WO2002050103A2 - Tumour-related antigens - Google Patents

Abstract

CASB88 polypeptides and polynucleotides and methods for producing such polypeptides by recombinant techniques are disclosed. Also disclosed are methods for utilising CASB88 polypeptides and polynucleotides in diagnostics and vaccines for prophylactic and therapeutic treatment of cancers, Crohn's disease, Colitis ulcerosa, colorectal cancer, lung cancer and preneoplasic lesions, breast, brain, uterus, muscle, eye and germ cell cancers, Wilm's tumour, retinoblastoma, rabdomyosarcoma, leimyosarcoma and synovial sarcoma, autoimmune diseases, and related conditions.

Description

Novel Compounds

The present invention relates to pharmaceutical compositions and methods for inducing an immune response against tumour-related antigens. The present invention relates to polynucleotides, herein referred to as CASB88 polynucleotides, polypeptides encoded thereby (referred to herein as CASB88 polypeptides), recombinant materials and methods for their production. In another aspect, the invention relates to methods for using such polypeptides and polynucleotides, including the treatment and prevention of cancer and autoimmune diseases, including prevention of metastasis, more particularly colorectal cancer, lung cancer and preneoplasic lesions, Crohn's disease and Colitis ulcerosa, breast, brain, uterus, muscle, eye and germ cell cancers, Wilm's tumour, retinoblastoma, rabdomyosarcoma, leimyosarcoma and synovial sarcoma, and other related conditions. In another aspect, the invention relates to pharmaceutical compositions containing CASB88 polypeptides and polynucleotides, to methods of manufacture of such compositions and to their use in medicine. In a further aspect, the invention relates to methods for identifying agonists and antagonists/inhibitors using the materials provided by the invention, and treating conditions associated with CASB88 polypeptide imbalance with the identified compounds. In a still f rther aspect, the invention relates to diagnostic assays for detecting diseases associated with inappropriate CASB88 polypeptide activity or levels.

Polypeptides and polynucleotides ofthe present invention are believed to be important immunogens for specific prophylactic or therapeutic immunization against tumours, because they are specifically expressed or highly over-expressed in tumours compared to normal cells and can thus be targeted by antigen-specific immune mechanisms leading to the destruction ofthe tumour cell. They can also be used to diagnose the occurrence of tumour cells. Furthermore, their inappropriate expression in certain circumstances can cause an induction of autoimmune, inappropriate immune responses, which could be corrected through appropriate vaccination using the same polypeptides or polynucleotides. In this respect the most important biological activities to our purpose are the antigenic and immunogenic activities ofthe polypeptide ofthe present invention. A polypeptide ofthe present invention may also exhibit at least one other biological activity of a CASB88 polypeptide, which could qualify it as a target for therapeutic or prophylactic intervention different from that linked to the immune response.

In a first aspect, the present invention relates to CASB88 polypeptides. Such peptides include isolated polypeptides comprising an amino acid sequence which has at least 70% identity, preferably at least 80% identity, more preferably at least 85% identity, still more preferably at least 90% identity, yet more preferably at least 95% identity, most preferably at least 97-99% identity, to that of SEQ ID NO:2 over the entire length of SEQ ID NO:2. Such polypeptides include those comprising the amino acid of SEQ ID NO:2.

Further peptides ofthe present invention include isolated polypeptides in which the amino acid sequence has at least 70% identity, preferably at least 80% identity, more preferably at least 85% identity, still more preferably at least 90% identity, yet more preferably at least 95% identity, most preferably at least 97-99% identity, to the amino acid sequence of SEQ ID NO:2 over the entire length of SEQ ID NO:2. Such polypeptides include the polypeptide of SEQ ID NO:2.

Preferably the aforementioned polypeptides are recombinantly produced. Most preferably the polypeptides according to the invention are purified, and are substantially free of any other proteins or contaminating host-originating material.

Further peptides ofthe present invention include isolated polypeptides encoded by a polynucleotide comprising the sequence contained in SEQ ID NO:l.

The invention also provides an immunogenic fragment of a CASB88 polypeptide, that is a contiguous portion ofthe CASB88 polypeptide which has the same or similar immunogenic properties to the polypeptide comprising the amino acid sequence of SEQ ID NO:2. That is to say, the fragment (if necessary when coupled to a carrier) is capable of raising an immune response which recognizes the CASB88 polypeptide. Such an immunogenic fragment may include, for example, the CASB88 polypeptide lacking an N-terminal leader sequence, a transmembrane domain or a C-terminal anchor domain. In a preferred aspect the immunogenic fragment of CASB88 according to the invention comprises substantially all ofthe extracellular domain of a polypeptide which has at least 70% identity, preferably at least 80% identity, more preferably at least 85% identity, still more preferably at least 90% identity, yet more preferably at least 95% identity, most preferably at least 97-99% identity, to that of SEQ ID NO:2 over the entire length of SEQ ID NO:2. Preferably an immunogenic fragment according to the invention comprises at least one epitope.

Peptide fragments incorporating an epitope of CASB88 typically will comprise at least 7, preferably 9 or 10 contiguous amino acids from SEQ ID NO:2. Preferred epitopes are shown in SEQ ID NOJ-102 .Peptides that incorporate these epitopes form a preferred aspect ofthe present invention. Mimotopes which have the same characteristics as these epitopes, and immunogens comprising such mimotopes which generate an immune response which cross-react with an epitope in the context ofthe CASB88 molecule, also form part ofthe present invention.

The present invention, therefore, includes isolated peptides encompassing these epitopes themselves, and any mimotope thereof. The meaning of mimotope is defined as an entity which is sufficiently similar to the native CASB88 epitope so as to be capable of being recognized by antibodies which recognize the native molecule; (Gheysen, H.M., et al., 1986, Synthetic peptides as antigens. Wiley, Chichester, Ciba foundation symposium 119, pl30-149; Gheysen, H.M., 1986, Molecular Immunology, 23,7, 709-715); or are capable of raising antibodies, when coupled to a suitable carrier, which antibodies cross- react with the native molecule.

Peptide mimotopes ofthe above-identified epitopes may be designed for a particular purpose by addition, deletion or substitution of elected amino acids. Thus, the peptides of the present invention may be modified for the purposes of ease of conjugation to a protein carrier. For example, it may be desirable for some chemical conjugation methods to include a terminal cysteine to the epitope. In addition it may be desirable for peptides conjugated to a protein carrier to include a hydrophobic terminus distal from the conjugated terminus ofthe peptide, such that the free unconjugated end ofthe peptide remains associated with the surface ofthe carrier protein. This reduces the conformational degrees of freedom ofthe peptide, and thus increases the probability that the peptide is presented in a conformation which most closely resembles that ofthe peptide as found in the context ofthe whole molecule. For example, the peptides may be altered to have an N-terminal cysteine and a C-terminal hydrophobic amidated tail. Alternatively, the addition or substitution of a D-stereoisomer form of one or more ofthe amino acids may be performed to create a beneficial derivative, for example to enhance stability ofthe peptide. Those skilled in the art will realise that such modified peptides, or mimotopes, could be a wholly or partly non-peptide mimotope wherein the constituent residues are not necessarily confined to the 20 naturally occurring amino acids. In addition, these may be cyclised by techniques known in the art to constrain the peptide into a conformation that closely resembles its shape when the peptide sequence is in the context ofthe whole molecule. A preferred method of cyclismg a peptide comprises the addition of a pair of cysteine residues to allow the formation of a disulphide bridge.

Further, those skilled in the art will that mimotopes or immunogens ofthe present invention may be larger than the above-identified epitopes, and as such may comprise the sequences disclosed herein. Accordingly, the mimotopes ofthe present invention may consist of addition of N and/or C terminal extensions of a number of other natural residues at one or both ends. The peptide mimotopes may also be retro sequences ofthe natural sequences, in that the sequence orientation is reversed; or alternatively the sequences may be entirely or at least in part comprised of D-stereo isomer amino acids (inverso sequences). Also, the peptide sequences may be retro-inverso in character, in that the sequence orientation is reversed and the amino acids are ofthe D-stereoisomer form. Such retro or retro-inverso peptides have the advantage of being non-self, and as such may overcome problems of self-tolerance in the immune system.

Alternatively, peptide mimotopes may be identified using antibodies which are capable themselves of binding to the epitopes ofthe present invention using techniques such as phage display technology (EP 0 552 267 BI). This technique, generates a large number of peptide sequences which mimic the structure ofthe native peptides and are, therefore, capable of binding to anti-native peptide antibodies, but may not necessarily themselves share significant sequence homology to the native peptide. This approach may have significant advantages by allowing the possibility of identifying a peptide with enhanced immunogenic properties, or may overcome any potential self-antigen tolerance problems which may be associated with the use ofthe native peptide sequence. Additionally this technique allows the identification of a recognition pattern for each native-peptide in terms of its shared chemical properties amongst recognised mimotope sequences.

The covalent coupling ofthe peptide to the immunogenic carrier can be carried out in a manner well known in the art. Thus, for example, for direct covalent coupling it is possible to utilise a carbodiimide, glutaraldehyde or (N-[γ-maleimidobutyryloxy] succinimide ester, utilising common commercially available heterobifunctional linkers such as CDAP and SPDP (using manufacturers instructions). After the coupling reaction, the immunogen can easily be isolated and purified by means of a dialysis method, a gel filtration method, a fractionation method etc.

The types of carriers used in the immunogens ofthe present invention will be readily known to the man skilled in the art. The function ofthe carrier is to provide cytokine help in order to help induce an iinmune response against the peptide. A non-exhaustive list of carriers which may be used in the present invention include: Keyhole limpet

Haemocyanin (KLH), serum albumins such as bovine serum albumin (BSA), inactivated bacterial toxins such as tetanus or diphtheria toxins (TT and DT), or recombinant fragments thereof (for example, Domain 1 of Fragment C of TT, or the translocation domain of DT), or the purified protein derivative of tuberculin (PPD). Alternatively the mimotopes or epitopes may be directly conjugated to liposome carriers, which may additionally comprise immunogens capable of providing T-cell help. Preferably the ratio of mimotopes to carrier is in the order of 1 : 1 to 20 : 1 , and preferably each carrier should carry between 3-15 peptides.

In an embodiment ofthe invention a preferred carrier is Protein D from Haemophilus influenzae (EP 0 594 610 BI). Protein D is an IgD-binding protein from Haemophilus influenzae and has been patented by Forsgren (WO 91/18926, granted EP 0 594 610 BI). In some circumstances, for example in recombinant immunogen expression systems it may be desirable to use fragments of protein D, for example Protein D l/3rd (comprising the N-terminal 100-110 amino acids of protein D (GB 9717953.5)).

Another preferred method of presenting the peptides ofthe present invention is in the context of a recombinant fusion molecule. For example, EP 0 421 635 B describes the use of chimaeric hepadnavirus core antigen particles to present foreign peptide sequences in a virus-like particle. As such, immunogens ofthe present invention may comprise peptides presented in chimaeric particles consisting of hepatitis B core antigen. Additionally, the recombinant fusion proteins may comprise the mimotopes ofthe present invention and a carrier protem, such as NS1 ofthe influenza virus. For any recombinantly expressed protein which forms part ofthe present invention, the nucleic acid which encodes said immunogen also forms an aspect ofthe present invention.

