide as defined herein may be useful as antigenic material, and may be used in the production of vaccines for treatment or prophylaxis of a disease through the regulation of apoptosis. Such material can be "antigenic" and/or "immunogenic". Generally, "antigenic" is taken to mean that the protein is capable of being used to raise antibodies or indeed is capable of inducing an antibody response in a subject. "Immunogenic" is taken to mean that the protein is capable of eliciting a protective immune response in a subject. Thus, in the latter case, the protein may be capable of not only generating an antibody response but, in addition, non-antibody based immune responses. It is well known that it is possible to screen an antigenic protein or polypeptide to identify epitopic regions, i.e. those regions which are responsible for the protein or polypeptide's antigenicity or immunogenicity. Methods well known to the skilled person can be used to test fragments for antigenicity. Thus, the fragments of the present invention may include one or more such epitopic regions or be sufficiently similar to such regions to retain their antigenic/immunogenic properties. Thus, for fragments according to the present invention the degree of identity is perhaps irrelevant, since they may be 100% identical to a particular part of a protein or polypeptide, homologue or derivative as described herein. The key issue may be that the fragment retains the antigenic/immunogenic properties of the polypeptide from which it is derived.

Fragments may possess at least a degree of the antigenicity/immunogenicity of the polypeptide from which they are derived.

In a twelfth aspect, the present invention provides the use of a polypeptide as defined in the eleventh aspect in the manufacture of a medicament for the treatment and/or prophylaxis of a disease through the regulation of apoptosis, wherein the medicament is a vaccine. The vaccine optionally comprises one or more suitable adjuvants. Examples of adjuvants well-known in the art include inorganic gels, such as aluminium hydroxide, and water-in-oil emulsions, such as incomplete Freund's adjuvant. Other useful adjuvants will be well known to the skilled person.

In yet further aspects, the present invention provides:

(a) the use of a polypeptide as defined herein in the preparation of an immunogenic composition, preferably a vaccine;

(b) the use of such an immunogenic composition in inducing an immune response in a subject; and

(c) a method for the treatment and/or prophylaxis of a disease through the regulation of apoptosis in a subject, or of vaccinating a subject against a disease involving apoptosis, which comprises the step of administering to the subject an effective amount of a polypeptide as defined herein, preferably as a vaccine. A thirteenth aspect provides a method of diagnosis of a disease involving apoptosis in a subject, the method comprising detecting and/or quantifying the amount of a polypeptide as defined above in a biological sample obtained from said subject.

Preferably, an antibody is used for detecting and/or quantifying the amount of a polypeptide as defined in the eleventh aspect of the invention in a biological sample obtained from said subject.

In one embodiment, binding of antibody in tissue sections can be used to detect aberrant polypeptide localisation or an aberrant level of polypeptide. In a specific embodiment, antibody to a polypeptide as defined herein can be used to assay a patient tissue for the level of the polypeptide where an aberrant level of polypeptide is indicative of a disease involving apoptosis. As used herein, an "aberrant level" means a level that is increased or decreased compared with the level in a subject free from the disease involving regulation of apoptosis or a reference level. If desired, the comparison can be performed with a matched sample from the same subject, taken from a portion of the body not affected by the disease involving regulation of apoptosis.

In one embodiment, tissue from a subject is analysed for quantitative detection of a polypeptide as defined in the tenth aspect, wherein a change in abundance of the polypeptide in the tissue from the subject relative to tissue from a subject or subjects free from a disease involving apoptosis (e.g., a control sample or a previously determined reference range) indicates the presence of a disease involving apoptosis.

Suitable immunoassays include, without limitation, competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays and protein A immunoassays. In another embodiment, symptoms of a disease involving apoptosis, maybe ameliorated by decreasing the level or activity of a polypeptide as defined in the tenth aspect by using nucleic acid sequences of the first aspect in conjunction with well- known gene "knock-out," ribozyme or triple helix methods to decrease expression of the polypeptide. In this approach, ribozyme or triple helix molecules are used to modulate the activity, expression or synthesis of the nucleic acid sequence, and thus to ameliorate the symptoms of the disease involving apoptosis. Such molecules may be designed to reduce or inhibit expression of the identified genes. Techniques for the production and use of such molecules are well known to those of skill in the art.

Endogenous polypeptide expression can also be reduced by inactivating or "knocking out" an identified gene as defined herein, or the promoter of such a gene, using targeted homologous recombination (e.g., see Smithies, et ah, 1985, Nature 317:230- 234; Thomas & Capecchi, 1987, Cell 51:503-512; Thompson et al., 1989, Cell 5:313- 321; and Zijlstra et ah, 1989, Nature 342:435-438). For example, an identified gene encoding a non-functional polypeptide (or a completely unrelated DNA sequence) flanked by DNA homologous to the endogenous gene (either the coding regions or regulatory regions of the gene encoding the polypeptide) can be used, with or without a selectable marker and/or a negative selectable marker, to transfect cells that express the target gene in vivo. Insertion of the DNA construct, via targeted homologous recombination, results in inactivation of the target gene. Such approaches are particularly suited in the agricultural field where modifications to ES (embryonic stem) cells can be used to generate animal offspring with an inactive target gene (e.g., see Thomas & Capecchi, 1987 and Thompson, 1989, supra). However this approach can be adapted for use in humans provided the recombinant DNA constructs are directly administered or targeted to the required site in vivo using appropriate viral vectors.

Alternatively, and as discussed above, RNA interference (RNAi) may be used to silence or inhibit expression of a nucleic acid molecule of the invention. In a further aspect, the present invention provides an antibody which binds to at least one polypeptide as defined in the eleventh aspect.

Preferably the antibody binds specifically to a polypeptide as defined in the eleventh aspect.

In a further aspect, the present invention provides the use of an antibody of the invention for screening for and/or diagnosis of a disease involving regulation of apoptosis.

In a further aspect, the present invention provides a method for the screening for and/or diagnosis of a disease involving apoptosis in a subject, which comprises detecting and/or quantifying the amount of a polypeptide as defined in the eleventh aspect in a biological sample obtained from said subject using an antibody of the invention.

In a further aspect, the present invention provides a method for the prophylaxis and/or treatment of a disease involving apoptosis in a subject, which comprises administering to said subject a therapeutically effective amount of an antibody of the invention.

In a yet further aspect, the present invention provides the use of an antibody of the invention in the preparation of a medicament for use in the prophylaxis and/or treatment of a disease involving apoptosis.

Preferred antibodies bind specifically to polypeptides of the present invention so that they can be used to purify and/or inhibit the activity of such polypeptides. The antibodies may be monoclonal or polyclonal.

Thus, the polypeptide of the present invention may be used as an immunogen to generate antibodies which immunospecifically bind such an immunogen. Antibodies of the invention include, but are not limited to polyclonal, monoclonal, bispecific, humanised or chimeric antibodies, single chain antibodies, Fab fragments and F(ab')₂ fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above. The term "antibody" as used herein refers to immunoglobulin molecules and immunologically-active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that specifically binds an antigen. The immunoglobulin molecules of the invention can be of any class (e.g., IgG, IgE, IgM, IgD and IgA) or subclass of immunoglobulin molecule.

In the production of antibodies, screening for the desired antibody can be accomplished by techniques known in the art, e.g. ELISA (enzyme-linked immunosorbent assay). For example, to select antibodies which recognise a specific domain of a polypeptide used in the invention, one may assay generated hybridomas for a product which binds to a polypeptide fragment containing such domain. For selection of an antibody that specifically binds a first polypeptide homologue but which does not specifically bind to (or binds less avidly to) a second polypeptide homologue, one can select on the basis of positive binding to the first polypeptide homologue and a lack of binding to (or reduced binding to) the second polypeptide homologue.

For preparation of monoclonal antibodies (mAbs) directed toward a polypeptide used in the invention, any technique which provides for the production of antibody molecules by continuous cell lines in culture may be used. For example, the hybridoma technique originally developed by Kohler and Milstein (1975, Nature 256:495-497), as well as the trioma technique, the human B-cell hybridoma technique (Kozbor et ah, 1983, Immunology Today 4:72), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et ah, 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof. The hybridoma producing the mAbs used in the invention may be cultivated in vitro or in vivo. In an additional embodiment of the invention, monoclonal antibodies can be produced in germ-free animals utilising known technology (PCT/US90/02545). The monoclonal antibodies include but are not limited to human monoclonal antibodies and chimeric monoclonal antibodies (e.g., human-mouse chimeras). A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a human immunoglobulin constant region and a variable region derived from a murine mAb. (See, e.g., U.S. Patent No. 4,816,567; and U.S. Patent No. 4,816397) Humanised antibodies are antibody molecules from non-human species having one or more complementarity determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule. (See, e.g., U.S. Patent No. 5,585,089).

Chimeric and humanised monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in WO 87/02671; EP-A-184,187; EP-A-171,496; EP-A-173,494; WO 86/01533; U.S. Patent No. 4,816,567; EP-A-125,023; Better et al, 1988, Science 240:1041-1043; Liu et al, 1987, Proc. Natl. Acad. Sd. USA 84:3439-3443; Liu et al, 1987, J. Immunol.

139:3521-3526; Sun et al, 1987, Proc. Natl. Acad. Sd. USA 84:214-218; Nishimura et al, 1987, Cane. Res. 47:999-1005; Wood et al, 1985, Nature 314:446-449; Shaw et al, 1988, J Natl. Cancer Inst. 80:1553-1559; Morrison, 1985, Science 229:1202-1207; Oi et al, 1986, Bio/Techniques 4:214; U.S. Patent 5,225,539; Jones et al, 1986, Nature 321:552-525; Verhoeyan et al (1988) Science 239:1534; and Beidler et al, 1988, J. Immunol. 141:4053-4060.

Completely human antibodies are particularly desirable for therapeutic treatment of human patients. Such antibodies can be produced using transgenic mice which are incapable of expressing endogenous immunoglobulin heavy and light chain genes, but which can express human heavy and light chain genes. The transgenic mice are immunised in the normal fashion with a selected antigen, e.g., all or a portion of a polypeptide used in the invention. Monoclonal antibodies directed against the antigen can be obtained using conventional hybridoma technology. The human immunoglobulin transgenes harboured by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg & Huszar (1995), Int. Rev. Immunol. 13:65-93. For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., U.S. Patent 5,625,126; U.S. Patent 5,633,425; U.S. Patent 5,569,825; U.S. Patent 5,661,016; and U.S. Patent 5,545,806. In addition, companies such as Abgenix, Inc. (Freemont, CA) and Genpharm (San Jose, CA) can be engaged to provide human antibodies directed against a selected antigen using technology similar to that described above.

Completely human antibodies which recognise a selected epitope can be generated using a technique referred to as "guided selection." hi this approach a selected non- human monoclonal antibody, e.g., a mouse antibody, is used to guide the selection of a completely human antibody recognising the same epitope. (Jespers et al. (1994) Bio/technology 12:899-903).

The antibodies used in the present invention can also be generated using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them. In a particular, such phage can be utilised to display antigen binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine). Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified with antigen, e.g., using labelled antigen or antigen bound or captured to a solid surface or bead. Phage used in these methods are typically filamentous phage including fd and Ml 3 binding domains expressed from phage with Fab, Fv or disulphide stabilised Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein. Phage display methods that can be used to make the antibodies used in the present invention include those disclosed in Brinkman et al, J. Immunol. Methods 182: 41-50 (1995); Ames et al, J. Immunol. Methods 184:177-186 (1995); Kettleborough et al, Eur. J. Immunol 24:952-958 (1994); Persic et al, Gene 187 9- 18 (1997); Burton et al, Advances in Immunology 57:191-280 (1994);. PCT/GB91/01134; WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Patent Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108.

As described in the above references, after phage selection, the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described in detail below. For example, techniques to recombinantly produce Fab, Fab' and F(ab')2 fragments can also be employed using methods known in the art such as those disclosed in WO 92/22324; Mullinax et al, BioTechniques 12(6):864-869 (1992); and Sawai et al, AJRI 34:26-34 (1995); and Better et al, Science 240:1041-1043 (1988).