Peptides used in the present invention can be readily synthesised by solid phase procedures well known in the art. Suitable syntheses may be performed by utilising "T- boc" or "F-moc" procedures. Cyclic peptides can be synthesised by the solid phase procedure employing the well-known "F-moc" procedure and polyamide resin in the fully automated apparatus. Alternatively, those skilled in the art will know the necessary laboratory procedures to perform the process manually. Techniques and procedures for solid phase synthesis are described in 'Solid Phase Peptide Synthesis: A Practical

Approach' by E. Atherton and R.C. Sheppard, published by iRL at Oxford University Press (1989). Alternatively, the peptides may be produced by recombinant methods, including expressing nucleic acid molecules encoding the mimotopes in a bacterial or mammalian cell line, followed by purification ofthe expressed mimotope. Techniques for recombinant expression of peptides and proteins are known in the art, and are described in Maniatis, T., Fritsch, E.F. and Sambrook et al., Molecular cloning, a laboratory manual, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989).

The polypeptides or immunogenic fragment ofthe invention may be in the form ofthe "mature" protein or may be a part of a larger protein such as a precursor or a fusion protein. It is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in purification such as multiple histidine residues, or an additional sequence for stability during recombinant production. Furthermore, addition of exogenous polypeptide or lipid tail or polynucleotide sequences to increase the immunogenic potential ofthe final molecule is also considered. In one aspect, the invention relates to genetically engineered soluble fusion proteins comprising a polypeptide ofthe present invention, or a fragment thereof, and various portions ofthe constant regions of heavy or light chains of immunoglobulins of various subclasses (IgG, IgM, IgA, IgE). Preferred as an immunoglobulin is the constant part of the heavy chain of human IgG, particularly IgGl , where fusion takes place at the hinge region. In a particular embodiment, the Fc part can be removed simply by incorporation of a cleavage sequence which can be cleaved with blood clotting factor Xa. Furthermore, this invention relates to processes for the preparation of these fusion proteins by genetic engineering, and to the use thereof for drug screening, diagnosis and therapy. A further aspect ofthe invention also relates to polynucleotides encoding such fusion proteins. Examples of fusion protein technology can be found in International Patent Application Nos. WO94/29458 and WO94/22914.

The proteins may be chemically conjugated, or expressed as recombinant fusion proteins allowing increased levels to be produced in an expression system as compared to non- fused protein. The fusion partner may assist in providing T helper epitopes (immunological fusion partner), preferably T helper epitopes recognised by humans, or assist in expressing the protein (expression enhancer) at higher yields than the native recombinant protein. Preferably the fusion partner will be both an immunological fusion partner and expression enhancing partner.

Fusion partners include protein D from Haemophilus influenza B and the non-structural protein from influenzae virus, NS1 (hemagglutinin). Another immunological fusion partner is the protein known as LYTA. Preferably the C terminal portion ofthe molecule is used. Lyta is derived from Streptococcus pneumoniae which synthesize an N-acetyl-L- alanine amidase, amidase LYTA, (coded by the lytA gene {Gene, 43 (1986) page 265- 272} an autolysin that specifically degrades certain bonds in the peptidoglycan backbone. The C-terminal domain ofthe LYTA protein is responsible for the affinity to the choline or to some choline analogues such as DEAE. This property has been exploited for the development of E.coli C-L YTA expressing plasmids useful for expression of fusion proteins. Purification of hybrid proteins containing the C-L YTA fragment at its amino terminus has been described {Biotechnology: 10, (1992) page 795-798}. It is possible to use the repeat portion ofthe Lyta molecule found in the C terminal end starting at residue 178, for example residues 188 - 305.

The present invention also includes xenogeneic forms (also termed ortholog forms) ofthe aforementioned polypeptides, said xenogeneic forms referring to an antigen having substantial sequence identity to the human antigen (also termed autologous antigen) which serves as a reference antigen but which is derived from a different non-human species. In this context the substantial identity refers to concordance of an amino acid sequence with another amino acid sequence or of a polynucleotide sequence with another polynucleotide sequence when such sequence are arranged in a best fit alignment in any of a number of sequence alignment proteins known in the art. By substantial identity is meant at least 70-95%, and preferably at least 85-95%, most preferably at least 90%-95%, sequence identity between the compared sequences. Therefore according to the invention the xenogeneic CASB88 polypeptide will be a CASB88 polypeptide which is xenogeneic with respect to human CASB88, in other words which is isolated from a species other than human. In a preferred embodiment, the polypeptide is isolated from mouse, rat, pig, or rhesus monkey, most preferably from mouse or rat. Accordingly the present invention also provides a method of inducing an immune response against human CASB88 having an amino acid sequence as set forth in SEQ ID NO:2 in a human, comprising administering to the subject an effective dosage of a composition comprising a xenogeneic form of said human CASB88 as described herein. A preferred embodiment is a method of inducing an immune response against human CASB88 using the xenogeneic CASB88 isolated from mouse, rat, pig or rhesus monkey, more preferably from mouse. Another preferred method of inducing an immune response according to the present invention is using an antigen composition including a live viral expression system which expresses said xenogeneic antigen.

The preferred isolated xenogeneic CASB88 polypeptide will generally share substantial sequence similarity, and include isolated polypeptides comprising an amino acid sequence which has at least 70% identity, preferably at least 80% identity, more preferably at least 85% identity, still more preferably at least 90% identity, yet more preferably at least 95% identity, most preferably at least 97-99% identity, to that of SEQ ID NO: 3 over the entire length of SEQ ID NO:3. Accordingly the xenogeneic polypeptide will comprise an immunogenic fragment ofthe polypeptide of SEQ ID NO:3 in which the immunogenic activity ofthe immunogenic fragment is substantially the same as the polypeptide of SEQ ID NO:3. In addition the xenogeneic CASB88 polypeptide can be a fragment of at least about 20 consecutive amino acids, preferably about 30, more preferably about 50, yet more preferably about 100, most preferably about 150 contiguous amino acids selected from the amino acid sequences as shown in SEQ ID NO:3. More particularly xenogeneic CASB88 fragments will retain some functional property, preferably an immunological activity, ofthe larger molecule set forth in SEQ ID NO:3, and are useful in the methods described herein (e.g. in pharmaceutical and vaccine compositions, in diagnostics, etc.). In particular the fragments will be able to generate an immune response against the human counterpart, such as the generation of cross-reactive antibodies which react with the autologous human form of CASB88 as set forth in any ofthe SEQ ID NO:2. In a specific embodiment, the xenogeneic polypeptide ofthe invention may be part of a larger fusion, comprising the xenogeneic CASB88 polypeptide or fragment thereof and a heterologous protein or part of a protein acting as a fusion partner as described hereabove.

The present invention also includes variants ofthe aforementioned polypeptides, that is polypeptides that vary from the referents by conservative amino acid substitutions, whereby a residue is substituted by another with like characteristics. Typical such substitutions are among Ala, Nal, Leu and He; among Ser and Thr; among the acidic residues Asp and Glu; among Asn and Gin; and among the basic residues Lys and Arg; or aromatic residues Phe and Tyr. Particularly preferred are variants in which several, 5-10, 1-5, 1-3, 1-2 or 1 amino acids are substituted, deleted, or added in any combination.

Polypeptides ofthe present invention can be prepared in any suitable manner. Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for preparing such polypeptides are well understood in the art.

In a further aspect, the present invention relates to CASB88 polynucleotides. Such polynucleotides include isolated polynucleotides comprising a nucleotide sequence encoding a polypeptide which has at least 70% identity, preferably at least 80% identity, more preferably at least 85% identity, still more preferably at least 90% identity, yet more preferably at least 95% identity, to the amino acid sequence of SEQ ID NO:2, over the entire length of SEQ ID NO:2. In this regard, polypeptides which have at least 97% identity are highly preferred, whilst those with at least 98-99% identity are more highly preferred, and those with at least 99% identity are most highly preferred. Such polynucleotides include a polynucleotide comprising the nucleotide sequence contained in SEQ ID NO:l encoding the polypeptide of SEQ ID NO:2.

Further polynucleotides of the present invention include isolated polynucleotides comprising a nucleotide sequence that has at least 70% identity, preferably at least 80% identity, more preferably at least 85°Λ> identity, still more preferably at least 90% identity, yet more preferably at least 95% identity, to a nucleotide sequence encoding a polypeptide of SEQ ID NO:2, over the entire coding region. In this regard, polynucleotides which have at least 97% identity are highly preferred, whilst those with at least 98-99% identity are more highly preferred, and those with at least 99% identity are most highly preferred.

Further polynucleotides ofthe present invention include isolated polynucleotides comprising a nucleotide sequence which has at least 70% identity, preferably at least 80% identity, more preferably at least 85% identity, still more preferably at least 90% identity, yet more preferably at least 95% identity, to SEQ ID NO:l or to the coding sequence of SEQ ID NO: 1 over the entire length of SEQ ID NO: 1 or over the entire length ofthe coding sequence of SEQ ID NO:l respectively. In this regard, polynucleotides which have at least 97% identity are highly preferred, whilst those with at least 98-99% identity are more highly preferred, and those with at least 99% identity are most highly preferred. Such polynucleotides include a polynucleotide comprising the polynucleotide of SEQ ID NO: 1 as well as the polynucleotide of SEQ ID NO: 1 or the coding region of SEQ ID NO: 1. Said polynucleotide can be inserted in a suitable plasmid or recombinant microorganism vector and used for immunization ( see for example Wolff et. al., Science 247:1465-1468 (1990); Corr et. al., J. Exp. Med. 184:1555-1560 (1996); Doe et. al., Proc. Natl. Acad. Sci. 93:8578-8583 (1996)). The present invention also provides a nucleic acid encoding the aforementioned xenogeneic proteins ofthe present invention and their use in medicine. In a preferred embodiment, the xenogeneic CASB88 polynucleotide for use in pharmaceutical compositions has the sequence set forth in SEQ ID N°4. The isolated xenogeneic CASB88 polynucleotides according to the invention may be single-stranded (coding or antisense) or double-stranded, and may be DNA (genomic, cDNA or synthetic) or RNA molecules. Additional coding or non-coding sequences may, but need not, be present within a polynucleotide ofthe present invention. In other related embodiments, the present invention provides polynucleotide variants having substantial identity to the sequences disclosed herein in SEQ ID N°4 for example those comprising at least 70% sequence identity, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher, sequence identity compared to a polynucleotide sequence of this invention using the methods described herein, (e.g., BLAST analysis using standard parameters). In a related embodiment, the isolated xenogeneic polynucleotide ofthe invention will comprise a nucleotide sequence encoding a polypeptide that has at least 90%, preferably 95% and above, identity to the amino acid sequence of SEQ ID NO:4, over the entire length of SEQ ID NO:4; or a nucleotide sequence complementary to said isolated polynucleotide.

The invention also provides polynucleotides which are complementary to all the above- described polynucleotides.

The invention also provides a fragment of a CASB88 polynucleotide which when administered to a subject has the same immunogenic properties as the polynucleotide of SEQ ID NO: 1.

The invention also provides a polynucleotide encoding an immunological fragment of a CASB88 polypeptide as hereinbefore defined.

The fragments have a level of immunogenic activity of at least about 50%, preferably at least about 70% and more preferably at least about 90% ofthe level of immunogenic activity of a polypeptide sequence set forth in SEQ ID NO:2 or a polypeptide sequence encoded by a polynucleotide sequence set forth in SEQ ID NO: 1. The polypeptide fragments according to the invention preferably comprise at least about 5, 10, 15, 20, 25, 50, or 100 contiguous amino acids, or more, including all intermediate lengths, of a polypeptide composition set forth herein, such as those set forth in SEQ ID NO:2, or those encoded by a polynucleotide sequence set forth in a sequence of SEQ ID NO:l.

The nucleotide sequence of SEQ ID NO:l is a cDNA sequence which comprises a polypeptide encoding sequence (nucleotide 51 to 1646) encoding a pol peptide of 532 amino acids, the polypeptide of SEQ ID NO:2. The nucleotide sequence encoding the polypeptide of SEQ ID NO:2 may be identical to the polypeptide encoding sequence contained in SEQ ID NO:l or it may be a sequence other than the one contained in SEQ ID NO:l, which, as a result ofthe redundancy (degeneracy) ofthe genetic code, also encodes the polypeptide of SEQ ID NO:2. The polypeptide ofthe SEQ ID NO:2 is related to proteins ofthe M33-CBX2 gene family, having homology and/or structural similarity with Mus Musculus Chromobox Protein Homolog-2 (Modifier 3 Protein, M33; SwissProt accession : P30658|MOD3_MOUSE).

Preferred polypeptides and polynucleotides ofthe present invention are expected to have, inter alia, similar biological functions/properties to their homologous polypeptides and polynucleotides. Furthermore, preferred polypeptides, immunological fragments and polynucleotides ofthe present invention have at least one activity of either SEQ ID NO:l or SEQ ID NO:2, as appropriate.

The present invention also relates to partial or other incomplete polynucleotide and polypeptide sequences which were first identified prior to the determination ofthe corresponding full length sequences of SEQ ID NO:l and SEQ ID NO:2.

Accordingly, in a further aspect, the present invention provides for an isolated polynucleotide which: (a) comprises a nucleotide sequence which has at least 70% identity, preferably at least 80% identity, more preferably at least 85% identity, still more preferably at least 90% identity, yet more preferably at least 95% identity, even more preferably at least 97-99% identity to SEQ ID NO:5 over the entire length of SEQ ID NO:5; (b) has a nucleotide sequence which has at least 70% identity, preferably at least 80% identity, more preferably at least 85% identity, still more preferably at least 90% identity, yet more preferably at least 95% identity, even more preferably at least 97-99% identity, to SEQ ID NO: 1 over the entire length of SEQ ID NO:5; (c) the polynucleotide of SEQ ID NO:5; or (d) a nucleotide sequence encoding a polypeptide which has at least 70% identity, preferably at least 80% identity, more preferably at least 85% identity, still more preferably at least 90% identity, yet more preferably at least 95% identity, even more preferably at least 97-99% identity, to the amino acid sequence of SEQ ID NO:6, over the entire length of SEQ ID NO: 6, respectively, as well as the polynucleotide of SEQ ID NO:5.

The present invention further provides for a polypeptide which:

(a) comprises an amino acid sequence which has at least 70% identity, preferably at least 80% identity, more preferably at least 85% identity, still more preferably at least 90% identity, yet more preferably at least 95% identity, most preferably at least 97-99% identity, to that of SEQ ID NO:6 over the entire length of SEQ ID NO:6;

(b) has an amino acid sequence which is at least 70% identity, preferably at least 80% identity, more preferably at least 85% identity, still more preferably at least 90% identity, yet more preferably at least 95% identity, most preferably at least 97-99% identity, to the amino acid sequence of SEQ ID NO:6 over the entire length of SEQ ID NO:6;

(c) comprises the amino acid of SEQ ID NO:6; and

(d) is the polypeptide of SEQ ID NO:6; as well as polypeptides encoded by a polynucleotide comprising the sequence contained in SEQ ID NO:5.

Polynucleotides ofthe present invention may be obtained, using standard cloning and screening techniques, from a cDNA library derived from mRNA in cells of human fetus, normal testis, T-cell, colorectal cancer, lung cancer and preneoplasic lesions, breast, brain, uterus, muscle, eye and germ cell cancers, Wilm's tumour, retinoblastoma, rabdomyosarcoma, leimyosarcoma and synovial sarcoma, (for example Sarnbrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring harbor Laboratory Press, Cold Spring harbor, N.Y. (1989)). Polynucleotides ofthe invention can also be obtained from natural sources such as genomic DNA libraries or can be synthesized using well- known and commercially available techniques.

When polynucleotides ofthe present invention are used for the recombinant production of polypeptides ofthe present invention, the polynucleotide may include the coding sequence for the mature polypeptide, by itself; or the coding sequence for the mature polypeptide in reading frame with other coding sequences, such as those encoding a leader or secretory sequence, a pre-, or pro- or prepro- protein sequence, or other fusion peptide portions. For example, a marker sequence which facilitates purification ofthe fused polypeptide can be encoded. In certain preferred embodiments of this aspect ofthe invention, the marker sequence is a hexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) and described in Gentz et al, Proc Natl Acad Sci USA (1989) 86:821-824, or is an HA tag. The polynucleotide may also contain non-coding 5' and 3' sequences, such as transcribed, non-translated sequences, splicing and polyadenylation signals, ribosome binding sites and sequences that stabilize mRNA.

Further embodiments ofthe present invention include polynucleotides encoding polypeptide variants which comprise the amino acid sequence of SEQ ID NO:2 and in which several, for instance from 5 to 10, 1 to 5, 1 to 3, 1 to 2 or 1, amino acid residues are substituted, deleted or added, in any combination.

Polynucleotides which are identical or sufficiently identical to a nucleotide sequence contained in SEQ ID NO:l, may be used as hybridization probes for cDNA and genomic DNA or as primers for a nucleic acid amplification (PCR) reaction, to isolate full-length cDNAs and genomic clones encoding polypeptides ofthe present invention and to isolate cDNA and genomic clones of other genes (including genes encoding paralogs from human sources and orthologs and paralogs from species other than human) that have a high sequence similarity to SEQ ID NO:l. Typically these nucleotide sequences are 70% identical, preferably 80% identical, more preferably at least 85% identity, still more preferably 90% identical, most preferably 95% identical to that ofthe referent. The probes or primers will generally comprise at least 15 nucleotides, preferably, at least 30 nucleotides and may have at least 50 nucleotides. Particularly preferred probes will have between 30 and 50 nucleotides. Particularly preferred primers will have between 20 and 25 nucleotides. In particular, polypeptides or polynucleotides derived from sequences from homologous animal origin could be used as immunogens to obtain a cross-reactive immune response to the human gene.

A polynucleotide encoding a polypeptide ofthe present invention, including homologs from species other than human, may be obtained by a process which comprises the steps of screening an appropriate library (human or not) under stringent hybridization conditions with a labeled probe having the sequence of SEQ ID NO:l or a fragment thereof; and isolating full-length cDNA and genomic clones containing said polynucleotide sequence. Such hybridization techniques are well known to the skilled artisan. Preferred stringent hybridization conditions include overnight incubation at 42oC in a solution comprising: 50% formamide, 5xSSC (150mM NaCI, 15mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5x Denhardt's solution, 10 % dextran sulfate, and 20 microgram/ml denatured, sheared salmon sperm DNA; followed by washing the filters in OJx SSC at about 65oC. Thus the present invention also includes polynucleotides obtainable by screening an appropriate library (human or not) under stingent hybridization conditions with a labeled probe having the sequence of SEQ ID NO: 1 or a fragment thereof.

The skilled artisan will appreciate that, in many cases, an isolated cDNA sequence will be incomplete, in that the region coding for the polypeptide is short at the 5' end ofthe cDNA.

There are several methods available and well known to those skilled in the art to obtain full-length cDNAs, or extend short cDNAs, for example those based on the method of Rapid Amplification of cDNA ends (RACE) (see, for example, Frohman et al., PNAS USA 85, 8998-9002, 1988). Recent modifications ofthe technique, exemplified by the Marathon™ technology (Clontech Laboratories Inc.) for example, have significantly simplified the search for longer cDNAs. In the Marathon™ technology, cDNAs have been prepared from mRNA extracted from a chosen tissue and an 'adaptor' sequence ligated onto each end. Nucleic acid amplification (PCR) is then carried out to amplify the 'missing' 5' end ofthe cDNA using a combination of gene specific and adaptor specific oligonucleotide primers. The PCR reaction is then repeated using 'nested' primers, that is, primers designed to anneal within the amplified product (typically an adaptor specific primer that anneals further 3' in the adaptor sequence and a gene specific primer that anneals further 5' in the known gene sequence). The products of this reaction can then be analysed by DNA sequencing and a full-length cDNA constructed either by joining the product directly to the existing cDNA to give a complete sequence, or carrying out a separate full-length PCR using the new sequence information for the design ofthe 5' primer.

Recombinant polypeptides ofthe present invention may be prepared by processes well known in the art from genetically engineered host cells comprising expression systems. Accordingly, in a further aspect, the present invention relates to an expression system which comprises a polynucleotide ofthe present invention, to host cells which are genetically engineered with such expression systems and to the production of polypeptides ofthe invention by recombinant techniques. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs ofthe present invention.

For recombinant production, host cells can be genetically engineered to incorporate expression systems or portions thereof for polynucleotides ofthe present invention. Introduction of polynucleotides into host cells can be effected by methods described in many standard laboratory manuals, such as Davis et al. , Basic Methods in Molecular

Biology (1986) and Sarnbrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989). Preferred such methods include, for instance, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection.

Preferably the proteins ofthe invention are coexpressed with thioredoxin (TIT or Trx) in trans. Coexpression of thioredoxin in trans versus in cis is preferred to keep antigen free of thioredoxin without the need for protease. Thioredoxin coexpression eases the solubilisation ofthe proteins ofthe invention. Thioredoxin coexpression has also a significant impact on protein purification yield, on purified-protein solubility and quality.

Representative examples of appropriate hosts include bacterial cells, such as Streptococci, Staphylococci, E. coli, Streptomyces and Bacillus subtilis cells; fungal cells, such as yeast cells and Aspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells; and plant cells.

A great variety of expression systems can be used, for instance, chromosomal, episomal and virus-derived systems, e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SN40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids. The expression systems may contain control regions that regulate as well as engender expression. Generally, any system or vector which is able to maintain, propagate or express a polynucleotide to produce a polypeptide in a host may be used. The appropriate nucleotide sequence may be inserted into an expression system by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sarnbrook et al., Molecular Cloning, A Laboratory Manual (supra). Appropriate secretion signals may be incorporated into the desired polypeptide to allow secretion ofthe translated protein into the lumen ofthe endoplasmic reticulum, the periplasmic space or the extracellular environment. These signals may be endogenous to the polypeptide or they may be heterologous signals.