Examples of techniques which can be used to produce single-chain Fvs and antibodies include those described in U.S. Patents 4,946,778 and 5,258,498; Huston et al, Methods in Enzymology 203:46-88 (1991); Shu et al, PNAS 90:7995-7999 (1993); and Skerra et al, Science 240:1038-1040 (1988).

The invention further provides for the use of bispecific antibodies, which can be made by methods known in the art. Traditional production of full length bispecific antibodies is based on the coexpression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (Milstein et al, 1983, Nature 305:537-539). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, which is usually done by affinity chromatography steps, is rather cumbersome, and the product yields are low. Similar procedures are disclosed in WO 93/08829, and in Traunecker et al, 1991, EMBO J. 10:3655-3659. According to a different and more preferred approach, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CHl) containing the site necessary for light chain binding, present in at least one of the fusions. DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism. This provides for great flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments when unequal ratios of the three polypeptide chains used in the construction provide the optimum yields. It is, however, possible to insert the coding sequences for two or all three polypeptide chains in one expression vector when the expression of at least two polypeptide chains in equal ratios results in high yields or when the ratios are of no particular significance.

In a preferred embodiment of this approach, the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. It was found that this asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides for a facile way of separation. This approach is disclosed in WO 94/04690. For further details for generating bispecific antibodies see, for example, Suresh et al, Methods in Enzymology, 1986, 121:210.

The invention provides for the use of functionally-active fragments, derivatives or analogues of the anti-polypeptide immunoglobulin molecules. "Functionally-active" means that the fragment, derivative or analogue is able to elicit anti-anti-idiotype antibodies (i.e., tertiary antibodies) that recognise the same antigen that is recognised by the antibody from which the fragment, derivative or analogue is derived. Specifically, in a preferred embodiment, the antigenicity of the idiotype of the immunoglobulin molecule maybe enhanced by deletion of framework and CDR sequences that are C-terminal to the CDR sequence that specifically recognises the antigen. To determine which CDR sequences bind the antigen, synthetic peptides containing the CDR sequences can be used in binding assays with the antigen by any binding assay method known in the art.

The present invention provides antibody fragments such as, but not limited to, F(ab')₂ fragments and Fab fragments. Antibody fragments which recognise specific epitopes may be generated by known techniques. F(ab')₂ fragments consist of the variable region, the light chain constant region and the CHl domain of the heavy chain and are generated by pepsin digestion of the antibody molecule. Fab fragments are generated by reducing the disulphide bridges of the F(ab')₂ fragments. The invention also provides heavy chain and light chain dimmers of the antibodies of the invention, or any minimal fragment thereof such as Fvs or single chain antibodies (SCAs) (e.g., as described in U.S. Patent 4,946,778; Bird, 1988, Science 242:423-42; Huston et al., 1988, Proc. Natl. Acad. ScL USA 85:5879-5883; and Ward et al., 1989, Nature 334:544-54), or any other molecule with the same specificity as the antibody of the invention. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide. Techniques for the assembly of functional Fv fragments in E. coli may be used (Skerra et al., 1988, Science 242:1038-1041).

In other embodiments, the invention provides fusion proteins of the immunoglobulins of the invention (or functionally active fragments thereof), for example in which the immunoglobulin is fused via a covalent bond (e.g., a peptide bond), at either the N- terminus or the C-terminus to an amino acid sequence of another protein (or portion thereof, preferably at least 10, 20 or 50 amino acid portion of the protein) that is not the immunoglobulin. Preferably the immunoglobulin, or fragment thereof, is covalently linked to the other protein at the N-terminus of the constant domain. As stated above, such fusion proteins may facilitate purification, increase half-life in vivo, and enhance the delivery of an antigen across an epithelial barrier to the immune system. The immunoglobulins used in the invention include analogues and derivatives that are either modified, i.e., by the covalent attachment of any type of molecule as long as such covalent attachment that does not impair immunospecific binding. For example, but not by way of limitation, the derivatives and analogues of the immunoglobulins include those that have been further modified, e.g., by glycosylation, acetylation, pegylation, phosphylation, amidation, derivatisation by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, etc.

Additionally, the analogue or derivative may contain one or more non-classical amino acids.

The foregoing antibodies can be used in methods known in the art relating to the localisation and activity of the polypeptides of the invention, e.g., for imaging or radioimaging these proteins, measuring levels thereof in appropriate physiological samples, in diagnostic methods, etc. and for radiotherapy.

The antibodies of the invention can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or by recombinant expression, and are preferably produced by recombinant expression technique.

Recombinant expression of antibodies, or fragments, derivatives or analogues thereof, requires construction of a nucleic acid that encodes the antibody. If the nucleotide sequence of the antibody is known, a nucleic acid encoding the antibody may be assembled from chemically synthesised oligonucleotides (e.g., as described in Kutmeier et ah, 1994, BioTechniques 17:242), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding antibody, annealing and ligation of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR. Alternatively, the nucleic acid encoding the antibody may be obtained by cloning the antibody. If a clone containing the nucleic acid encoding the particular antibody is not available, but the sequence of the antibody molecule is known, a nucleic acid encoding the antibody may be obtained from a suitable source (e.g., an antibody cDNA library, or cDNA library generated from any tissue or cells expressing the antibody) by PCR amplification using synthetic primers hybridisable to the 3' and 5' ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence.

If an antibody molecule that specifically recognises a particular antigen is not available (or a source for a cDNA library for cloning a nucleic acid encoding such an antibody), antibodies specific for a particular antigen may be generated by any method known in the art, for example, by immunising an animal, such as a rabbit, to generate polyclonal antibodies or, more preferably, by generating monoclonal antibodies. Alternatively, a clone encoding at least the Fab portion of the antibody may be obtained by screening Fab expression libraries (e.g., as described in Huse et ah, 1989, Science 246:1275-1281) for clones of Fab fragments that bind the specific antigen or by screening antibody libraries (See, e.g., Clackson et ah, 1991, Nature 352:624; Hane et ah, 1997 Proc. Natl. Acad. ScL USA 94:4937).

Once a nucleic acid encoding at least the variable domain of the antibody molecule is obtained, it may be introduced into a vector containing the nucleotide sequence encoding the constant region of the antibody molecule (see, e.g., WO 86/05807; WO 89/01036; and U.S. Patent No. 5,122,464). Vectors containing the complete light or heavy chain for co-expression with the nucleic acid to allow the expression of a complete antibody molecule are also available. Then, the nucleic acid encoding the antibody can be used to introduce the nucleotide substitution(s) or deletion(s) necessary to substitute (or delete) the one or more variable region cysteine residues participating in an intrachain disulphide bond with an amino acid residue that does not contain a sulphydryl group. Such modifications can be carried out by any method known in the art for the introduction of specific mutations or deletions in a nucleotide sequence, for example, but not limited to, chemical mutagenesis, in vitro site directed mutagenesis (Hutchinson et al., 1978, J Biol. Chem. 253:6551), PCR based methods, etc.

In addition, techniques developed for the production of "chimeric antibodies" (Morrison et al., 1984, Proc. Natl. Acad. ScL 81:851-855; Neuberger et al., 1984, Nature 312:604-608; Takeda et al, 1985, Nature 314:452-454) by splicing genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used. As described supra, a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine niAb and a human antibody constant region, e.g., humanised antibodies.

Once a nucleic acid encoding an antibody molecule has been obtained, the vector for the production of the antibody molecule may be produced by recombinant DNA technology using techniques well known in the art. Thus, methods for preparing the polypeptides used in the invention by expressing nucleic acid containing the antibody molecule sequences are described herein. Methods which are well known to those skilled in the art can be used to construct expression vectors containing an antibody molecule coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. See, for example, the techniques described in Sambrook et al. (1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) and Ausubel et al. (eds., 1998, Current Protocols in Molecular Biology, John Wiley & Sons, NY).

The expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody of the invention. The host cells used to express a recombinant antibody of the invention may be either bacterial cells such as Escherichia coli, or, preferably, eukaryotic cells, especially for the expression of whole recombinant antibody molecule. In particular, mammalian cells, such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for antibodies (Foecking et al., 198, Gene 45:101; Cockett et al, 1990, Bio/Technology 8:2).

A variety of host-expression vector systems may be utilised to express an antibody molecule of the invention. Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express the antibody molecule of the invention in situ. These include but are not limited to microorganisms such as bacteria (e.g., E. coli, B. siibtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces, Pichiά) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing the antibody coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3 cells) harbouring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter).

Li bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the antibody molecule being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions comprising an antibody molecule, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include, but are not limited, to the E. coli expression vector ρUR278 (Ruther et al, 1983, EMBO J. 2:1791), in which the antibody coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Lαouye & Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol. Chem. 24:5503-5509); and the like. pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to a matrix glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhidrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The antibody coding sequence may be cloned individually into non-essential regions (for example, the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example, the polyhedrin promoter). In mammalian host cells, a number of viral-based expression systems (e.g., an adenovirus expression system) may be utilised.

As discussed above, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein.

For long-term, high-yield production of recombinant antibodies, stable expression is preferred. For example, cells lines that stably express an antibody of interest can be produced by transfecting the cells with an expression vector comprising the nucleotide sequence of the antibody and the nucleotide sequence of a selectable (e.g., neomycin or hygromycin), and selecting for expression of the selectable marker. Such engineered cell lines may be particularly useful in screening and evaluation of compounds that interact directly or indirectly with the antibody molecule.

The expression levels of the antibody molecule can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol.3. (Academic Press, New York, 1987)). When a marker in the vector system expressing antibody is amplifiable, increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the antibody gene, production of the antibody will also increase (Grouse et ah, 1983, MoI. Cell. Biol. 3:257).

The host cell may be co-transfected with two expression vectors of the invention, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide. The two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides. Alternatively, a single vector may be used which encodes both heavy and light chain polypeptides. In such situations, the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, 1986, Nature 322:52; Kohler, 1980, Proc. Natl. Acad. ScL USA 77:2197). The coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.

Once the antibody molecule used in the invention has been recombinantly expressed, it may be purified by any method known in the art for purification of an antibody molecule, for example, by chromatography (e.g., ion exchange chromatography, affinity chromatography such as with protein A or specific antigen, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins.

Alternatively, any fusion protein may be readily purified by utilising an antibody specific for the fusion protein being expressed. For example, a system described by Janknecht et al. allows for the ready purification of non-denatured fusion proteins expressed in human cell lines (Janknecht et al., 1991, Proc. Natl. Acad. Sci. USA 88:8972-897). In this system, the gene of interest is subcloned into a vaccinia recombination plasmid such that the open reading frame of the gene is translationally fused to an amino-terminal tag consisting of six histidine residues. The tag serves as a matrix binding domain for the fusion protein. Extracts from cells infected with recombinant vaccinia virus are loaded onto Ni²⁺ nitriloacetic acid-agarose columns and histidine-tagged proteins are selectively eluted with imidazole-containing buffers.

In a preferred embodiment, antibodies of the invention or fragments thereof are conjugated to a diagnostic or therapeutic moiety. The antibodies can be used for diagnosis or to determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive nuclides, positron emitting metals (for use in positron emission tomography), and nonradioactive paramagnetic metal ions. See generally U.S. Patent No. 4,741,900 for metal ions which can be conjugated to antibodies for use as diagnostics according to the present invention. Suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; suitable prosthetic groups include streptavidin, avidin and biotin; suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride and phycoerythrin; suitable luminescent materials include luminol; suitable bioluminescent materials include luciferase, luciferin, and aequorin;

125 131 111 99 and suitable radioactive nuclides include I, I, In and Tc.

An antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described in U.S. Patent No. 4,676,980.

An antibody can be used as a therapeutic that is administered alone. As discussed herein, certain polypeptides, nucleic acid molecules and antibodies rind use in the treatment and/or prophylaxis of diseases through the regulation of apoptosis.

Thus, in a further aspect, the present invention provides a pharmaceutical formulation comprising at least one polypeptide, nucleic acid molecule or antibody of the invention, optionally together with one or more pharmaceutically acceptable excipients, carriers or diluents. Preferably, the pharmaceutical formulation is for use as a vaccine and so any additional components will be acceptable for vaccine use. In addition, the skilled person will appreciate that one or more suitable adjuvants may be added to such vaccine preparations.

The medicament will usually be supplied as part of a sterile, pharmaceutical composition which will normally include a pharmaceutically acceptable carrier. This pharmaceutical composition may be in any suitable form (depending upon the desired method of administering it to a patient).

It may be provided in unit dosage form, will generally be provided in a sealed container and may be provided as part of a kit. Such a kit would normally (although not necessarily) include instructions for use. It may include a plurality of said unit dosage forms.

The pharmaceutical composition may be adapted for administration by any appropriate route, for example by the oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual or transdermal), vaginal or parenteral (including subcutaneous, intramuscular, intravenous or intradermal) route. Such compositions may be prepared by any method known in the art of pharmacy, for example by admixing the active ingredient with the carrier(s) or excipient(s) under sterile conditions.

Pharmaceutical compositions adapted for oral administration may be presented as discrete units such as capsules or tablets; as powders or granules; as solutions, syrups or suspensions (in aqueous or non-aqueous liquids; or as edible foams or whips; or as emulsions).

Suitable excipients for tablets or hard gelatine capsules include lactose, maize starch or derivatives thereof, stearic acid or salts thereof.

Suitable excipients for use with soft gelatine capsules include for example vegetable oils, waxes, fats, semi-solid, or liquid polyols etc.

For the preparation of solutions and syrups, excipients which may be used include for example water, polyols and sugars. For the preparation of suspensions, oils (e.g. vegetable oils) may be used to provide oil-in-water or water in oil suspensions.

Pharmaceutical compositions adapted for transdermal administration maybe presented as discrete patches intended to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. For example, the active ingredient may be delivered from the patch by iontophoresis as generally described in Pharmaceutical Research, 3(6):318 (1986).

Pharmaceutical compositions adapted for topical administration may be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils. For infections of the eye or other external tissues, for example mouth and skin, the compositions are preferably applied as a topical ointment or cream. When formulated in an ointment, the active ingredient may be employed with either a paraffmic or a water-miscible ointment base. Alternatively, the active ingredient may be formulated in a cream with an oil-in-water cream base or a water-in-oil base. Pharmaceutical compositions adapted for topical administration to the eye include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent. Pharmaceutical compositions adapted for topical administration in the mouth include lozenges, pastilles and mouth washes. Pharmaceutical compositions adapted for rectal administration maybe presented as suppositories or enemas.

Pharmaceutical compositions adapted for nasal administration wherein the carrier is a solid include a coarse powder having a particle size for example in the range 20 to 500 microns which is administered in the manner in which snuff is taken, i.e. by rapid inhalation through the nasal passage from a container of the powder held close up to the nose. Suitable compositions wherein the carrier is a liquid, for administration as a nasal spray or as nasal drops, include aqueous or oil solutions of the active ingredient.

Pharmaceutical compositions adapted for administration by inhalation include fine particle dusts or mists which maybe generated by means of various types of metered dose pressurised aerosols, nebulisers or insufflators.

Pharmaceutical compositions adapted for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations.

Pharmaceutical compositions adapted for parenteral administration include aqueous and non-aqueous sterile injection solution which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation substantially isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. Excipients which may be used for injectable solutions include water, alcohols, polyols, glycerine and vegetable oils, for example. The compositions may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carried, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.

The pharmaceutical compositions may contain preserving agents, solubilising agents, stabilising agents, wetting agents, emulsifiers, sweeteners, colourants, odourants, salts (substances of the present invention may themselves be provided in the form of a pharmaceutically acceptable salt), buffers, coating agents or antioxidants. They may also contain therapeutically active agents in addition to the substance of the present invention.

Dosages of the polypeptide, nucleic acid or antibody used in of the present invention can vary between wide limits, depending upon the disease to be treated, the age and condition of the individual to be treated, etc. and a physician will ultimately determine appropriate dosages to be used. This dosage maybe repeated as often as appropriate. If side effects develop the amount and/or frequency of the dosage can be reduced, in accordance with normal clinical practice.

In the context of the present invention, the biological sample can be obtained from any source, such as a serum sample or a tissue sample. The biological sample may be obtained using a small intestinal biopsy.

The invention also provides diagnostic kits, comprising an antibody against a polypeptide as defined in the eleventh aspect. In addition, such a kit may optionally comprise one or more of the following: (1) instructions for using the antibody for diagnosis, prognosis, therapeutic monitoring or any combination of these applications; (2) a labelled binding partner to the antibody; (3) a solid phase (such as a reagent strip) upon which the antibody is immobilised; and (4) a label or insert indicating regulatory approval for diagnostic, prognostic or therapeutic use or any combination thereof. If no labelled binding partner to the antibody is provided, the anti- polypeptide antibody itself can be labelled with a detectable marker, e.g., a chemiluminescent, enzymatic, fluorescent, or radioactive moiety.

The invention also provides a kit comprising a nucleic acid probe capable of hybridising to RNA as defined in the first aspect. The invention provides methods for identifying agents, candidate compounds or test compounds that bind to a polypeptide as defined in the eleventh aspect or have a stimulatory or inhibitory effect on the expression or activity of a polypeptide as defined herein.

The compounds of the invention include but are not limited to any compound, e.g., a small organic molecule, protein, peptide, antibody, nucleic acid, etc. that restores the profile towards normal with the proviso that such compounds or treatments include, but are not limited to, taxol, cyclophosphamide, tamoxifen, and doxorubacin.

A further aspect provides a method for identifying agents (e.g. drug candidates or test compounds) that have an inhibitory effect on the expression of a nucleic acid molecule of the first aspect, or activity of a polypeptide of the eleventh aspect, comprising contacting a nucleic acid molecule of the first aspect, or a polypeptide of the eleventh aspect with a candidate agent, and determining if the agent inhibits expression of the nucleic acid molecule or the activity of the polypeptide

In a further aspect, the present invention provides a method of screening for an agent that interacts with a polypeptide of the present invention, the method comprising contacting a polypeptide of the invention with a candidate agent and determining whether the agent interacts with the polypeptide.

In a further aspect, the present invention provides the use of an agent identified using the above methods of screening in the manufacture of a medicament for the treatment and/or prophylaxis of a disease through the regulation of apoptosis.

In a yet further aspect, the present invention provides a method for the treatment and/or prophylaxis of a disease through the regulation of apoptosis, the method comprising inducing expression of one or more of the genes listed in Figure 27 in a subject. The disease may be cancer. Alternatively, the present invention in another aspect provides a method for the treatment and/or prophylaxis of a disease throught the regulation of apoptosis, the method comprising blocking and/or reducing the activity of one or more of the genes listed in Figure 27 in a subject. The disease may be an autoimmune disease or a neurodegenerative disorder.

The method may comprise decreasing expression of the genes using well-known gene "knock-out," ribozyme or triple helix methods to decrease expression of a polypeptide. Alternatively, endogenous polypeptide expression can also be reduced by inactivating or "knocking out" a gene, or the promoter of such a gene, using targeted homologous recombination (e.g., see Smithies, et al, 1985, Nature 317:230- 234; Thomas & Capecchi, 1987, Cell 51:503-512; Thompson et al, 1989, Cell 5:313- 321; and Zijlstra et al, 1989, Nature 342:435-438). RNA interference (RNAi) or antisense techniques may also be used to suppress expression of the genes.

The invention is concerned with the treatment of mammalian patients, and preferably human patients.

Although the invention is primarily described herein in relation to the treatment and/or prophylaxis of cancer through the regulation of apoptosis, the invention may equally be applied in treating and/or preventing diseases through the regulation of apoptosis including neurodegenerative disorders such as Alzheimer's disease, vascular dementia, frontotemporal dementia and Cortical Lewy Body Disease Parkinson's Disease and Huntington's Disease, and autoimmune diseases.

Preferred features of each aspect of the invention are as for each of the other aspects mutatis mutandis. The prior art documents mentioned herein are incorporated to the fullest extent permitted by law.

Examples The invention will now be described with reference to the following examples, which should not in any way be construed as limiting the scope of the present invention. The examples refer to the figures in which:

Figure 1 provides the nucleic acid sequence of the gene C22ORF23.

Figure 2 provides the amino acid sequence encoded by the nucleic acid sequence of Figure 1.

Figure 3 provides the nucleic acid sequence of the gene XBPl.

Figure 4 provides the amino acid sequence encoded by the nucleic acid sequence of Figure 3.

Figure 5 provides the nucleic acid sequence of the gene CSTB.

Figure 6 provides the amino acid sequence encoded by the nucleic acid sequence of Figure 5.

Figure 7 provides the nucleic acid sequence of the gene MGC_5439.

Figure 8 provides the amino acid sequence encoded by the nucleic acid sequence of Figure 7.

Figure 9 provides the nucleic acid sequence of the gene STK-3.

Figure 10 provides the amino acid sequence encoded by the nucleic acid sequence of Figure 9.

Figure 11 provides the nucleic acid sequence of the gene ACOl . Figure 12 provides the amino acid sequence encoded by the nucleic acid sequence of Figure 11.

Figure 13 provides the nucleic acid sequence of the gene AFlQ.

Figure 14 provides the amino acid sequence encoded by the nucleic acid sequence of Figure 13.

Figure 15 provides the nucleic acid sequence of the gene CCBP2.

Figure 16 provides the amino acid sequence encoded by the nucleic acid sequence of Figure 15.

Figure 17 provides the nucleic acid sequence of the gene LOC134285.

Figure 18 provides the amino acid sequence encoded by the nucleic acid sequence of Figure 17.

Figure 19 provides the nucleic acid sequence of the gene EXOC7.

Figure 20 provides the amino acid sequence encoded by the nucleic acid sequence of Figure 19.

Figure 21 is an overview of the design and use of a high-density cell-based microarray. a. Representative agarose gel image of plasmids prepared from 2,976 MGC (DRAT) clones, b. Array was designed such that each clone was printed in quadruplicate (yellow and red squares) surrounded by columns of GFP vector (green squares). The position of GFP -tagged positive control genes is shown by small white boxes, c. 1,959 plasmids in 0.3% gelatin were printed on to a glass slide in to form an array with 9,888 features. The image is of an array scanned directly after printing

(Agilent microarray scanner), d. An array cultured with HEK293T cells and scanned with a fluorescent imager (GE Healthcare, Typhoon) to show lines of GFP positive cells, e. Arrays were subjected to a functional assay to detect changes in the cell after over-expression of proteins. The image is of TUNEL positive cells, scale bar = 10 μm.

Figure 22 illustrated the percentage of apoptotic cells after over-expression of the 10 pro-apototic genes, STS treatment and mock transfection in 6 well plate CASP3 assays.

Figure 23 shows ACOl, STK3 and XBPl expression following transfection in six- well plates. ACOl, STK3 and XBPl probe set PvMA normalised signal intensities were plotted over the time course for each combined sample. Results for replicate samples were averaged, error bars indicate the individual replicate measurements.

Figure 24 provides Venn diagrams prepared with the list of differentials (genes that appeared in more than two sample comparisons only). IT and JD, number of genes increased or decreased in expression respectively, compared to the mock transfection control at that time point. Venn, diagrams, 12 hours (red), 24 hours (blue), 48 hours (white). Boxes; No. genes - gene number increasing or decreasing at that time point, % I+D - percentage of genes increasing or decreasing at that time point.

Figure 25 provides a gene tree prepared with the list of 997 differentially expressed transcripts (genes that appeared in more than two sample comparisons only). The tree was generated using the Spearman correlation algorithm within GeneSpring. Expression is shown in fold change compared to the appropriate mock transfection control. Red - genes increased in expression; Green - genes decreased in expression; Black - unchanged.