The expression system may also be a recombinant live microorganism, such as a virus or bacterium. The gene of interest can be inserted into the genome of a live recombinant virus or bacterium. Inoculation and in vivo infection with this live vector will lead to in vivo expression ofthe antigen and induction of immune responses. Therefore, in certain embodiments, polynucleotides encoding immunogenic polypeptides ofthe present invention are introduced into suitable mammalian host cells for expression using any of a number of known viral-based systems. In one illustrative embodiment, retroviruses provide a convenient and effective platform for gene delivery systems. A selected nucleotide sequence encoding a polypeptide ofthe present invention can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to a subject. A number of illustrative retroviral systems have been described (e.g., U.S. Pat. No. 5,219,740; Miller and Rosman (1989) BioTechniques 7:980-990; Miller, A. D. (1990) Human Gene Therapy 1 :5-14; Scarpa et al. (1991) Nirology 180:849-852; Burns et al. (1993) Proc. Νatl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie and Temin (1993) Cur. Opin. Genet. Develop. 3 : 102- 109.

In addition, a number of illustrative adenovirus-based systems have also been described. Unlike retroviruses which integrate into the host genome, adeno viruses persist extrachromosomally thus minimizing the risks associated with insertional mutagenesis

(Haj-Ahmad and Graham (1986) J. Nirol. 57:267-274; Bett et al. (1993) J. Nirol.

67:5911-5921; Mittereder et al. (1994) Human Gene Therapy 5:717-729; Seth et al.

(1994) J. Nirol. 68:933-940; Barr et al. (1994) Gene Therapy 1:51-58; Berkner, K. L. (1988) BioTechniques 6:616-629; and Rich et al. (1993) Human Gene Therapy 4:461-

476).

Various adeno-associated virus (AAN) vector systems have also been developed for polynucleotide delivery. AAN vectors can be readily constructed using techniques well known in the art. See, e.g., U.S. Pat. Νos. 5,173,414 and 5,139,941; International.

Publication Νos. WO 92/01070 and WO 93/03769; Lebkowski et al. (1988) Molec. Cell.

Biol. 8:3988-3996; Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory

Press); Carter, B. J. (1992) Current Opinion in Biotechnology 3:533-539; Muzyczka, Ν.

(1992) Current Topics in Microbiol. and Immunol. 158:97-129; Kotin, R. M. (1994) Human Gene Therapy 5:793-801; Shelling and Smith (1994) Gene Therapy 1:165-169; and Zhou et al. (1994) J. Exp. Med. 179:1867-1875. Additional viral vectors use ul for delivering the nucleic acid molecules encoding polypeptides ofthe present invention by gene transfer include those derived from the pox family of viruses, such as vaccinia virus and avian poxvirus. By way of example, vaccinia virus recombinants expressing the novel molecules can be constructed as follows. The DNA encoding a polypeptide is first inserted into an appropriate vector so that it is adjacent to a vaccinia promoter and flanking vaccinia DNA sequences, such as the sequence encoding thymidine kinase (TK). This vector is then used to transfect cells which are simultaneously infected with vaccinia. Homologous recombination serves to insert the vaccinia promoter plus the gene encoding the polypeptide of interest into the viral genome. The resulting TK.sup.(-) recombinant can be selected by culturing the cells in the presence of 5-bromodeoxyuridine and picking viral plaques resistant thereto.

A vaccinia-based infection/transfection system can be conveniently used to provide for inducible, transient expression or coexpression of one or more polypeptides described herein in host cells of an organism. In this particular system, cells are first infected in vitro with a vaccinia virus recombinant that encodes the bacteriophage T7 RNA polymerase. This polymerase displays exquisite specificity in that it only transcribes templates bearing T7 promoters. Following infection, cells are transfected with the polynucleotide or polynucleotides of interest, driven by a T7 promoter. The polymerase expressed in the cytoplasm from the vaccinia virus recombinant transcribes the transfected DNA into RNA which is then translated into polypeptide by the host translational machinery. The method provides for high level, transient, cytoplasmic production of large quantities of RNA and its translation products. See, e.g., Elroy-Stein and Moss, Proc. Natl. Acad. Sci. USA (1990) 87:6743-6747; Fuerst et al. Proc. Natl. Acad. Sci. USA (1986) 83:8122-8126.

Alternatively, avipoxviruses, such as the fowlpox and canarypox viruses, can also be used to deliver the coding sequences of interest. Recombinant avipox viruses, expressing immunogens from mammalian pathogens, are known to confer protective immunity when administered to non-avian species. The use of an Avipox vector is particularly desirable in human and other mammalian species since members ofthe Avipox genus can only productively replicate in susceptible avian species and therefore are not infective in mammalian cells. Methods for producing recombinant Avipoxviruses are known in the art and employ genetic recombination, as described above with respect to the production of vaccinia viruses. See, e.g., WO 91/12882; WO 89/03429; and WO 92/03545.

Any of a number of alphavirus vectors can also be used for delivery of polynucleotide compositions ofthe present invention, such as those vectors described in U.S. Patent Nos. 5,843,723; 6,015,686; 6,008,035 and 6,015,694. Certain vectors based on Venezuelan Equine Encephalitis (VEE) can also be used, illustrative examples of which can be found in U.S. Patent Nos. 5,505,947 and 5,643,576.

Moreover, molecular conjugate vectors, such as the adenovirus chimeric vectors described in Michael et al. J. Biol. Chem. (1993) 268:6866-6869 and Wagner et al. Proc. Natl. Acad. Sci. USA (1992) 89:6099-6103, can also be used for gene delivery under the invention.

Additional illustrative information on these and other known viral-based delivery systems can be found, for example, in Fisher-Hoch et al, Proc. Natl. Acad. Sci. USA 86:317-321, 1989; Flexner et al., Ann. N.Y. Acad. Sci. 5<59:86-103, 1989; Flexner et al., Vaccine 8:11-21, 1990; U.S. Patent Nos. 4,603,112, 4,769,330, and 5,017,487; WO 89/01973; U.S. Patent No. 4,777,127; GB 2,200,651; EP 0,345,242; WO 91/02805; Berkner, Biotechniques 6:616-621, 1988; Rosenfeld et al., Science 252:431-434, 1991; KoUs et al., Proc. Natl. Acad. Sci. USA 91 :215-219, 1994; Kass-Eisler et al, Proc. Natl. Acad. Sci. USA 90:11498-11502, 1993; Guzman et al., Circulation 55:2838-2848, 1993; and Guzman et al., Cir. Res. 73:1202-1207, 1993.

The recombinant live microorganisms described above can be virulent, or attenuated in various ways in order to obtain live vaccines. Such live vaccines also form part ofthe invention.

In certain embodiments, a polynucleotide may be integrated into the genome of a target cell. This integration may be in the specific location and orientation via homologous recombination (gene replacement) or it may be integrated in a random, non-specific location (gene augmentation). In yet further embodiments, the polynucleotide may be stably maintained in the cell as a separate, episomal segment of DNA. Such polynucleotide segments or "episomes" encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle. The manner in which the expression construct is delivered to a cell and where in the cell the polynucleotide remains is dependent on the type of expression construct employed.

In another embodiment ofthe invention, a polynucleotide is administered/delivered as "naked" DNA, for example as described in Ulmer et al, Science 259:1145-1149, 1993 and reviewed by Cohen, Science 259:1691-1692, 1993. The uptake of naked DNA may be increased by coating the DNA onto biodegradable beads, which are efficiently transported into the cells.

In still another embodiment, a composition ofthe present invention can be delivered via a particle bombardment approach, many of which have been described. In one illustrative example, gas-driven particle acceleration can be achieved with devices such as those manufactured by Powderject Pharmaceuticals PLC (Oxford, UK) and Powderject

Vaccines Inc. (Madison, Wl), some examples of which are described in U.S. Patent Nos. 5,846,796; 6,010,478; 5,865,796; 5,584,807; and EP Patent No. 0500 799. This approach offers a needle-free delivery approach wherein a dry powder formulation of microscopic particles, such as polynucleotide or polypeptide particles, are accelerated to high speed within a helium gas jet generated by a hand held device, propelling the particles into a target tissue of interest.

In a related embodiment, other devices and methods that may be useful for gas-driven needle-less injection of compositions ofthe present invention include those provided by Bioject, Inc. (Portland, OR), some examples of which are described in U.S. Patent Nos. 4,790,824; 5,064,413; 5,312,335; 5,383,851; 5,399,163; 5,520,639 and 5,993,412.

Polypeptides ofthe present invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, ion metal affinity chromatography (IMAC) is employed for purification. Well-known techniques for refolding proteins may be employed to regenerate active conformation when the polypeptide is denatured during intracellular synthesis, isolation and or purification.

Another important aspect ofthe invention relates to a method for inducing , re-inforcing or modulating an immunological response in a mammal which comprises inoculating the mammal with a fragment or the entire polypeptide or polynucleotide ofthe invention, adequate to produce antibody and/or T cell immune response for prophylaxis or for therapeutic treatment of cancer and autoimmune diseases and related conditions. Yet another aspect of the invention relates to a method of inducing, re-inforcing or modulating immunological response in a mammal which comprises, delivering a polypeptide ofthe present invention via a vector or cell directing expression ofthe polynucleotide and coding for the polypeptide in vivo in order to induce such an immunological response to produce immune responses for prophylaxis or treatment of said mammal from diseases.

A further aspect ofthe invention relates to an immunological/vaccine formulation (composition) which, when introduced into a mammalian host, induces, re-inforces or modulates an immunological response in that mammal to a polypeptide ofthe present invention wherein the composition comprises a polypeptide or polynucleotide ofthe invention or an immunological fragment thereof as herein before defined. The vaccine formulation may further comprise a suitable carrier. Since a polypeptide may be broken down in the stomach, it is preferably administered parenterally (for instance, subcutaneous, intramuscular, intravenous, or intradermal injection). Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood ofthe recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials and may be stored in a freeze-dried condition requiring only the addition ofthe sterile liquid carrier immediately prior to use. A further aspect ofthe invention relates to the in vitro induction of immune responses to a fragment or the entire polypeptide or polynucleotide ofthe present invention or a molecule comprising the polypeptide or polynucleotide ofthe present invention, using cells from the immune system of a mammal, and reirrfusing these activated immune cells ofthe mammal for the treatment of disease. Activation ofthe cells from the immune system is achieved by in vitro incubation with the entire polypeptide or polynucleotide of the present invention or a molecule comprising the polypeptide or polynucleotide ofthe present invention in the presence or absence of various immunomodulator molecules. A further aspect ofthe invention relates to the immunization of a mammal by administration of antigen presenting cells modified by in vitro loading with part or the entire polypeptide ofthe present invention or a molecule comprising the polypeptide of the present invention and administered in vivo in an immunogenic way. Alternatively, antigen presenting cells can be transfected in vitro with a vector containing a fragment or the entire polynucleotide ofthe present invention or a molecule comprising the polynucleotide ofthe present invention, such as to express the corresponding polypeptide, and administered in vivo in an immunogenic way.

According to another embodiment, the pharmaceutical compositions described herein will comprise one or more immunostimulants in addition to the immunogenic polynucleotide, polypeptide, antibody, T-cell and/or antigen presenting cell (APC) compositions of this invention. An immunostimulant refers to essentially any substance that enhances or potentiates an immune response (antibody and/or cell-mediated) to an exogenous antigen. One preferred type of immunostimulant comprises an adjuvant. Many adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and a stimulator of immune responses, such as lipid A, Bortadella pertussis or Mycobacterium tuberculosis derived proteins. Certain adjuvants are commercially available as, for example, Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, MI); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, NJ); AS-2 (GlaxoSmithKline, Philadelphia, PA); aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; salts of calcium, iron or zinc; an insoluble suspension of acylated tyrosine; acylated sugars; cationically or anionically derivatized polysaccharides; polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A and quil A. Cytokines, such as GM-CSF, interleukin-2, -7, -12, and other like growth factors, may also be used as adjuvants.