Figure 26 shows how RMA normalised data was used to prepare a list of differentially expressed genes by comparing replicate data using an ANOVA analysis from each of the four test conditions (three over-expressed genes plus STS treatment) at each time point with the appropriate negative mock transfected control. Overall, 3,791 gene transcripts were observed to be significantly differentially expressed in at least one of the 12 comparisons. To minimise the false discovery rate only the 997 gene transcripts that showed a fold change greater than 1.4 and appeared in more than two of the 12 individual comparisons were analysed further (shown here).

Figure 27 shows the Apoptosis Differentials List. Genes from Differentials Lists that were present in the University of Michigan list of apoptosis regulators and apoptosis GO ontologies (GO genes indicated in light yellow if additional to the Univeristy of Michigan list). Genes were ranked depending on the number of times they occurred in the 12 time course samples compared to the negative mock transfection control at that time point. Affymetrix probe sets for the same gene were grouped together. Dark blue = genes decreased in expression by 2-fold or more. Light blue = genes decreased in expression between 1.4 and 2 fold. Dark pink = genes increased in expression 2 fold or over. Light pink = genes increased in expression between 1.4 and 2 fold. White = no change in expression compared to the negative control. APOP red = when over expressed, genes increase apoptosis according to the literature. APOP green = when over expressed, genes decrease apoptosis according to the literature. APOP white = no confirmation via literature whether an increase or decrease in apoptosis is caused by the gene change in expression. EXPT = consequent action in this experiment dependent on whether gene expression is increased or decreased. Red = increases apoptosis. Green = decreases apoptosis. White = No confirmation via literature of apoptotic effect therefore unable to deduce role in this experiment. If genes only occurred in one sample at one time point, they were only included if the fold change compared to the appropriate mock transfection control was more than 1.6.

Figure 28 shows the ACOl over-expression effect on apoptotic pathway. Adapted from the KEGG apoptotic pathway (light blue) and BD Biosciences apoptotic pathway (light orange). Genes coloured red potentially increase apoptosis, genes coloured green potentially decrease apoptosis dependent upon their expression (red and green genes may be increased or decreased in expression, see Figure 27). White writing indicates the changed expression in the gene was only observed in ACOl . Figure 29 STK3 over-expression effect on apoptotic pathway. Adapted from the KEGG apoptotic pathway (light blue) and BD Biosciences apoptotic pathway (light orange). Genes coloured red potentially increase apoptosis, genes coloured green potentially decrease apoptosis dependent upon their expression (red and green genes may be increased or decreased in expression, see Figure 27. White writing indicates the changed expression in the gene was only observed in STK3).

Figure 30 XBPl over-expression effect on apoptotic pathway. Adapted from the KEGG apoptotic pathway (light blue) and BD Biosciences apoptotic pathway (light orange). Genes coloured red potentially increase apoptosis, genes coloured green potentially decrease apoptosis dependent upon their expression (red and green genes may be increased or decreased in expression, see Figure 27. White writing indicates the changed expression in the gene was only observed in XBPl).

Figure 31 STS over-expression effect on apoptotic pathway. Adapted from the KEGG apoptotic pathway (light blue) and BD Biosciences apoptotic pathway (light orange). Genes coloured red potentially increase apoptosis, genes coloured green potentially decrease apoptosis dependent upon their expression (red and green genes may be increased or decreased in expression, see Figure 27. White writing indicates the changed expression in the gene was only observed in STS).

Figure 32 summarises the effects of gene over-expression on the mRNA levels for genes associated with apoptotic pathways. Central pathway has been adapted from the KEGG apoptotic pathways (light blue) and BD Biosciences apoptotic pathway (light orange). A gene is shown on the pathway if present in at least one of the time points in at least three of the 4 individual treatments. Arrows within the gene mRNA boxes indicate genes with increased or decreased expression. Genes coloured red potentially increase apoptosis, genes coloured green potentially decrease apoptosis according to the literature.

Figure 33 illustrates the transfection efficiencies of eGFP-Cl in Example 2. Figure 34 illustrates the percentage of apoptosis in cells at 54 hours.

Figure 35 illustrates apoptosis at 48 and 60 hours in HEK293T cells.

Figure 36 illustrates apoptosis in HEK293T cells. Blue DAPI stained nuclei and CASP3 antibody with green fluorescence from Alexa488 fluor secondary antibody indicating apoptotic bodies. Bar, 100 μm.

Figure 37 illustrates apoptosis in HeLa cells. Blue DAPI stained nuclei and CASP3 antibody with green fluorescence from Alexa488 fluor secondary antibody indicating apoptotic bodies. Bar, 100 μm.

Figure 38 illustrates apoptosis in HEPG2 cells. Blue DAPI stained nuclei and CASP3 antibody with green fluorescence from Alexa488 fluor secondary antibody indicating apoptotic bodies. Bar, 100 μm.

Figure 39 illustrates apoptosis in OVCAR3 cells. Blue DAPI stained nuclei and CASP3 antibody with green fluorescence from Alexa488 fluor secondary antibody indicating apoptotic bodies. Bar, 100 μm.

Figure 40 illustrates apoptosis in HUH7 cells. Blue DAPI stained nuclei and CASP3 antibody with green fluorescence from Alexa488 fluor secondary antibody indicating apoptotic bodies. Bar, 100 μm.

Figure 41 illustrates apoptosis in PANCl cells. Blue DAPI stained nuclei and CASP3 antibody with green fluorescence from Alexa488 fluor secondary antibody indicating apoptotic bodies. Bar, 100 μm.

Example 1 A cell-based microarray technology was used to screen for novel pro-apoptotic genes. The activity of a number of the genes identified was then characterised, following the transcriptional response of cell cultures undergoing increased levels of apoptosis due to the over-expression of the transgene.

Cell-based microarray technology was first described by Ziauddin and Sabatini in 2001 (Ziauddin and Sabatini 2001) for use in performing high throughput transfection studies. The technique entails printing the full-length ORF of genes inserted into an expression vector onto a glass microscope slide to form a microarray. The array is treated with transfection reagent and cells grown over the top of the array until confluent. Cells growing in the vicinity of the spots of packaged genes undergo transfection and the encoded protein is over-expressed. Arrays can then be examined for alterations in cellular function, as manifested in localised changes to the cells' biochemistry or morphology. If the expression vector contains a 'tag', the sub-cellular localisation of the protein can also be analysed (Palmer and Freeman 2004; Ziauddin and Sabatini 2001). Due to the techniques potential for high throughput analyses and economy of reagents, cell-base-d microarrays have now been adopted by a number of groups for a variety of applications. Cell-based microarray transfection studies have been used to discover new members of signalling pathways (Webb et al. 2003), to identify novel G-protein coupled receptor (GPCR) targets, (Mishina et al. 2004) and to screen single-chain antibody fragments (Delehanty et al. 2004). Comprehensive reviews on cell-based microarrays are available elsewhere (Palmer and Freeman 2005; Wheeler et al. 2005).

Whilst in principle cell-based microarrays provide a powerful platform for performing high throughput transfection screens, to date no studies have reported the use of high- density arrays and analyses have tended to focus on a relatively small number of genes. One factor that has limited the use of the technology to date has been the availability of suitable clone sets that contain tagged full length ORF 's in mammalian expression vectors, as described in the original paper (Ziauddin and Sabatini 2001). Such clone collections are beginning to become available from commercial sources, but for most their use is prohibited by their expense and restrictions on their use. In a previous study (Palmer and Freeman 2004), the use of GFP tagged genes in Gateway expression vectors in the fabrication of reverse transfection arrays was explored. Whilst this work demonstrated the utility of using tagged clones in visualising the sub-cellular localisation of the transfected protein, it also highlighted certain limitations with this approach. For example, apart from the considerable expense and time involved in sub-cloning genes into the Gateway cloning system, there is the possibility of introducing errors into the ORF during the initial PCR of the cDNA insert. It has also now been demonstrated that tagging a gene can cause the protein to mis-localise and therefore disrupt the function of the native protein.

In the present invention, a strategy was adopted for the construction of a high-density cell-based microarray using human clones from the Mammalian Gene Collection (MGC) (Gerhard et al. 2004; Strausberg et al. 2002). There are currently a non- redundant set of over 13,000 sequence verified, full length ORF human clones in the MGC collection. A high density reverse transfection array containing plasmid DNA from 1,959 of these clones has been constructed, with each clone printed in quadruplicate. A GFP vector (pEGFP-Cl) was also printed to act as a transfection control and also to provide a positional address for the untagged MGC clones.

Therefore, an approach to the construction of high-density cell-based microarrays was developed in order to screen for proteins that induce apoptosis when over-expressed.

METHODS Clone purification

MGC clones (Gerhard et al. 2004; Strausberg et al. 2002) in IRAT plates 1-21 and 36- 45 were purchased from MRC geneservice (http://www.geneservice.co.uk/home/). Replicate working plates were prepared by adding 1 μl of HRAT plate clones to 2X TY media containing 8% glycerol (Sigma, Gillingham, Dorset, UK) and 50 μg/ml ampicillin (Sigma), grown at 37⁰C overnight and stored at -2O⁰C. Clones from each IRAT plate were grown and purified four times. Clones were grown by adding 10 μl of the working plate clones to 1 ml 2X TY media containing 50 μg/ml ampicillin and grown for 26 h at 37⁰C in a shaking incubator at 320 rpm. Clones were purified as described in the MultiScreeng₆ PLASMDD plate kit protocol (Millipore, Watford, UK).

pEGFP-Cl (Clontech, Cowley, Oxford, UK) was propagated as described in the

DH5cϋE. coli kit protocol, (Invitrogen, Paisley, UK), glycerol stocks were prepared from 850 μl of the culture and 150 μl of glycerol and stored at -7O⁰C. 10 μl of pEGFP-Cl glycerol stock was added to 5 ml 2X TY media with 100 μg/ml kanamycin (Sigma) and 10 μl glycerol stocks of CXADR, MARKLl, TGIF, CDK9, NFDB, IL17BR, TNFRSFlOB Gateway C-terminal GFP destination vector pcDNA- DEST47 (Invitrogen) prepared as described previously (Palmer and Freeman 2004) were added to 5 ml 2X TY media with 100 μg/ml ampicillin and grown in a shaking incubator at 37⁰C for 8 h. The pEGFP-Cl was transferred to flasks containing 100 ml 2X TY media with 100 μg/ml kanamycin and pcDNA-DEST47 and pCMV-SPORT6 clones were transferred to flasks containing 100 ml 2X TY media with 100 μg/ml ampicillin and grown at 37⁰C for 16 h in a shaking incubator, the clones were purified as described in the plasmid midiprep kit protocol (Qiagen, Crawley, West Sussex, UK).

IRAT clones for the 10 apoptotic inducing genes; XBPl, CSTB, MGC5439, STK3, C22ORF23, ACOl, AFlQ, CCBP2, LOC134285 and EXOC7 were streaked out on 2X TY agar plates containing 100 μg/ml ampicillin and incubated overnight at 37⁰C. A single colony was picked from each. XBPl, CSTB, MGC5439, STK3 and C22ORF23 were grown up and purified as the pcDNA-DEST47 clones described above. ACOl, AFlQ, CCBP2, LOC134285 and EXOC7 were grown up in 5 ml 2X TY media with 100 μg/ml ampicillin at 37⁰C for 12-16 h with vigorous shaking and plasmids were prepared as described in the Wizard miniprep kit protocol (Promega, Southampton, UK). The 96-well Picogreen dsDNA Quantitation Kit (Molecular probes, Paisley, UK) and the Cytofluor 4000 with Cytofluor software (Applied Biosystems, Warrington, UK) and Cytocalc (Applied Biosystems) were used to quantify 96-well miniprep purified IRAT clones. Concentrations were adjusted to a 200 ng/ml control. A Jenway Spectrophotometer (Genova Lifescience, UK) was used to measure all other clone purifications. AU samples were electrophoresed on 1% ethidium bromide agarose gels.

Re-array plates for reverse transfection Plasmid DNA from IRAT plates 1-21 and 36-45 with concentrations over 2 μg were re-arrayed into 21 fresh 96-well flat bottomed plates. The DNA was dried via a heated vacuum centrifuge (Eppendorf, Westbury, US), H₂O was added back to a 0.5 μg/μl concentration and the plates were stored at -2O⁰C.