Within certain embodiments ofthe invention, the adjuvant composition is preferably one that induces an immune response predominantly ofthe Thl type. High levels of Thl-type cytokines (e.g., IFN-γ, TNFα, IL-2 and IL-12) tend to favor the induction of cell mediated immune responses to an administered antigen. In contrast, high levels of Th2- type cytokines (e.g., IL-4, IL-5, IL-6 and IL-10) tend to favor the induction of humoral immune responses. Following application of a vaccine as provided herein, a patient will support an immune response that includes Thl- and Th2-type responses. Within a preferred embodiment, in which a response is predominantly Thl-type, the level of Thl- type cytokines will increase to a greater extent than the level of Th2-type cytokines. The levels of these cytokines may be readily assessed using standard assays. For a review of the families of cytokines, see Mosmann and Coffman, Ann. Rev. Immunol. 7:145-173, 1989.

Certain preferred adjuvants for eliciting a predominantly Thl-type response include, for example, a combination of monophosphoryl lipid A, preferably 3-de-O-acylated monophosphoryl lipid A, together with an aluminum salt. MPL® adjuvants are available from Corixa Corporation (Seattle, WA; see, for example, US Patent Nos. 4,436,727; 4,877,611 ; 4,866,034 and 4,912,094). CpG-containing oligonucleotides (in which the CpG dinucleotide is unmethylated) also induce a predominantly Thl response. Such oligonucleotides are well known and are described, for example, in WO 96/02555, WO 99/33488 and U.S. Patent Nos. 6,008,200 and 5,856,462. Immunostimulatory DNA sequences are also described, for example, by Sato et al, Science 273:352, 1996. Another preferred adjuvant comprises a saponin, such as Quil A, or derivatives thereof, including QS21 and QS7 (Aquila Biopharmaceuticals Inc., Framingham, MA); Escin; Digitonin; or Gypsophila or Chenopodium quinoa saponins . Other preferred formulations include more than one saponin in the adjuvant combinations ofthe present invention, for example combinations of at least two ofthe following group comprising QS21, QS7, Quil A, β- escin, or digitonin.

Alternatively the saponin formulations may be combined with vaccine vehicles composed of chitosan or other polycationic polymers, polylactide and polylactide-co-glycolide particles, poly-N-acetyl glucosamine-based polymer matrix, particles composed of polysaccharides or chemically modified polysaccharides, liposomes and lipid-based particles, particles composed of glycerol monoesters, etc. The saponins may also be formulated in the presence of cholesterol to form particulate structures such as liposomes or ISCOMs. Furthermore, the saponins may be formulated together with a polyoxyethylene ether or ester, in either a non-particulate solution or suspension, or in a particulate structure such as a paucilamelar liposome or ISCOM. The saponins may also be formulated with excipients such as CarbopolR to increase viscosity, or may be formulated in a dry powder form with a powder excipient such as lactose.

In one preferred embodiment, the adjuvant system includes the combination of a monophosphoryl lipid A and a saponin derivative, such as the combination of QS21 and 3D-MPL® adjuvant, as described in WO 94/00153, or a less reactogenic composition where the QS21 is quenched with cholesterol, as described in WO 96/33739. Other preferred formulations comprise an oil-in-water emulsion and tocopherol. Another particularly preferred adjuvant formulation employing QS21, 3D-MPL® adjuvant and tocopherol in an oil-in-water emulsion is described in WO 95/17210. Another enhanced adjuvant system involves the combination of a CpG-containing oligonucleotide and a saponin derivative particularly the combination of CpG and QS21 as disclosed in WO 00/09159 or in WO 00/62800. Preferably the formulation additionally comprises an oil in water emulsion and tocopherol.

Additional illustrative adjuvants for use in the pharmaceutical compositions ofthe invention include Montanide ISA 720 (Seppic, France), SAF (Chiron, California, United States), ISCOMS (CSL), MF-59 (Chiron), the SBAS series of adjuvants (e.g., SBAS-2 or SBAS-4, available from GlaxoSmithKline Biologicals, Rixensart, Belgium), Detox ^• (Enhanzyn®) (Corixa, Hamilton, MT), RC-529 (Corixa, Hamilton, MT) and other aminoalkyl glucosaminide 4-phosphates (AGPs), such as those described in pending U.S. Patent Application Serial Nos. 08/853,826 and 09/074,720, the disclosures of which are incorporated herein by reference in their entireties, and polyoxyethylene ether adjuvants such as those described in WO 99/52549A1. Other preferred adjuvants include adjuvant molecules ofthe general formula (I):

HO(CH₂CH₂O)_n-A-R Wherein, n is 1-50, A is a bond or -C(O)-, R is Cl-50 alkyl or Phenyl Cl-50 alkyl. One embodiment ofthe present invention consists of a vaccine formulation comprising a polyoxyethylene ether of general formula (I), wherein n is between 1 and 50, preferably 4-24, most preferably 9; the R component is Cl-50, preferably C4-C20 alkyl and most preferably C12 alkyl, and A is a bond. The concentration ofthe polyoxyethylene ethers should be in the range 0.1-20%, preferably from 0.1-10%, and most preferably in the range 0.1-1%. Preferred polyoxyethylene ethers are selected from the following group: polyoxyethylene-9-lauryl ether, polyoxyethylene-9-steoryl ether, polyoxyethylene-8- steoryl ether, polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether, and polyoxyethylene-23-lauryl ether. Polyoxyethylene ethers such as polyoxyethylene lauryl ether are described in the Merck index (12th edition: entry 7717). These adjuvant molecules are described in WO 99/52549. The polyoxyethylene ether according to the general formula (I) above may, if desired, be combined with another adjuvant. For example, a preferred adjuvant combination is preferably with CpG as described in the pending UK patent application GB 9820956.2. Preferably a carrier is also present in the vaccine composition according to the invention. The carrier may be an oil in water emulsion, or an aluminium salt, such as aluminium phosphate or aluminium hydroxide.

A preferred oil-in-water emulsion comprises a metabolisible oil, such as squalene, alpha tocopherol and Tween 801 In a particularly preferred aspect the antigens in the vaccine composition according to the invention are combined with QS21 and 3D-MPL in such an emulsion. Additionally the oil in water emulsion may contain span 85 and or lecithin and/or tricaprylin.

Typically for human administration QS21 and 3D-MPL will be present in a vaccine in the range of lμg - 200μg, such as 10-100μg, preferably lOμg - 50μg per dose. Typically the oil in water will comprise from 2 to 10% squalene, from 2 to 10% alpha tocopherol and from 0.3 to 3% tween 80. Preferably the ratio of squalene: alpha tocopherol is equal to or less than 1 as this provides a more stable emulsion. Span 85 may also be present at a level of 1%. In some cases it may be advantageous that the vaccines ofthe present invention will further contain a stabiliser.

Non-toxic oil in water emulsions preferably contain a non-toxic oil, e.g. squalane or squalene, an emulsifier, e.g. Tween 80, in an aqueous carrier. The aqueous carrier may be, for example, phosphate buffered saline.

A particularly potent adjuvant formulation involving QS21, 3D-MPL and tocopherol in an oil in water emulsion is described in WO 95/17210.

The present invention also provides a polyvalent vaccine composition comprising a vaccine formulation ofthe invention in combination with other antigens, in particular antigens useful for treating cancers, autoimmune diseases and related conditions. Such a polyvalent vaccine composition may include a TH-1 inducing adjuvant as hereinbefore described.

According to another embodiment of this invention, an immunogenic composition described herein is delivered to a host via antigen presenting cells (APCs), such as dendritic cells, macrophages, B cells, monocytes and other cells that may be engineered to be efficient APCs. Such cells may, but need not, be genetically modified to increase the capacity for presenting the antigen, to improve activation and/or maintenance ofthe T cell response, to have anti-tumor effects per se and or to be immunologically compatible with the receiver (i.e., matched HLA haplotype). APCs may generally be isolated from any of a variety of biological fluids and organs, including tumour and peritumoural tissues, and may be autologous, allogeneic, syngeneic or xenogeneic cells.

Certain preferred embodiments ofthe present invention use dendritic cells or progenitors thereof as antigen-presenting cells. Dendritic cells are highly potent APCs (Banchereau and Steinman, Nature 392:245-251, 1998) and have been shown to be effective as a physiological adjuvant for eliciting prophylactic or therapeutic antitumour immunity (see Timmerman and Levy, Ann. Rev. Med. 5(7:507-529, 1999). In general, dendritic cells may be identified based on their typical shape (stellate in situ, with marked cytoplasmic processes (dendrites) visible in vitro), their ability to take up, process and present antigens with high efficiency and their ability to activate naϊve T cell responses. Dendritic cells may, of course, be engineered to express specific cell-surface receptors or ligands that are not commonly found on dendritic cells in vivo or ex vivo, and such modified dendritic cells are contemplated by the present invention. As an alternative to dendritic cells, secreted vesicles antigen-loaded dendritic cells (called exosomes) may be used within a vaccine (see Zitvogel et al., Nature Med. 4:594-600, 1998). Dendritic cells and progenitors may be obtained from peripheral blood, bone marrow, tumour-infiltrating cells, peritumoural tissues-infiltrating cells, lymph nodes, spleen, skin, umbilical cord blood or any other suitable tissue or fluid. For example, dendritic cells may be differentiated ex vivo by adding a combination of cytokines such as GM-CSF, IL- 4, IL-13 and/or TNFα to cultures of monocytes harvested from peripheral blood. Alternatively, CD34 positive cells harvested from peripheral blood, umbilical cord blood or bone marrow may be differentiated into dendritic cells by adding to the culture medium combinations of GM-CSF, IL-3, TNFα, CD40 ligand, LPS, flt3 ligand and/or other compound(s) that induce differentiation, maturation and proliferation of dendritic cells. Dendritic cells are conveniently categorized as "immature'' and "mature" cells, which allows a simple way to discriminate between two well characterized phenotypes.

However, this nomenclature should not be construed to exclude all possible intermediate stages of differentiation. Immature dendritic cells are characterized as APC with a high capacity for antigen uptake and processing, which correlates with the high expression of Fcγ receptor and mannose receptor. The mature phenotype is typically characterized by a lower expression of these markers, but a high expression of cell surface molecules responsible for T cell activation such as class I and class II MHC, adhesion molecules (e.g., CD54 and GDI 1) and costimulatory molecules (e.g., CD40, CD80, CD86 and 4- 1BB).

APCs may generally be transfected with a polynucleotide ofthe invention (or portion or other variant thereof) such that the encoded polypeptide, or an immunogenic portion thereof, is expressed on the cell surface. Such transfection may take place ex vivo, and a pharmaceutical composition comprising such transfected cells may then be used for therapeutic purposes, as described herein. Alternatively, a gene delivery vehicle that targets a dendritic or other antigen presenting cell may be administered to a patient, resulting in transfection that occurs in vivo. In vivo and ex vivo transfection of dendritic cells, for example, may generally be performed using any methods known in the art, such as those described in WO 97/24447, or the gene gun approach described by Mahvi et al., Immunology and cell Biology 75:456-460, 1997. Antigen loading of dendritic cells may be achieved by incubating dendritic cells or progenitor cells with the tumour polypeptide, DNA (naked or within a plasmid vector) or RNA; or with antigen-expressing recombinant bacterium or viruses (e.g., vaccinia, fowlpox, adeno virus or lentivirus vectors). Prior to loading, the polypeptide may be covalently conjugated to an immunological partner that provides T cell help (e.g., a carrier molecule). Alternatively, a dendritic cell may be pulsed with a non-conjugated immunological partner, separately or in the presence of the polypeptide.