Printing of high-density cell-based microarrays

1 μg IRAT plasmids, pEGFP-Cl vector (Clontech) and TNFRSFlOB, IL17BR, NFIB, CDKNlB, NFIL3 and PTPNl 1 in the pcDNA-DEST47 vector (Invitrogen) were made up to 30 μl with 0.3% gelatin (Sigma) and transferred into 384-well plates. Clones were printed onto poly-lysine slides (Sigma) using a Biorobotics MicroGrid II Microarrayer (Biorobotics, Cambridge, UK) with a 48 pin head (Quill pins 2500,

Biorobotics). The microarrayer was programmed to print 16 single pre-spots, 12 spots with a 25 ms dwell with two 7 sec, 6O⁰C wash and dries between picking up clones. Printed arrays were scanned with Cy3 and Cy5 lasers on a DNA microarrayer scanner (Agilent Technologies, West Lothian, UK), the resultant .tif files were split using Tiff splitter A5.1.1.1 (Agilent Technologies) and viewed with the software program Image Analysis A.5.1.1 (Agilent Technologies). Each spot was -140 μm in diameter. Arrays were stored desiccated at 4⁰C.

Reverse transfection 16 μl Enhancer (Effectene transfection reagent kit, Qiagen) and 150 μl EC buffer (Qiagen) per array were incubated at RT for 5 min, 25 μl Effectene (Qiagen) was added and the total volume pipetted onto the array. Parafϊlm (Teklab, Durham, UK) was cut to the size of the slide was lowered onto the array and the transfection reagent incubated on the array at RT for 20 min. The parafϊlm and transfection reagent was removed, and arrays were placed in a 10 x 10 cm square dish (BD biosciences,

Cowley, Oxford, UK). Human embryonic kidney (HEK293T) cells were grown and maintained in 500 ml DMEM with 0.11 g/1 NA PYR with pyroxidine containing 50 ml FCS, 100 U/ml penicillin, 100 μg/ml streptomycin (Invitrogen) at 37⁰C and 5% CO₂. 1 x 10⁷ HEK293T cells were incubated at 37⁰C, 5% CO₂ for 24 h before reverse transfection, after 24 h, 1 x 10⁷ cells in a total of 20 ml culture medium were carefully poured into the 10 x 10 cm dish and incubated at 37⁰C, 5% CO₂ for 40 h.

6-well plate transfections

22 mm coverslips (VWR) were coated with 0.01% poly-1-lysine solution (Sigma) and placed in 6-well plates. The 79 positive clones from the reverse transfection screen were added in Effectene transfection reagent (Qiagen) according to manufacturer's recommendations to 2 x 10⁵ HEK293T cells and incubated for 40 h. Cover slips were removed and the TUNEL assay performed (see below). For the time course, cells were seeded into 5 wells per treatment/time point, then the 10 verified positive apoptotic IRAT clones, a negative control (transfection reagent only) and a positive control (final concentration 1 μm staurosporine (STS)) were added to the cells for 12, 24, 36, 48 and 60 h, then the TUNEL and cleaved CASP3 assay applied (see below). Cells were removed and frozen from three wells per treatment/condition for the time course and stored at -7O⁰C for subsequent Affymetrix microarray sample preparation.

TUNEL and cleaved CASP3 assays For the TUNEL assay, cells were fixed with 1 % paraformaldehyde (38% paraformaldehyde (VWH, Dorset, UK), diluted with PBS) for 10 min and the protocol followed as described in the Apoptag Apoptosis Detection System kit protocol (Flowgen, Nottingham, UK). 200 μl TdT enzyme/reaction buffer, anti-digoxigenin antibody/blocking solution and blocking solution were added per cell-based microarray and 50 μl per 6-well plate coverslip.

For the cleaved CASP3 assay, cells were fixed with 3.8% paraformaldehyde for 20 min and the protocol followed as described for the cleaved caspase-3 (Asp 175) antibody with fluorescein conjugate (Cell Signalling Technology, Beverly, Massachusetts, USA). 50 μl of diluted cleaved CASP3 antibody was added per 6-well plate coverslip, a circle of parafilm (Teklab, Durham, UK) was applied to ensure even coverage of antibody and incubated shielded from light at 4⁰C for 8 h. Cell visualisation and counting positive cell fluorescence

A drop of mounting medium containing DAPI stain (Vector, Peterborough, UK) was applied to a glass slide coverslip (Agilent) for the cell-based microarrays or to the 6- well plate coverslip and lowered onto the cell-based microarray or a standard glass microscope slide for the 6-well plate coverslips (Amersham Biosciences, Buckinghamshire, UK). Fluorescence was visualised with an Eclipse E800 microscope (Nikon, Kingston Upon Thames, UK) with a confocal attachment (BioRad, Hemel Hempstead, UK) or using a Typhoon scanner (Amersham Biosciences). Reverse transfection positives were recorded as a quadruplicate clone patch with one or more fluorescent cells. Each cell-based microarray was scored twice and the genes were ranked in Excel according to the number of positives. For the TUNEL and cleaved CASP3 6-well plate assays, cells were scored as positive if clear fluorescent apoptotic bodies were observed in or near the cells. The number of cells in patches of cleared cells were estimated and included in the total percentage of apoptotic cells. Slides were stored at 4⁰C.

Statistical analysis of reverse transfection assays

The distribution of probabilities was calculated based on constant probability.

The number of measurable array positions for each plasmid was divided by the probability distribution and the number of positive's expected by chance for each plasmid was calculated. This number was compared to the actual number observed on the arrays.

To calculate the sensitivity of the arrays, the equation True Positive (TP)/(True Positive (TP) + False Negative (FN)) was used. TP was calculated as the number of positives in the 6-well assay follow up experiments. FN was calculated as the number of known tyrosine apoptosis inducing genes (determined using Gene Ontology) on the reverse transfection array that should have been positive, but were not found to be positive. The equation True Positive TP/True Positive (TP)+False Positive (FP) was used to calculate the Positive Predictive Value of the arrays. False Positive (FP) was calculated as the number of genes that were found to be positive in half of more of the reverse transfection arrays, but in the follow up 6-well plate assay were not found to be positive.

Sample preparation for Affymetrix GeneChip microarrays

RNA was extracted as described in the RNeasy mini kit protocol (Qiagen) from the three frozen time course samples for STK3, ACOl, XBPl transfections and the positive and negative control. cDNA was prepared using the T7-(dT)₂₄ primer

(5'ggccagtgaattgtaatacgactcatagggaggcgg-(dT)₂₄3') (SigmaGenosys) as described in the Superscript DoubleStranded cDNA synthesis kit (hwitrogen) from the two samples per condition with the highest RNA yields. cDNA was purified as described in the 1.5 ml Heavy Phase lock Gel (PLG) tubes protocol (Fisher Scientific, Loughborough, UK). Biotin labelled cRNA was prepared by in vitro transcription (rVT) from the cDNA as described in the Bioarray High Yield RNA transcript labelling kit (Enzo, New York, USA) and cleaned up as described in the RNeasy Mini kit (Qiagen). The cRNA was fragmented as described in the GeneChip Expression Analysis Technical Manual (http://www.affvmetrix.com/support/technical/manual/expression manual.affx).

RNA, cRNA and fragmented cRNA was checked on the Agilent 2100 Bioanalyzer using the RNA 6000 assay (Agilent, West Lothian, UK) with universal RNA as a control (Stratagene, Cambridge, UK) and RNA 6000 ladder (Ambion, Huntingdon, UK). cDNA and RNA was quantified with the Nanodrop (Labtech International Ltd, East Sussex, UK). The hybridisation cocktail was prepared and the GeneChips hybridised and washed as the GeneChip eukaryotic control kit (Affymetrix) and the GeneChip Expression Analysis Technical Manual.

(http://www.affyiΩetiix.com/support/technical/manual/expression_manual.affx) The GeneChips were scanned using the Scanner 3000 (Affymetrix) and the GeneChip Operating System (GCOS) (Affymetrix).

Data analysis The .eel data was normalised using GCOS (Affymetrix) and Robust Multichip Analysis (RMA) which is available as a programme called 'affy' from Bioconductor (Hornik 2004). The .tort GCOS and RMA normalised data were loaded into GeneSpring 7 (Silicon Genetics, Redwood City, California). A list of genes differentially expressed between each condition time point and the negative control sample at the relevant time point was prepared in 3 stages: 1. the GCOS normalised data was filtered to remove any gene not flagged as being present (P) in both replicate samples in at least one condition/time point. 2. The GCOS Present lists (1.) were used to filter the RMA normalised data. The cross gene error model (CGEM) and a parametric analysis of variance between groups (ANOVA) statistical test was applied and the false discovery rate was set at 0.05. 3. ANOVA lists (2.) were further filtered to remove any gene whose change was less than 1.4 fold were removed. The Differentials gene lists were entered into Excel and a pivot table of genes was prepared and ordered according to the number of times the genes appeared over the whole condition set (see Figure 26). Differentially regulated genes present in more than two conditions were visualised using Venn diagrams for each condition at each time point. The HUGO approved names were obtained for the University of Michigan list of apoptosis regulators (mohara 2004), combined with the lists of differentially changed genes and a list of apoptotic genes was obtained (see Figure 27). Genes from the apoptosis list with an apoptotic pathway recorded from the literature were mapped onto a simplified Kyoto Encyclopedia of Genes and Genomes (KEGG)/BD Biosciences pathway (Kanehisa and Goto 2000; Wilson 2002).

RESULTS Cell-based microarrays were constructed using plasmid DNA extracted from 1 ,959 human MGC clones in pCMV-SPORT6 vector, seven genes in the Gateway pcDNA- DEST47 C-terminal GFP fusion vector and a clone for GFP in the pEGFP-Cl vector. AU plasmids were printed in quadruplicate within grids (Figure 21b) except GFP, which was printed in columns to demarcate the grids and to act as a control for transfection. The arrays were incubated with HEK293T cells for approximately 40 hours, after which time clear grids of the transfection control pEGFP-Cl could be observed (Figure 2Id). Four separate arrays on two separate occasions were subjected to the Terminal Deoxynucleotide Transferase dUTP Nick End Label (TUNEL) assay, which allows the detection of free 3'-OH termini present in the fragmented DNA of apoptotic cells (Figure 2Ie). Replication of assays was found to be crucial for stringent screening as the ability to obtain a result for an individual gene was often compromised by imperfections in the cell monolayer, either as a result of differences in cell growth or post-culture treatment of the slides. A protein was scored as positive for apoptosis if one TUNEL-positive cell was observed over one or more of the four replicate spots. This was a deliberately low threshold for a positive readout. The small number of cells over each spot, and the fact that apoptotic cells could be cleared in the late stages of apoptosis meant that the signal for even a strong pro-apoptotic gene was potentially weak. 79 of the 1,959 genes (4%) represented on the array were scored positive in two of the 4 replicate assays and were therefore considered to be potentially pro-apoptotic.

The 79 genes were then scrutinised further by examining their activity when transfected in 6-well plates. The results from the 6-well experiment indicated that out of the 79 positives from the array, 69 (87.3%) showed no higher rates of cell death in cultures following transfection than the controls, with 10 (12.7%) being true positives (Table 1).

There were 11 genes on the array annotated as being involved in cell death according to gene ontology (GO) categorisation, only one of which was observed in the final list often positive genes. Assuming that all of these genes were capable of inducing apoptosis when over-expressed, the false negative rate of the array based assay can be estimated as 91%. From these numbers the sensitivity could be estimated as 60%, with an assay specificity of 12.1% (see Methods for details of calculations). However, a high false negative rate from this assay is not unexpected due to the relatively weak signal produced by the TUNEL assay. An additional problem when scoring the arrays manually was that it was necessary to view the arrays at a relatively low magnification (xlO objective) in order to maintain the positional address of the positive signals relative to the marker gene (GFP) transfections. At this resolution, weakly positive cells may have been overlooked. To determine the dynamics of apoptosis induction brought about by the over- expression of the 10 pro-apoptotic proteins, all were analysed in plate-based assays, using both the TUNEL and cleaved-CASP3 assays at 12, 24, 36, 48 and 60 hours following transfection. A mock transfection and a well-characterised inducer of apoptosis, staurosporine (STS) were assayed in parallel at each time point, as negative and positive controls respectively. Higher levels of apoptosis were observed in cell cultures over-expressing each of the 10 proteins and the positive (STS) control relative to the negative control, with levels of apoptosis rising at later time points.