While any suitable carrier known to those of ordinary skill in the art may be employed in the pharmaceutical compositions of this invention, the type of carrier will typically vary depending on the mode of administration. Compositions ofthe present invention may be formulated for any appropriate manner of administration, including for example, topical, oral, nasal, mucosal, intravenous, intracranial, intraperitoneal, subcutaneous and intramuscular administration. Carriers for use within such pharmaceutical compositions are biocompatible, and may also be biodegradable. In certain embodiments, the formulation preferably provides a relatively constant level of active component release. In other embodiments, however, a more rapid rate of release immediately upon administration may be desired. The formulation of such compositions is well within the level of ordinary skill in the art using known techniques. Illustrative carriers useful in this regard include microparticles of poly(lactide-co-glycolide), polyacrylate, latex, starch, cellulose, dextran and the like. Other illustrative delayed-release carriers include supramolecular biovectors, which comprise a non-liquid hydrophilic core (e.g., a cross-linked polysaccharide or oligosaccharide) and., optionally, an external layer comprising an amphiphilic compound, such as a phospholipid (see e.g., U.S. Patent No. 5,151,254 and PCT applications WO 94/20078, WO/94/23701 and WO 96/06638). The amount of active compound contained within a sustained release formulation depends upon the site of implantation, the rate and expected duration of release and the nature ofthe condition to be treated or prevented.

In another illustrative embodiment, biodegradable microspheres (e.g., polylactate polyglycolate) are employed as carriers for the compositions of this invention. Suitable biodegradable microspheres are disclosed, for example, in U.S. Patent Nos. 4,897,268; 5,075,109; 5,928,647; 5,811,128; 5,820,883; 5,853,763; 5,814,344, 5,407,609 and 5,942,252. Modified hepatitis B core protein carrier systems, such as described in WO/99 40934, and references cited therein, will also be useful for many applications. Another illustrative carrier/delivery system employs a carrier comprising particulate-protein complexes, such as those described in U.S. Patent No. 5,928,647, which are capable of inducing a class I-restricted cytotoxic T lymphocyte responses in a host. The pharmaceutical compositions ofthe invention will often further comprise one or more buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannose, sucrose or dextrans), mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, bacteriostats, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), solutes that render the formulation isotonic, hypotonic or weakly hypertonic with the blood of a recipient, suspending agents, thickening agents and/or preservatives. Alternatively, compositions ofthe present invention may be formulated as a lyophilizate. The pharmaceutical compositions described herein may be presented in unit-dose or multi-dose containers, such as sealed ampoules or vials. Such containers are typically sealed in such a way to preserve the sterility and stability ofthe formulation until use. In general, formulations may be stored as suspensions, solutions or emulsions in oily or aqueous vehicles. Alternatively, a pharmaceutical composition may be stored in a freeze- dried condition requiring only the addition of a sterile liquid carrier immediately prior to use.

The development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens, including e.g., oral, parenteral, intravenous, intranasal, and intramuscular administration and formulation, is well known in the art, some of which are briefly discussed below for general purposes of illustration.

In certain applications, the pharmaceutical compositions disclosed herein may be delivered via oral administration to an animal. As such, these compositions may be formulated with an inert diluent or with an assimilable edible carrier, or they may be enclosed in hard- or soft-shell gelatin capsule, or they may be compressed into tablets, or they may be incorporated directly with the food ofthe diet.

The active compounds may even be incorporated with excipients and used in the form of ingestible tablets, buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like (see, for example, Mathiowitz et al, Nature 1997 Mar 27;386(6623):410-4; Hwang et al, Crit Rev Ther Drug Carrier Syst 1998;15(3):243-84; U. S. Patent 5,641 ,515; U. S. Patent 5,580,579 and U. S. Patent 5,792,451). Tablets, troches, pills, capsules and the like may also contain any of a variety of additional components, for example, a binder, such as gum tragacanth, acacia, cornstarch, or gelatin; excipients, such as dicalcium phosphate; a disintegrating agent, such as corn starch, potato starch, alginic acid and the like; a lubricant, such as magnesium stearate; and a sweetening agent, such as sucrose, lactose or saccharin may be added or a flavoring agent, such as peppermint, oil of wintergreen, or cherry flavoring. When the dosage unit form is a capsule, it may contain, in addition to materials ofthe above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form ofthe dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar, or both. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compounds may be incorporated into sustained-release preparation and formulations.

Typically, these formulations will contain at least about 0.1% ofthe active compound or more, although the percentage ofthe active ingredient(s) may, of course, be varied and may conveniently be between about 1 or 2% and about 60% or 70% or more ofthe weight or volume ofthe total formulation. Naturally, the amount of active compound(s) in each therapeutically useful composition may be prepared is such a way that a suitable dosage will be obtained in any given unit dose ofthe compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.

For oral administration the compositions ofthe present invention may alternatively be incoφorated with one or more excipients in the form of a mouthwash, dentifrice, buccal tablet, oral spray, or sublingual orally-administered formulation. Alternatively, the active ingredient may be incoφorated into an oral solution such as one containing sodium borate, glycerin and potassium bicarbonate, or dispersed in a dentifrice, or added in a therapeutically-effective amount to a composition that may include water, binders, abrasives, flavoring agents, foaming agents, and humectants. Alternatively the compositions may be fashioned into a tablet or solution form that may be placed under the tongue or otherwise dissolved in the mouth.

In certain circumstances it will be desirable to deliver the pharmaceutical compositions disclosed herein parenterally, intravenously, intramuscularly, or even intraperitoneally. Such approaches are well known to the skilled artisan, some of which are further described, for example, in U. S. Patent 5,543,158; U. S. Patent 5,641,515 and U. S. Patent 5,399,363. In certain embodiments, solutions ofthe active compounds as free base or pharmacologically acceptable salts may be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations generally will contain a preservative to prevent the growth of microorganisms.

Illustrative pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (for example, see U. S. Patent 5,466,468). In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and/or by the use of surfactants. The prevention of the action of microorganisms can be facilitated by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absoφtion ofthe injectable compositions can be brought about by the use in the compositions of agents delaying absoφtion, for example, aluminum monostearate and gelatin.

In one embodiment, for parenteral administration in an aqueous solution, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, a sterile aqueous medium that can be employed will be known to those of skill in the art in light ofthe present disclosure. For example, one dosage may be dissolved in 1 ml of isotonic NaCI solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition ofthe subject being treated. Moreover, for human administration, preparations will of course preferably meet sterility, pyrogenicity, and the general safety and purity standards as required by FDA Office of Biologies standards.

In another embodiment ofthe invention, the compositions disclosed herein may be formulated in a neutral or salt form. Illustrative pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups ofthe protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.

The carriers can further comprise any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absoφtion delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incoφorated into the compositions. The phrase "pharmaceutically-acceptable" refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human.

In certain embodiments, the pharmaceutical compositions may be delivered by intranasal sprays, inhalation, and/or other aerosol delivery vehicles. Methods for delivering genes, nucleic acids, and peptide compositions directly to the lungs via nasal aerosol sprays has been described, e.g., in U. S. Patent 5,756,353 and U. S. Patent 5,804,212. Likewise, the delivery of drugs using intranasal microparticle resins (Takenaga et al., J Controlled Release 1998 Mar 2;52(l-2):81-7) and lysophosphatidyl-glycerol compounds (U. S. Patent 5,725,871) are also well-known in the pharmaceutical arts. Likewise, illustrative transmucosal drug delivery in the form of a polytetrafluoroetheylene support matrix is described in U. S. Patent 5,780,045.

In certain embodiments, liposomes, nanocapsules, microparticles, lipid particles, vesicles, and the like, are used for the introduction ofthe compositions ofthe present invention into suitable host cells/organisms. In particular, the compositions ofthe present invention may be formulated for delivery either encapsulated in a lipid particle, a liposome, a vesicle, a nanosphere, or a nanoparticle or the like. Alternatively, compositions ofthe present invention can be bound, either covalently or non-covalently, to the surface of such carrier vehicles.

The formation and use of liposome and liposome-like preparations as potential drug carriers is generally known to those of skill in the art (see for example, Lasic, Trends Biotechnol 1998 Jul;16(7):307-21; Takakura, Nippon Rinsho 1998 Mar;56(3):691-5; Chandran et al, Indian J Exp Biol. 1997 Aug;35(8):801-9; Margalit, Crit Rev Ther Drug Carrier Syst. 1995;12(2-3):233-61; U.S. Patent 5,567,434; U.S. Patent 5,552,157; U.S. Patent 5,565,213; U.S. Patent 5,738,868 and U.S. Patent 5,795,587, each specifically incoφorated herein by reference in its entirety).

Liposomes have been used successfully with a number of cell types that are normally difficult to transfect by other procedures, including T cell suspensions, primary hepatocyte cultures and PC 12 cells (Renneisen et al, J Biol Chem. 1990 Sep 25;265(27):16337-42; Muller et al, DNA Cell Biol. 1990 Apr;9(3):221-9). In addition, liposomes are free ofthe DNA length constraints that are typical of viral-based delivery systems. Liposomes have been used effectively to introduce genes, various drugs, radiotherapeutic agents, enzymes, viruses, transcription factors, allosteric effectors and the like, into a variety of cultured cell lines and animals. Furthermore, he use of liposomes does not appear to be associated with autoimmune responses or unacceptable toxicity after systemic delivery.

In certain embodiments, liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs). Alternatively, in other embodiments, the invention provides for pharmaceutically- acceptable nanocapsule formulations ofthe compositions ofthe present inventiomNanocapsules can generally entrap compounds in a stable and reproducible way (see, for example, Quintanar-Guerrero et al, Drug Dev Ind Pharm. 1998

Dec;24(12):l 113-28).To avoid side effects due to intracellular polymeric overloading, such ultrafine particles (sized around 0J μm) may be designed using polymers able to be degraded in vivo. Such particles can be made as described, for example, by Couvreur et al, Crit Rev Ther Drug Carrier Syst. 1988;5(l):l-20; zur Muhlen et al, Eur J Pharm Biopharm. 1998 Mar;45(2): 149-55; Zambaux et al. J Controlled Release. 1998 Jan 2;50(l-3):31-40; and U. S. Patent 5,145,684.

This invention also relates to the use of polynucleotides, in the form of primers derived from the polynucleotides ofthe present invention, and of polypeptides, in the form of antibodies or reagents specific for the polypeptide ofthe present invention, as diagnostic reagents.

The identification of genetic or biochemical markers in blood or tissues that will enable the detection of very early changes along the carcinogenesis pathway will help in determining the best treatment for the patient. Surrogate tumour markers, such as polynucleotide expression, can be used to diagnose different forms and states of cancer. The identification of expression levels ofthe polynucleotides ofthe invention will be useful in both the staging ofthe cancerous disorder and grading the nature ofthe cancerous tissue. The staging process monitors the advancement ofthe cancer and is determined on the presence or absence of malignant tissue in the areas biopsied. The polynucleotides ofthe invention can help to perfect the staging process by identifying markers for the aggresivity of a cancer, for example the presence in different areas ofthe body. The grading ofthe cancer describes how closely a tumour resembles normal tissue of its same type and is assessed by its cell moφhology and other markers of differentiation. The polynucleotides of the invention can be useful in determining the tumour grade as they can help in the determination ofthe differentiation status ofthe cells of a tumour. The diagnostic assays offer a process for diagnosing or determining a susceptibility to cancers, autoimmune disease and related conditions through diagnosis by methods comprising determining from a sample derived from a subject an abnormally decreased or increased level of polypeptide or mRNA. This method of diagnosis is -known as differential expression. The expression of a particular gene is compared between a diseased tissue and a normal tissue. A difference between the polynucleotide-related gene, mRNA, or protein in the two tissues is compared, for example in molecular weight, amino acid or nucleotide sequence, or relative abundance, indicates a change in the gene, or a gene which regulates it, in the tissue ofthe human that was suspected of being diseased.

Decreased or increased expression can be measured at the RNA level. PolyA RNA is first isolated from the two tissues and the detection of mRNA encoded by a gene corresponding to a differentially expressed polynucleotide ofthe invention can be detected by, for example, in situ hybridization in tissue sections, reverse trascriptase- PCR, using Northern blots containing poly A+ mRNA, or any other direct or inderect RNA detection method. An increased or decreased expression of a given RNA in a diseased tissue compared to a normal tissue suggests that the transcript and/or the expressed protein has a role in the disease. Thus detection of a higher or lower level of mRNA corresponding to SEQ ID NO: 1 relative to normal level is indicative ofthe presence of cancer in the patient.

mRNA expression levels in a sample can be determined by generation of a library of expressed sequence tags (ESTs) from the sample. The relative representation of ESTs in the library can be used to assess the relative representation ofthe gene transcript in the starting sample. The EST analysis ofthe test can then be compared to the EST analysis of a reference sample to determine the relative expression levels ofthe polynucleotide of interest.

Other mRNA analyses can be carried out using serial analysis of gene expression (SAGE) methodology (Velculescu et. Al. Science (1995) 270:484) , differential display methodology (For example, US 5,776,683) or hybridization analysis which relies on the specificity of nucleotide interactions. Alternatively, the comparison could be made at the protein level. The protein sizes in the two tissues may be compared using antibodies to detect polypeptides in Western blots of protein extracts from the two tissues. Expression levels and subcellular localization may also be detected immunologically using antibodies to the corresponding protein. Further assay techniques that can be used to determine levels of a protein, such as a polypeptide ofthe present invention, in a sample derived from a host are well-known to those of skill in the art. A raised or decreased level of polypeptide expression in the diseased tissue compared with the same protein expression level in the normal tissue indicates that the expressed protein may be involved in the disease.

In the assays ofthe present invention, the diagnosis can be determined by detection of gene product expression levels encoded by at least one sequence set forth in SEQ ID NO: 1. A comparison ofthe mRNA or protein levels in a diseased versus normal tissue may also be used to follow the progression or remission of a disease.

A large number of polynucleotide sequences in a sample can be assayed using polynucleotide arrays. These can be used to examine differential expression of genes and to determine gene function. For example, arrays ofthe polynucleotide sequences SEQ ID NO: 1 can be used to determine if any ofthe polynucleotides are differentially expressed between a normal and cancer cell. In one embodiment ofthe invention, an array of oligonucleotides probes comprising the SEQ ID NO:l nucleotide sequence or fragments thereof can be constructed to conduct efficient screening of e.g., genetic mutations. Array technology methods are well known and have general applicability and can be used to address a variety of questions in molecular genetics including gene expression, genetic linkage, and genetic variability (see for example: M.Chee et al., Science, Vol 274, pp 610-613 (1996)).

"Diagnosis" as used herein includes determination of a subject's susceptibility to a disease, determination as to whether a subject presently has the disease, and also the prognosis of a subject affected by the disease. The present invention, further relates to a diagnostic kit for performing a diagnostic assay which comprises:

(a) a polynucleotide ofthe present invention, preferably the nucleotide sequence of SEQ ID NO: 1 , or of SEQ ID NO:5 or a fragment thereof ;

(b) a nucleotide sequence complementary to that of (a);

(c) a polypeptide ofthe present invention, preferably the polypeptide of SEQ ID NO:2, or a fragment thereof; or

(d) an antibody to a polypeptide ofthe present invention, preferably to the polypeptide of SEQ ID NO:2 or SEQ ID NO:6.

The nucleotide sequences ofthe present invention are also valuable for chromosomal localisation. The sequence is specifically targeted to, and can hybridize with, a particular location on an individual human chromosome. The mapping of relevant sequences to chromosomes according to the present invention is an important first step in correlating those sequences with gene associated disease. Once a sequence has been mapped to a precise chromosomal location, the physical position ofthe sequence on the chromosome can be correlated with genetic map data. Such data are found in, for example, V. McKusick, Mendelian Inheritance in Man (available on-line through Johns Hopkins University Welch Medical Library). The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheritance of physically adjacent genes).The differences in the cDNA or genomic sequence between affected and unaffected individuals can also be determined.

The polypeptides ofthe invention or their fragments or analogs thereof, or cells expressing them, can also be used as irnmunogens to produce antibodies immunospecific for polypeptides ofthe present invention. The term "immunospecific" means that the antibodies have substantially greater affinity for the polypeptides ofthe invention than their affinity for other related polypeptides in the prior art.

In a further aspect the invention provides an antibody immunospecific for a polypeptide according to the invention or an immunological fragment thereof as hereinbefore defined. Preferably the antibody is a monoclonal antibody Antibodies generated against polypeptides ofthe present invention may be obtained by administering the polypeptides or epitope-bearing fragments, analogs or cells to an animal, preferably a non-human animal, using routine protocols. For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler, G. and Milstein, C, Nature (1975) 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al, Immunology Today (1983) 4:72) and the EBV- hybridoma technique (Cole et al, Monoclonal Antibodies and Cancer Therapy, 77-96, Alan R. Liss, Inc., 1985).

Techniques for the production of single chain antibodies, such as those described in U.S. Patent No. 4,946,778, can also be adapted to produce single chain antibodies to polypeptides of this invention. Also, transgenic mice, or other organisms, including other mammals, may be used to express humanized antibodies.

The above-described antibodies may be employed to isolate or to identify clones expressing the polypeptide or to purify the polypeptides by affinity chromatography. The antibody ofthe invention may also be employed to prevent or treat cancer, particularly Crohn's disease, Colitis ulcerosa, colorectal cancer, lung cancer and preneoplasic lesions, breast, brain, uterus, muscle, eye and germ cell cancers, Wilm's tumour, retinoblastoma, rabdomyosarcoma, leimyosarcoma and synovial sarcoma, autoimmune disease and related conditions.

Another aspect ofthe invention relates to a method for inducing or modulating an immunological response in a mammal which comprises inoculating the mammal with a polypeptide ofthe present invention, adequate to produce antibody and/or T cell immune response to protect or ameliorate the symptoms or progression ofthe disease. Yet another aspect ofthe invention relates to a method of inducing or modulating immunological response in a mammal which comprises, delivering a polypeptide ofthe present invention via a vector directing expression ofthe polynucleotide and coding for the polypeptide in vivo in order to induce such an immunological response to produce antibody to protect said animal from diseases. It will be appreciated that the present invention therefore provides a method of treating and preventing metastasis associated with abnormal conditions such as, for instance, cancer and autoimmune diseases, and in particular, Crohn's disease and Colitis ulcerosa, colorectal cancer, lung cancer and preneoplasic lesions, breast, brain, uterus, muscle, eye and germ cell cancers, Wilm's tumour, retinoblastoma, rabdomyosarcoma, leimyosarcoma and synovial sarcoma, and other related conditions, related to either a presence of, an excess of, or an under-expression of, CASB88 polypeptide activity.

The present invention further provides for a method of screening compounds to identify those which stimulate or which inhibit the function ofthe CASB88 polypeptide. In general, agonists or antagonists may be employed for therapeutic and prophylactic puφoses for such diseases as hereinbefore mentioned. Compounds may be identified from a variety of sources, for example, cells, cell-free preparations, chemical libraries, and natural product mixtures. Such agonists, antagonists or inhibitors so-identified may be natural or modified substrates, ligands, receptors, enzymes, etc., as the case may be, of the polypeptide; or may be structural or functional mimetics thereof (see Coligan et al. , Current Protocols in Immunology l(2):Chapter 5 (1991)). Screening methods will be known to those skilled in the art. Further screening methods may be found in for example D. Bennett et al, J Mol Recognition, 8:52-58 (1995); and K. Johanson et al, J Biol Chem, 270(16):9459-9471 (1995) and references therein.

Thus the invention provides a method for screening to identify compounds which stimulate or which inhibit the function ofthe polypeptide ofthe invention which comprises a method selected from the group consisting of: (a) measuring the binding of a candidate compound to the polypeptide (or to the cells or membranes bearing the polypeptide) or a fusion protein thereof by means of a label directly or indirectly associated with the candidate compound;

(b) measuring the binding of a candidate compound to the polypeptide (or to the cells or , membranes bearing the polypeptide) or a fusion protein thereof in the presense of a labeled competitior;

(c) testing whether the candidate compound results in a signal generated by activation or inhibition ofthe polypeptide, using detection systems appropriate to the cells or cell membranes bearing the polypeptide; (d) mixing a candidate compound with a solution containing a polypeptide of claim 1, to form a mixture, measuring activity ofthe polypeptide in the mixture, and comparing the activity ofthe mixture to a standard; or

(e) detecting the effect of a candidate compound on the production of mRNA encoding said polypeptide and said polypeptide in cells, using for instance, an ELISA assay.

The polypeptide ofthe invention may be used to identify membrane bound or soluble receptors, if any, through standard receptor binding techniques known in the art. Well known screening methods may also be used to identify agonists and antagonists ofthe polypeptide of the invention which compete with the binding of the polypeptide of the invention to its receptors, if any.

Thus, in another aspect, the present invention relates to a screening kit for identifying agonists, antagonists, ligands, receptors, substrates, enzymes, etc. for polypeptides ofthe present invention; or compounds which decrease or enhance the production of such polypeptides, which comprises:

(a) a polypeptide ofthe present invention;

(b) a recombinant cell expressing a polypeptide ofthe present invention;

(c) a cell membrane expressing a polypeptide ofthe present invention; or (d) antibody to a polypeptide ofthe present invention; which polypeptide is preferably that of SEQ ID NO:2 or that of SEQ ID NO:6.