Three of the 10 proteins, ACOl, STK3 and XBPl were chosen for further characterisation, by expression profiling the cell cultures over-expressing the genes. Following transfection, 12, 24 and 48 hour time points were chosen from the time- course study, in order to cover early, mid and late gene transcriptional events associated with apoptosis progression (Figure 23). Three separate cultures of cells were prepared for each time point, RNA was prepared from each and the two best quality RNA samples were used in further analysis. AU cell cultures were set up at the same time and transfections or STS treatment were performed at different points during culture, so all cell cultures were at the same growth phase when harvested. RNA samples were labelled and hybridised to the Affymetrix HG-Ul 33 GeneChip and the data was normalised using both the GeneChip Operating System (GCOS) and Robust Multivariate Analysis (RMA). AU raw and normalised data has been submitted to ArrayExpress (Dataset AcNo.: E-MEXP-421). The data was of high quality with the chips showing little variation in the quality control parameters recorded in the GCOS report file. Box plots also showed data distributions to be relatively similar across all chips. Non-supervised clustering of the data using the conditions tree function within GeneSpring (Agilent Technologies) suggested that there was little or no treatment or time-specific clustering of the data. ACOl, STK3 and XBPl appeared to be constitutively expressed within the cells as assessed by the GCOS software, with these genes being reported as being present (P) in all the samples at all the time points. XBPl appeared to have the highest constitutive expression i.e. gave the greatest signal, followed by ACOl, then STK3. The signal intensities for all three transcripts (ACOl, STK3 and XBPl) increased up to 41, 70 and 12 times, respectively, within cells where the genes were over-expressed compared with the average expression level in cell cultures in which the genes had not been transfected. Mock transfected and STS treated cells showed no rise in any of the three gene transcripts above constitutive levels. Despite apparent differences in the baseline level of expression of the three gene transcripts, all genes showed similar expression levels (signal) following transfection (Figure 23). These three genes exhibited the most significant fold changes in their expression following transfection relative of any of the genes represented on the array.

Data normalised using the RMA method, was then used to prepare a list of differentially expressed genes by comparing replicate data using an ANOVA analysis from each of the four test conditions (three over-expressed genes plus STS treatment) at each time point with appropriate data from negative (mock transfected) control. Overall, 3,791 gene transcripts were observed to be significantly differentially expressed in at least one of the 12 pair- wise comparisons. To refine the list of differentially expressed transcripts and minimise the false discovery rate, only transcripts that showed a fold change greater than 1.4 and appeared in three or more of the 12 individual pair-wise comparisons were analysed further. This 'Differentials List' (see Figure 26) contained 997 transcripts. Based on this list, Venn diagrams were prepared from the individual comparisons such that the overlap in differentially genes under or over-expressed across time-points could be assessed (Figure 24). Overall, there was considerable overlap in the genes found to be differentially expressed across time-points but there also appeared to be many that were specific to individual times. Of all the transcripts found to be differentially expressed following transfection, an average of 78.2% across all time points, were of observed to decrease in expression. However, the overall number of gene transcripts that increased and decreased in expression was similar following treatment with STS. There were more transcripts in the XBPl differentials lists than with other treatments and there were no obvious time point-specific trends in the number of gene transcripts increasing or decreasing in expression across the conditions. Supervised clustering of the 997 transcripts present in three or more of the individual comparisons was used to assess the behaviour of these genes across the four experimental conditions. This revealed a surprising uniformity in the behaviour of genes across different time points and treatments (Figure 25). Whilst individual genes showed some variation across the data set, genes that were up or down regulated in one comparison tended to show the same behaviour across all comparisons, although this change didn't always reach the level of statistical significance.

In an effort to associate the genes found to be differentially expressed in this experiment with apoptosis, we utilised the University of Michigan list of apoptosis regulators (Inohara, 2004). Of the 1,099 apoptosis regulators listed on this site 130 were present in the differentials list generated by this experiment. This 'Apoptosis

Differentials List' was therefore a list of differentially expressed genes that have been previously associated with apoptosis. The Apoptosis Differentials List in Figure 27 contained genes that belonged to the same family and there were also genes that had known interaction partners.

In an effort to piece this information together, proteins within the Apoptosis Differentials List were marked red if they were likely to be pro-apoptotic and green if increasing the likelihood of cell survival, based on information from previous studies. If genes could be categorised in this way, we endeavoured to place them on a simplified Kyoto Encyclopedia of Genes and Genomes (KEGG) apoptosis pathway to give an indication of the portion of the pathway in which they are likely to be active (see Figures 28-32).

The following discussion aims to provide an explanation as to why these genes might be present in Apoptosis Differentials List by highlighting their known associations with cell survival and apoptosis.

The MAP kinase family had five members present within the Apoptosis Differentials List, MAP2K2, MAP2K3, MAPK8IP3, MAP4K5 and MAPK8 (JNK). The first four all target MAPK8 (JNK), but through slightly different pathways. MAP2K2 directly effects ERK and MAP2K3 binds YOPJ which subsequently acts on ERK (Orth et al. 1999; Zheng and Guan 1993). MAPK8IP3 acts directly on ASKl via the SEK1/MKK4 pathway (Matsuura et al. 2002). MAP4K5 acts throught the GCKR/SAPK pathway (Tung and Blenis 1997).

There were two genes present from the TNF receptor superfamily- TNFRSF12A and TNFRSFl OB. TNFRSF12A has been shown to increase growth (Polek et al. 2003; Tanabe et al. 2003), so if withdrawn (as it would appear to be in this experiment) it might enhance apoptosis. TNFRSF12A was observed to be down regulated in expression at the 12 hour time point only in each sample so could be an early activator of apoptosis, as also concluded from the GO Cell Death pathway analysis. TNFRSFlOB over-expression instigates a caspase dependent apoptotic pathway via FADD (Walczak et al. 1997) and appears over-expressed in this experiment at 48 hours after STS treatment so could be a late indicator of apoptosis. Sabatini in the original reverse transfection cell-based microarray experiment observed that the cells on their reverse transfection array growing over TNFRSFlOB appeared stressed (Ziauddin and Sabatini 2001).

Protein tyrosine phosphatase family members PTP4A1, PTP4A2, PTPNl 3, PTPRF and PTPRS were present in the Apoptosis Differentials List. Over-expression of PTP4A1 and PTP4A2 can cause tumour growth (Gates et al. 1996; Zeng et al. 2003), PTP4A1 expression is increased in this study, therefore potentially promoting cell survival, but PTP4A2 is decreased, therefore potentially increasing the balance towards apoptosis. PTPRF is down-regulated in tumour tissue (Liu et al. 2003) and is down-regulated in this study and is therefore potentially acting to decrease apoptosis. PTPNl 3 inhibits FAS-induced apoptosis (Inizawa et al. 1996) and is up-regulated in this study, therefore again potentially acting to decrease apoptosis.

Frizzled family members, FZD7 and FZD8 were both present in the apoptosis Differentials List and increased in expression at similar time points. Frizzled (FZD) genes encode WNT receptors which transduce WNT signals to the beta-catenin-TCF pathway, the INK pathway or the Ca²⁺ pathway (Herin and Sheng 2002; Kirikoshi and Katoh 2002) TCF7L2 (from the Differentials Lists) targets FZD (Thorstensen and Lothe 2003) genes and is present at the same time points. BIRC family members BIRC4 and BIRC5 were also observed to alter in their expression. BIRC5 is an inhibitor of apoptosis and may counteract a default induction of apoptosis in G2/M phase, it inhibits CASP3 and 7 (Li et al. 1998). It is decreased in this study, therefore potentially increasing apoptosis. BIRC4 is increased in this study, it directly inhibits CASP3, 7 (Devereux et al. 1997) and 9 (Srinivasula et al. 2001). BIRC5 may be down regulated and therefore increasing apoptosis to counteract the apoptotic inhibitory effect of BIRC4.

There were many RING finger proteins whose expression was observed to change in these studys. The RING finger proteins play crucial roles in cell-cycle progression, differentiation, development, oncogenesis, signal transduction, apoptosis and are also essential components of the cellular ubiquitin-proteasome system, which removes misfolded proteins (Borden and Freemont 1996).

Interactors BAX, BAGl and BAD were decreased in expression at similar time points. BAG and BAD induce apoptosis via BCL2/X, BAG is also associated with BCL2, but prevents apoptosis (Takayama et al. 1995; Willis et al. 2003; Yang et al. 1995) and is therefore potentially promoting apoptosis in this study since it's own expression is decreased. Interactors UTRN and BCAP31 were also observed to change in their expression.

CASP8 activates the BCAP31 fragment which recruits UTRN causing the scission of mitochondria (Breckenridge et al. 2003; Chandra et al. 2004). UTRN gene expression was increased and BCAP31 decreased, although in different samples to UTRN.

Interactors SIRTl, SIRT2 and FOXO3A were all present in the Apoptosis

Differentials List, (Brunet et al. 2004), demonstrated that in mammalian cells, SIRTl has a dual effect on FOXO3 function: SIRTl increased FOXCβ's ability to induce cell cycle arrest and resistance to oxidative stress but inhibited FOXCβ's ability to induce cell death (Brunet et al. 2004). In this study, SIRTl is increased and FOXO3A decreased, appearing to support this theory. SIRT2 expression was also decreased although the significance of this observation is unknown. APP and APLP2 were observed increased in expression, APP is directly and efficiently cleaved by caspases (mostly CASP3) during apoptosis resulting in elevated amyloid beta formation. (Gervais et al. 1999). APP produces ABETA and AID, AID lowers cellular threshold to apoptosis and represses NOTCH dependent gene expression, cleaved APLP2 acts in a similar way to AID through CASP3 and 9

(Cowan et al. 2001; Scheinfeld et al. 2002). Meractors JUN, JUNB, JUND and FOS are all present and can form heterodimers and API complexes, they can however act independently in apoptosis as appears to be occurring in this study (Wang et al. 2000).

Figure 32 attempts to summarise this information by showing genes/pathways that appear to be central to apoptosis induction in the current study. In all conditions, many of the genes that could potentially increase apoptosis were associated with the MAPK8/CASP3 pathway. However, in the case of XBPl over-expression and STS treatment, the lists of apoptosis differentials also contained genes that could indicate pro-apoptotic signals were being induced in the death receptor pathways. UTRN and RAD21 were differentially expressed in cells over-expressing ACOl and STK3 and in STS treated cells, potentially indicating apoptosis induction via CASP7. Indeed CASP7 itself was up-regulated in STS treated cells. Genes involved in DNA fragmentation and phagocytosis occurred in all of the samples, except cells over- expressing STK3. SLIT2 might potentially be increasing apoptosis via CASP3 and CASP9 in every sample. A similar number of genes over all the samples are potentially increasing and decreasing apoptosis via P53. Differentially regulated gene transcripts appeared to be acting very similarly and NR4A1, EGRl SLIT2, CASP9, ADM, MADH7, JUN and TIMPl were differentially expressed in every transfection/treatment. Two genes, JUND and AMH were observed only in cells over- expressing ACOl, three genes, DAXX, DADl and BCAP 31 were observed only in cells over-expressing STK3, six gene transcripts, TRAF4, RACl, NME6, BAD, FYN and LRDD were observed only in cells over-expressing XBPl protein and eight genes MAX, PBEFl, TNFRSFlOB, CASP7, GULP, CHUK, CBL and E2F3 were observed only in STS treated cells. A strong time course specific variation in the regulation of most differentially expressed genes did not appear to be evident from the data. DISCUSSION

The aim of this study was to adopt a functional genomics approach to screen for human genes that induce apoptosis. In order to do this, the strategy required the construction of a high-density cell-based microarray. In a previous study, GFP-tagged genes in Gateway expression vectors were used to examine the sub-cellular localisation of proteins over-expressed by reverse transfection (Palmer and Freeman 2004). In common with other studies, it was found that gene-tagging can disrupt the normal sub-cellular localisation and therefore presumably the function of the protein. In addition, sub-cloning of the gene inserts has the potential to introduce errors in the ORF during vector construction and inserting large numbers of genes into Gateway constructs is both costly and time consuming. Therefore, for the present study it was decided to construct a high-density cell-based microarray by direct use of the readily available full-length MGC clones (Gerhard et al. 2004; Strausberg et al. 2002). This strategy was possible as many of the MGC clones are already in the pCMV-SPORT6 vector, which contains a CMV promoter to drive expression of the ORF in mammalian cells. Plasmid preparations were attempted for 2,976 MGC clones but we found that many did not yield enough/any product, despite repeated attempts. However, more than 2 μg plasmid DNA was recovered from 1,959 clones. In order to maximise the likelihood of observing the effects of over-expression on the cells covering arrays without compromising the array content, each of the 1,959 purified MGC clones was printed in quadruplicate onto a glass slide to form the array. After inclusion of control features, the array possessed 9,888 features in total. As such, this represents the largest cell-based reverse transfection microarray to date.

Following growth of HEK293T cells over the array, the TUNEL assay was used to detect genes which had induced cell death when over-expressed. The assay was repeated on four separate arrays. When proteins only positive in two or more of the four assays were taken into account, 79 of the 1,959 genes (4%) appeared to be potentially inducing cell death. For verification, these 79 genes were then transfected in 6-well plates. The results from the 6-well plate assay indicated that out of the 79 positives from the array, 10 (12.7%) were true positives (Table 1). This would indicate that the arrays gave a fairly high false positive rate. This in part could be due to the manual scoring of the array, where the readout from the TUNEL assay is weak. Employing automated high resolution scanning and image analysis tools however, would not only make scoring of cell-based arrays a lot easier, but potentially do much to improve the false discovery rates. The necessity for spot recognition software and the storage and analysis of the images present a considerable challenge. These issues are currently being explored and a microscope-based screening platform is being developed with automated sample preparation, image acquisition and data analysis (Starkuviene et al. 2004; Liebel et al. 2003). Improved tools for automated image analysis of cell-based arrays are also being developed elsewhere (http://cellprofiler.org).

The function, GO categories and localisations were obtained for the 10 apoptosis inducing proteins identified by this study (Table 1). STK3 was already known to be involved in Fas-mediated apoptosis and is cleaved/activated by CASP3 (Lee, et al., 2001). In addition, it is possible to hypothesise mechanisms of action for the other proteins identified. XBPl binds to the X-box of the HLA-DR-alpha promoter (MHC human class II gene) (Liou et al. 1990) and responds to accumulation of unfolded proteins in the ER (Yoshida et al. 2003). It is possible that this change may trigger apoptosis, although this has not been demonstrated previously. There is other evidence that XBPl may be linked to apoptosis. In an expression profiling study of murine mammary epithelial cells expressing conditionally active STAT3 (which provides an essential death signal for mammary epithelial cells following weaning), XBPl was highly up-regulated following STAT3 activation (Clarkson et al. 2005). CSTB maintains appropriate equilibrium between free cysteine proteases and their complexes (Lennon-Demenil et al. 2002). Cathepsins can be inhibited by CSTB and therefore this could prevent degradation of peptides and proteins which in turn could possibly act as a pro-apoptotic stimulus. ACOl represses ferritin and increases TFR translation (Yu et al. 1992), and its over-expression is therefore likely to cause a build up of free-iron within the cell. Previous studies have shown that increased levels of intracellular free-iron can induce apoptosis (Hirling et al. 1992). EXOC7 is a component of the exocyst complex involved in the docking of exocystic vesicles with fusion sites on the plasma membrane (Lipschutz and Mostov 2002) and potentially excessive removal of internal cell contents may cause apoptosis to occur. AFlQ and CCBP2 are both found in tumours, but no other information is available to allow speculation on a possible mode of action for their induction of apoptosis, the same is true of the relatively uncharacterised genes C22ORF23, MGC5439 and LOC134285.

In an attempt to ascertain the timing and strength of apoptosis, a time course transfection study using HEK293T cells was undertaken on all 10 genes, a positive control STS and a mock transfected negative control at 12, 24, 36, 48 and 60 hours following transfection. Staurosporine (STS) was used as the positive control as it is an apoptotic effector classically linked to caspase activation (Alves da Costa et al. 2002). As the TUNEL assay has been shown to potentially detect necrotic death in addition to apoptotic death (Duan et al. 2003), a cleaved CASP3 assay was performed on the cultures in addition to the TUNEL assay. STS and all 10 genes identified by the reverse transfection screen led to cultures in 6-well plates exhibiting between 40-70% TUNEL and cleaved CASP3 positive cells after 60 hours compared with only 3-4% in the mock transfection cultures.

Apoptosis has been studied extensively and core pathways and events are generally well established. The regulation of apoptotic cell death is a complex interplay between proteins that promote cell survival and those that promote cell death. It is widely thought that the processes that control the balance between the life and death of a cell are regulated exclusively at the post-transcriptional level. As a result few observations have been made of the transcriptome during this process, although those that have (Johnson et al. 2004) suggested that this would be a useful approach to further characterise the action of these genes.

STK3, ACOl and XBPl were selected over the other seven genes for expression profiling studies (Figure 22). For the expression profiling study, samples were taken at 12, 24 and 48 hours following transfection to observe the early, mid and late transcriptional events associated with apoptosis. ACOl, STK3 and XBPl were all found to be constitutively expressed within the cells but at different levels. Transcript levels (signal intensities) of XBPl, ACOl and STK3 rose to a maximum of 12, 41 and 70 times respectively, compared to the average signal from cells not transfected with the genes. It was interesting to note that despite differences in constitutive expression, all genes appeared to reach a similar level of up-regulation following transfection and that the changes in expression observed for these transcripts were the largest of all the transcripts represented on the array.

It was envisaged that the expression profiling experiments might reveal a transcriptional response in these cultures that would provide clues as to the mechanism by which the over-expression of these genes induces apoptosis. Overall, analysis of the microarray expression data indicated that the changes observed at all time points and across all conditions were relatively subtle. There was no strong tendency for the data to cluster according to treatment or time-point. The lists of differentially expressed transcripts of genes prepared by comparing each time point with its respective mock transfection control, showed many genes to be significantly changing their expression, but on the whole these changes were relatively small i.e. the majority of changes were less than 2-fold in magnitude. For each transfection experiment, many more gene transcripts were down-regulated in expression than up- regulated during apoptosis progression. A similar observation was also reported in a previous study of the transcriptional events associated with apoptosis following removal of cell survival factors from cultures of HUVEC cells (Johnson et al. 2004). However, with STS treatment the number of gene transcripts that were significantly down-regulated and up-regulated in expression overall was similar. What this indicates with respect to the fundamental mode of action for STS in apoptosis induction as opposed to the gene over-expression is uncertain. The number of transcripts identified as significantly changing with each treatment and at each time point varied considerably. In order to compare the condition or time-specific changes in transcript expression between the genes, a gene tree was plotted of the 997 transcripts that had changed in expression more than 1.4-fold and that were present in 3 or more of the 12 pair-wise treatment comparisons (Figure 25). There was a surprising degree of uniformity in behaviour of these genes across all conditions and time points and genes that were up- or down-regulated in one comparison tended to show the same behaviour across all comparisons, although this change didn't always reach the level of statistical significance.. Indeed, it was not possible to find any convincing condition-specific changes in transcriptome activity that might give clues as the mechanism by which functionally distinct genes induce apoptosis when over- expressed. Whether this is because the events leading to the initiation of a pro- apoptotic response occurred post-transcriptionally or that the changes are too subtle to be recognised is unclear. Rather, these finding strongly indicate that the majority of the changes we observed were associated with a universal pattern of gene regulation during apoptosis, regardless of the initiating trigger.

In order to further explore the apoptotic signatures in this data, the University of Michigan list of apoptosis regulators (Inohara 2004) was used to identify other potentially interesting apoptosis-associated genes in the list of differentials. 130 genes were shared between this list and the list of genes found to be differentially expressed here. Many gene family members and known interactors that have been previously associated with either the pro-apoptotic or cell survival machinery were present within this new list of 130 apoptosis-associated genes. A literature search was performed on the 130 genes in the list to ascertain their function. Some apoptosis associated genes e.g. NR4A1, EGRl SLIT2, CASP9, ADM, MADH7, JUN and TIMPl genes significantly changed in their expression in every transfection/treatment compared to the negative control, others were only observed to change under certain conditions. In order to provide a simplified view of this data, these genes were mapped onto a modified version of the KEGG apoptosis pathway if they were present in at least three of the four experimental conditions. Their action in either increasing or decreasing the likelihood of apoptosis and their directional change in expression is indicated (Figure 26). Overall, this approach supported the hypothesis that the transfected genes and STS were ultimately acting through similar pathways to induce cell death. In each case, numerous genes were observed to change which were associated with the MAPK8(JNK)/CASP3 pathway and in addition there were clear indications of some suppression of the cell survival pathways also occurring. Whilst the KEGG pathway was helpful in visualising the apoptotic pathways, many of the genes on the University of Michigan list of apoptosis regulators had to be added onto the pathway. In addition, there are most likely other genes in the lists of differentials that will be influencing the progression of apoptosis, but have not yet been recognised as being involved with the regulation of cell death.

Overall, the expression profiling studies have provided valuable insights into the transcription changes associated with apoptosis, as the transcriptional changes associated with programmed cell death have not been studied extensively. This current study supports the notion that there are discrete changes in the mRNA abundance of certain genes during apoptosis (Johnson et al. 2004) and as many of these transcripts encode proteins that are known regulators of cell survival and death. it would seem likely that transcriptional regulation of these mediators contributes to a cell's decision to undergo cell death.

This study reports the design and use of the first truly high-density reverse transfection cell-based microarray. It has demonstrated the potential as well as the current limitations of this technology to screen large number of genes for those that induce a functional change in cellular physiology, in this case apoptosis. 10 genes have been identified in this repect. hi order to examine the functional activity of these genes, a time course expression profiling experiment was set up to follow the transcriptional changes associated with apoptosis induction for three of the genes. This revealed that apoptosis induction is associated with discrete changes in a cell's transcriptome and that many of these changes seem identical regardless of mechanism by which apoptosis has been induced. Furthermore, many of the genes observed to change in their expression level during cell death have previously been associated with apoptosis and in this study strongly indicate the activation of the MAPK8(JNK)/CASP3 and BCL2 pathways. Table 1. Summary of the 10 genes found in Example 1 to induce apoptosis when over-expressed. Function, sub-cellular localisations and GO categories of the genes are given.

Example 2

In Example 1, the reverse transfection technique was optimised and a high-density reverse transfection array containing 1,959 ORFs in expression constructs was constructed using a novel strategy employing un-tagged clones. The array was then was then screened for proteins that induce cell death. Ten proteins that induced apoptosis were identified. The timing of the cell death caused by the 10 apoptosis- inducing proteins was characterised and the apoptotic pathways that three of the proteins were acting through were explored using the Affymetrix GeneChip platform. This analysis provided new insights into the mechanisms of apoptosis induction.

The 10 apoptosis inducing proteins were shown to cause apoptosis in human embryonic kidney (HEK293T) cells. It is thought that these proteins could be of use in the gene therapy arena, particularly in the treatment of cancer, if their activity in inducing apoptosis is not restricted to the cell type in which they were identified. To this end, the 10 genes needed to be over-expressed in cancerous cell lines.

In the study described here, the 10 apoptotic inducing proteins have been over- expressed for 54 hours in six cell lines; the 'normal' HEK293T cell line used in previous studies and five cancerous cell lines; human cervix adenocarcinoma (HeLa), human liver hepatocellular carcinoma (HEPG2), human hepatoma (HUH7), human ovary adenocarcinoma (OVCAR3) and human pancreas duct epitheloid carcinoma (PANCl) cells.

Materials and methods

Cell passage Culture medium

50 ml FCS (Invitrogen) 100 U/ml penicillin (Invitrogen)

100 μg/ml streptomycin (Invitrogen)

500 ml DMEM with 0.11 g/1 NA PYR with pyroxidine (Invitrogen) The solution was mixed and stored at 4⁰C

1. Human embryonic kidney (HEK293T), human cervix adenocarcinoma (HeLa), human liver hepatocellular carcinoma (HEPG2), human hepatoma (HUH7), human ovary adenocarcinoma (OVCAR3), human pancreas duct epitheloid carcinoma (PANCl) cells were grown and maintained in culture medium in a T75 flask (Nalge Nunc, Hereford, UK) at 37⁰C and 5% CO₂.

2. Once confluent, the culture medium was removed, 2 ml of trypsin-EDTA (Invitrogen) added, swirled over the surface of the cells and removed.

3. 1 ml of trypsin-EDTA was added, the flask tapped to help detach the cells and Ieft for 2 min.

4. 9 ml of culture medium was added, the cells mixed thoroughly by pipetting up and down 10 times and 1 ml added to a fresh flask containing 14 ml of culture medium.

6-well transfections, Effectene

(Effectene Transfection Reagent kit, Qiagen)

Cell culture media with STS (100 ml)

1 ml 100 mM STS (final concentration 1 μm on cells) (Sigma, Gillingham, Dorset,

UK) 100 ml cell culture media (0)

The media was mixed and used immediately.

1. Coverslips were added to 6-well plates and 2 x 10⁵ of each cell line in 1.5 ml culture medium (0) were applied.

2. After 24 h the media was removed from the cells and 1.5 ml of new culture medium added.

3. 0.4 μg of DNA was added to 3.2 μl of Enhancer and 150 μl EC buffer vortexed and incubated at RT for 5 min.

4. 10 μl Effectene was added, vortexed and incubated at RT for 10 min.

5. A negative Effectene only control was prepared in the same way, but DNA was added.

6. 600 μl of culture medium was added, vortexed and carefully pipetted onto each well and the cells grown for 54 h. For the positive control, 2 ml of cell culture media with STS was added to the cells after removal of the media.

Cleaved CASP3 assay 5% BSA/PBS (20 ml)

1 g BSA (Sigma)

20 ml IX PBS with calcium and magnesium (Invitrogen)

The solution was mixed and stored at 4⁰C

3.8% Formaldehyde (100 ml) 10 ml 38% paraformaldehyde (VWR, Dorset, UK),

90 ml IX PBS with calcium and magnesium (Invitrogen)

The solution was mixed and used immediately.

0.2% Triton/PBS (500 ml)

1 ml Triton X-100 (Sigma, XlOO) 500 ml IX PBS with calcium and magnesium (Invitrogen)

The solution was mixed and stored at RT.

Cleaved CASP3 antibody (1:100)

50 μl Cleaved CASP3 (Asp 175) Antibody (Cell Signaling Technology, Beverly,

Massachusetts, USA) 5 ml 5% BSA/PBS

The solution was mixed and used immediately

Goat anti-rabbit-Alexa488 (1:400)

12 μl goat anti-rabbit- Alexa488

4,800 μl 5% BSA/PBS The solution was mixed and used immediately

1. The medium was removed from the cells.

2. 1 ml of 3.8% formaldehyde was added to the cells at RT for 10 min to fix the cells, then the cells washed with IX PBS.

3. 1 ml of -2O⁰C 100% methanol (Sigma) was added to the cells for 5 min to permeabilise the cells, then the cells were washed twice with IX PBS for 2 min.

4. 1 ml 0.2% triton 100/PBS was added to the cells for 1 min. 5. 1 ml of 5%BSA/PBS was added to the cells for 20 min. at RT.

6. 50 μl of diluted Cleaved CASP3 antibody was added per coverslip, a circle of parafilm (Teklab, Durham, UK) the size of the coverslip was carefully applied to ensure even coverage of antibody, incubated shielded from light at 4⁰C overnight.

7. The coverslips were washed twice in IX PBS for 10 min.

8. A drop of DAPI or propidium idodide mounting medium (Vector, Peterborough, UK) was added to the coverslip, placed on a glass microscope slide and sealed with nail varnish. 9. The fluorescence was observed via fluorescent microscopy and the slides were stored at 4⁰C.

Scoring positive apoptotic cells

Apoptotic cells were scored as positive if clear fluorescent apoptotic bodies were observed in or near the cells. The number of nuclei and apoptotic bodies were counted in a representative XlO field of view.

Results

Transfection efficiencies

The vector eGFP-Cl was transfected into each of the cell lines to determine transfection efficiencies. HEK293T and HeLa cells had the best transfection efficiencies of about 60%, followed by OVCAR3, about 40%, HUH7 and PANCl about 20%, then HEPG2 about 5% (Figure 33).

Over-expression of apoptosis inducing proteins

The 10 apoptotic proteins discovered from a previous reverse transfection study, plus a negative control protein ACY-I that was shown not to induce apoptosis in the previous reverse transfection assay, a positive control staurosporine (STS) and the eGFP-Cl vector were added to the 6 cell lines for 54 hours, then the CASP3 assay applied to detect apoptosis. Apoptosis had been observed in HEK293T cells in previous experiments. However in this study no apoptosis was detectable except a small amount in cells over-expressing the STK3 and AFlQ proteins (Figures 34 and 36). Baseline levels of apoptosis was observed in HELA cells over-expressed with the ACY-I protein and cells over-expressed with the EXOC7 protein showed the highest levels of apoptosis in this cell line (Figures 34 and 37). In HEPG2 cells over- expressing the ACY-I protein (the positive control), some apoptosis was observed. XBPl, MGC5439, CCBP2 and LOC134285 showed some apoptosis above this level (Figures 34 and 38). In OVCAR3 cells over-expressing ACY-I protein, about 50% apoptosis was observed. As none of the other proteins over-expressed in this cell-line showed more apoptosis than the positive control ACY-I, the results are inconclusive (Figures 34 and 39). Baseline levels of apoptosis was observed in HUH7 cells over- expressed with the ACY-I protein and cells over-expressed with CSTB protein showed the highest levels of apoptosis in this cell line (Figures 34 and 40). Baseline levels of apoptosis was observed in PANCl cells over-expressed with the ACY-I protein and cells over-expressed with XBPl showed the highest levels of apoptosis in this cell line (Figures 34 and 41). A small amount of apoptosis was observed in cell lines treated with STS, the most apoptosis was observed in OVCAR3 cells treated with STS.

Discussion

Ten apoptotic proteins had been identified as inducing apoptosis in a previous reverse transfection experiment. The experiments had been undertaken with HEK293T cells, which are not cancerous. It was thought that these proteins could be of use in cancer gene therapy. In experiments these ten proteins, the eGFP-Cl vector, ACY-I and STS were added to five cancerous cell lines, HEPG2, HELA, HUH7, OVCAR3 and PANCl.

GFP fluorescence was observed in all the cell lines at similar levels in the GFP transfection and apoptosis study. Apoptosis was clearly observed in previous experiments with HEK293T cells. However, apoptosis was not clear in this experiment with HEK293T cells. The reason for this is uncertain but it is know that cells susceptibility to transfection can change with passage number etc. The STS control did not show clear apoptotic bodies either, again for reasons unknown.

There were baseline levels of apoptosis in most cell lines over-expressed with the negative control protein ACY-I, however, in OVCAR3 and HEPG2 cells, up to 50% of the cells were observed to be apoptotic. Therefore, in the OVC AR3 and HEPG2 cell lines, any over-expressed protein with less apoptosis than the cells over-expressed with the ACY-I protein were not valid.

Over-expression of the EXOC7 protein caused the most apoptosis in HELA cells. Over-expression of the MGC5439 protein caused the most apoptosis in HEPG2 cells. Over-expression of the MGC5439 protein caused the most apoptosis in HUH7 cells. Over-expression of the XBPl protein caused the most apoptosis in PANCl cells.

Each cell line showed a different pattern of apoptosis depending upon the over- expressed protein and this is probably due to inherent differences within the cell lines. The results indicate the different over-expressed proteins cause apoptosis in different cancerous cell lines. Therefore, the identified genes and the encoded proteins are of use in the treatment and/or prophylaxis of cancer.

Claims

1. An isolated or recombinant nucleic acid molecule comprising: a) a nucleotide sequence comprising the sequence of Figures 1, 3, 5, 7, 9, 11, 13, 15, 17 or 19, or its RNA equivalent; b) a nucleotide sequence which is complementary to the sequence of (a); c) a nucleotide sequence which codes for the same polypeptide as the nucleotide sequence of (a) or (b); or d) a fragment of the nucleotide sequence of (a) for use in the treatment and/or prophylaxis of a disease through the regulation of apoptosis.

2. An isolated or recombinant nucleic acid molecule as claimed in claim 1, wherein the nucleic acid molecule is for use in the treatment and/or prophylaxis of cancer.

3. An isolated or recombinant nucleic acid molecule as claimed in claim 2, wherein the nucleic acid molecule is adapted to be over-expressed in cancer cells.

4. An isolated or recombinant nucleic acid molecule as claimed in claim 3, wherein the nucleic acid molecule further comprises an inducible promoter.

5. The use of an isolated or recombinant nucleic acid molecule of any one of claims 1 to 4 in the manufacture of a medicament for the treatment and/or prophylaxis of a disease through the regulation of apoptosis.

6. A method for the prophylaxis and/or treatment of a disease through the regulation of apoptosis, the method comprising administering an isolated or recombinant nucleic acid molecule of any one of claims 1 to 4 to a subject.

7. An expression vector comprising a nucleic acid molecule as claimed in any one of claims 1 to 4, for use in the treatment and/or prophylaxis of a disease through the regulation of apoptosis.

8. An expression vector as claimed in claim 7, wherein the vector is for use in the treatment and/or prophylaxis of cancer.

9. A nucleic acid molecule that is antisense to a portion of a nucleic acid molecule of any one of claims 1 to 4 for use in the manufacture of a medicament for the treatment and/or prophylaxis of a disease through the regulation of apoptosis.

10. A method for the prophylaxis and/or treatment of a disease through the regulation of apoptosis comprising administering a nucleic acid molecule that is antisense to a portion of a nucleic acid molecule of any one of claims 1 to 4 to a subject.

11. A RNA molecule comprising a double stranded structure which has a nucleotide sequence which is identical to a portion of the nucleotide sequence of Figures 1, 3, 5, 7, 9, 11, 13, 15, 17 or 19.

12. A nucleic acid molecule that when transcribed provides the RNA molecule as claimed in claim 11.

13. The use of a RNA molecule as claimed in claim 11 , or a nucleic acid molecule as claimed in claim 12, in the manufacture of a medicament for the prophylaxis and/or treatment of a disease through the regulation of apoptosis.

14 A method for the prophylaxis and/or treatment of a disease through the regulation of apoptosis, comprising administering to a subject a RNA molecule as claimed in claim 11, or a nucleic acid molecule as claimed in claim 12

15. An isolated or recombinant polypeptide comprising:

(a) the amino acid sequence shown in Figures 2, 4, 6, 8, 10, 12, 14, 16, 18 or 20; or

(b) a fragment of a polypeptide as defined in a), for use in the treatment and/or prophylaxis of a disease through the regulation of apoptosis.

16. The use of a polypeptide as claimed in claim 15 in the manufacture of a medicament for the treatment and/or prophylaxis of a disease through the regulation of apoptosis, wherein the medicament is a vaccine.

17. A method of diagnosis of a disease involving apoptosis in a subject, the method comprising detecting and/or quantifying the amount of a polypeptide as claimed in claim 15 in a biological sample obtained from said subject.

18. A method as claimed in claim 17, wherein an antibody is used for detecting and/or quantifying the amount of the polypeptide in a biological sample obtained from said subject.