It will be readily appreciated by the skilled artisan that a polypeptide ofthe present invention may also be used in a method for the structure-based design of an agonist, antagonist or inhibitor ofthe polypeptide, by:

(a) determining in the first instance the three-dimensional structure ofthe polypeptide;

(b) deducing the three-dimensional structure for the likely reactive or binding site(s) of an agonist, antagonist or inhibitor;

(c) synthesing candidate compounds that are predicted to bind to or react with the deduced binding or reactive site; and

(d) testing whether the candidate compounds are indeed agonists, antagonists or inhibitors. Gene therapy may also be employed to effect the endogenous production of CASB88 polypeptide by the relevant cells in the subject. For an overview of gene therapy, see Chapter 20, Gene Therapy and other Molecular Genetic-based Therapeutic Approaches, (and references cited therein) in Human Molecular Genetics, T Strachan and A P Read, BIOS Scientific Publishers Ltd (1996).

Vaccine preparation is generally described in Pharmaceutical Biotechnology, Vol.61 Vaccine Design - the subunit and adjuvant approach, edited by Powell and Newman, Plenum Press, 1995. New Trends and Developments in Vaccines, edited by Voller et al., University Park Press, Baltimore, Maryland, U.S.A. 1978. Encapsulation within liposomes is described, for example, by FuUerton, U.S. Patent 4,235,877. Conjugation of proteins to macromolecules is disclosed, for example, by Likhite, U.S. Patent 4,372,945 and by Armor et al., U.S. Patent 4,474,757.

The amount of protein in each vaccine dose is selected as an amount which induces an immunoprotective response without significant, adverse side effects in typical vaccinees. Such amount will vary depending upon which specific immunogen is employed. Generally, it is expected that each dose will comprise 1-lOOODg of protein, preferably 2-1 OODg, most preferably 4-40 Dg. An optimal amount for a particular vaccine can be ascertained by standard studies involving observation of antibody titres and other responses in subjects. Following an initial vaccination, subjects may receive a boost in about 4 weeks.

"Isolated" means altered "by the hand of man" from the natural state. If an "isolated" composition or substance occurs in nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide or a polypeptide naturally present in a living animal is not "isolated," but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated", as the term is employed herein.

"Polynucleotide" generally refers to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA including single and double stranded regions. "Variant" refers to a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, but retains essential properties. A typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence ofthe variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences ofthe reference polypeptide and the variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination. A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a polynucleotide or polypeptide may be a naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques or by direct synthesis.

"Identity," as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. "Identity" and "similarity" can be readily calculated by known methods, including but not limited to those described in (Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988;

Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math, 48: 1073 (1988). Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S.F. et al, J. Molec. Biol. 215: 403-410 (1990). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al, NCBI NLM NIH Bethesda, MD 20894; Altschul, S., et al, J. Mol. Biol. 215: 403-410 (1990). The well known Smith Waterman algorithm may also be used to determine identity.

The preferred algorithm used is FASTA. The preferred parameters for polypeptide or polynuleotide sequence comparison using this algorithm include the following:

Gap Penalty: 12

Gap extension penalty: 4

Word size: 2, max 6

Preferred parameters for polypeptide sequence comparison with other methods include the following:

1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970)

Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl. Acad. Sci. USA. 89:10915-10919 (1992)

Gap Penalty: 12

Gap Length Penalty: 4

A program useful with these parameters is publicly available as the "gap" program from Genetics Computer Group, Madison Wl. The aforementioned parameters are the default parameters for polypeptide comparisons (along with no penalty for end gaps).

Preferred parameters for polynucleotide comparison include the following: 1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970) Comparison matrix: matches = +10, mismatch = 0 Gap Penalty: 50 Gap Length Penalty: 3 A program useful with these parameters is publicly available as the "gap" program from Genetics Computer Group, Madison Wl. The aforementioned parameters are the default parameters for polynucleotide comparisons.

By way of example, a polynucleotide sequence ofthe present invention may be identical to the reference sequence of SEQ ID NO:l, that is be 100% identical, or it may include up to a certain integer number of nucleotide alterations as compared to the reference sequence. Such alterations are selected from the group consisting of at least one nucleotide deletion, substitution, including transition and transversion, or insertion, and wherein said alterations may occur at the 5' or 3' terminal positions ofthe reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence. The number of nucleotide alterations is determined by multiplying the total number of nucleotides in SEQ ID NO:l by the numerical percent ofthe respective percent identity(divided by 100) and subtracting that product from said total number of nucleotides in SEQ ID NO:l, or: nn < xn - (xn • y), wherein nn is the number of nucleotide alterations, xn is the total number of nucleotides in SEQ ID NO:l, and y is, for instance, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for 95%,etc, and wherem any non-integer product of xn and y is rounded down to the nearest integer prior to subtracting it from xn. Alterations of a polynucleotide sequence encoding the polypeptide of SEQ ID NO:2 may create nonsense, missense or frameshift mutations in this coding sequence and thereby alter the polypeptide encoded by the polynucleotide following such alterations.

Similarly, a polypeptide sequence ofthe present invention may be identical to the reference sequence of SEQ ID NO:2, that is be 100% identical, or it may include up to a certain integer number of amino acid alterations as compared to the reference sequence such that the % identity is less than 100%. Such alterations are selected from the group consisting of at least one amino acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the amino- or carboxy-terminal positions ofthe reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids in the reference sequence or in one or more contiguous groups within the reference sequence. The number of amino acid alterations for a given % identity is determined by multiplying the total number of amino acids in SEQ ID NO:2 by the numerical percent of the respective percent identity(divided by 100) and then subtracting that product from said total number of amino acids in SEQ ID NO:2, or: na≤xa - (xa • y), wherein na is the number of amino acid alterations, xa is the total number of amino acids in SEQ ID NO:2, and y is, for instance 0.70 for 70%, 0.80 for 80%, 0.85 for 85% etc., and wherein any non-integer product of xa and y is rounded down to the nearest integer prior to subtracting it from xa.

"Homolog" is a generic term used in the art to indicate a polynucleotide or polypeptide sequence possessing a high degree of sequence relatedness to a subject sequence. Such relatedness may be quntified by determining the degree of identity and/or similarity between the sequences being compared as hereinbefore described. Falling within this generic term are the terms "ortholog", meaning a polynucleotide or polypeptide that is the functional equivalent of a polynucleotide or polypeptide in another species and "paralog" meaning a functionally similar sequence when considered within the same species.

Figure Legends

Figure 1 : CASB88 RT-PCR results. Individual tumour samples (black bars) and matched normal adjacent tissue (white bars). Primer Pair 1 amplification (Sybr detection).

Figure 2: CASB88 RT-PCR results. Individual tumour samples (black bars) and matched normal adjacent tissue (white bars). Primer Pair 2 amplification (Sybr detection).

Figure 3: CASB88 RT-PCR results. Individual tumour samples (black bars) and matched normal adjacent tissue (white bars). Primer Pair 3 amplification (Sybr detection).

Figure 4: CASB88 RT-PCR results. Individual tumour samples (black bars) and matched normal adjacent tissue (white bars). Primer Pair 4 amplification (Sybr detection).

Figure 5: CASB88 RT-PCR results. Individual tumour samples (black bars) and matched normal adjacent tissue (white bars). Primer Pair 1 amplification (TaqMan detection).

Figure 6: CASB88 RT-PCR results on normal tissues. Primer Pair 1 amplification (Sybr detection).

Figure 7: CASB88 RT-PCR results on normal tissues. Primer Pair 2 amplification (Sybr detection).

Figure 8: CASB88 RT-PCR results on normal tissues. Primer Pair 3 amplification (Sybr detection).

Figure 9: CASB88 RT-PCR results on normal tissues. Primer Pair 4 amplification (Sybr detection).

Figure 10: CASB88 RT-PCR results on normal tissues. Primer Pair 1 amplification (TaqMan detection). Figure 11 : CASB88 RT-PCR results on normal tissues. Primer Pair 5 amplification (Sybr detection).

Examples

Example 1 : Subtractive cDNA cloning of tumour-associated antigen (TAA) candidates.

Subtractive cDNA libraries are produced using standard technologies. Briefly, total RNA is extracted from frozen (-70°C) tumour and matched normal samples using the TriPure reagent and protocol (Boehringer). Target RNA is prepared by pooling total RNA from various tumours samples (30 μg each). Driver RNA is prepared by pooling total RNA from various matched normal samples (10 μg each) and total RNA from various normal tissues 1 (0 μg each). Total RNA from normal tissues is purchased from InVitrogen.

Messenger RNA is purified from total RNA using oligo-dT magnetic bead technology (Dynal) and quantified by spectrofluorimetry (BioRad).

Target and driver mRNA are reverse transcribed into cDNA using one of two strategies: 1) Target sequences for PCR oligonucleotides are introduced onto the ends ofthe newly synthesised cDNA during reverse transcription using the template switching capability of reverse transcriptase (ClonTech SMART PCR cDNA synthesis kit). 2) Alternatively, the target and driver mRNA are reverse transcribed into cDNA using an oligo-dT primer and converted to double-strand cDNA; the cDNA is cleaved with Rsal and linkers for PCR amplification are ligated onto the extremities ofthe cDNA fragments. In both cases, target and driver cDNA are amplified by long range PCR (ClonTech SMART PCR Synthesis Kit and Advantage PCR Polymerase Mix) and used as starting material for subtractive cloning. For amplification, cycling conditions and optimisation ofthe number of PCR cycles are as described in the Advantage PCR protocol. Two subtractive cloning strategies are used: ClonTech PCR SELECT (see ClonTech kit protocol and N. Gurskaya et al. 1996. Analytical Biochemistry: 240, 90) and cRDA (M.

Hubank and D. Schatz. 1994. Nucleic Acids Research: 22, 5640) . When the PCR SELECT protocol is used, the primary PCR SELECT subtraction products are submitted to a supplementary round of cRDA subtraction. When the cRDA protocol is used, two consecutive cycles of cRDA subtraction are performed. In each case the products of both cycles of subtraction are cloned into pCR4-TOPO (InVitrogen) and transformed into E. coli to produce a subtracted cDNA plasmid library.

Example 2 : Differential Screening of cDNA arrays.

Identification of tumour-associated genes in the subtracted cDNA library is accomplished by differential screening :

Total bacterial DNA is extracted from 100 μl over-night cultures. Bacteria are lysed with guanidium isothiocyantate and the bacterial DNA is affinity purified using magnetic glass (Boehringer). Plasmid inserts are recovered from the bacterial DNA by Advantage PCR amplification (Clontech). The PCR products are dotted onto two nylon membranes to produce high-density cDNA arrays using the Biomek 96 HDRT tool (Beekman). The spotted cDNA is covalently linked to the membrane by UV irradiation. The first membrane is hybridised with a mixed cDNA probe prepared from the tumour of a single patient. The second membrane is hybridised with an equivalent amount of mixed cDNA probe prepared from corresponding normal tissue from the cancerous organ ofthe same patient. The probe cDNA is prepared by PCR amplification as described above and is labelled using the AlkPhos Direct System (Amersham). Hybridisation conditions and stringency washes are as described in the AlkPhos Direct kit. Hybridised probe is detected by chemiluminescence. Hybridisation intensities for each cDNA fragment on both blots are measured by film densitometry or direct measurement (BioRad Fluor-S Max). The ratio ofthe tumour to normal hybridisation intensities (T/N) is calculated for each gene to evaluate the degree of over-expression in the tumour. Genes that are significantly over-expressed in tumours are followed-up. Significance is arbitrarily defined as one standard deviation ofthe T/N frequency distribution. Differential screening experiments are repeated using RNA from multiple patient donors to estimate the frequency of over-expressing tumours in the patient population.

In addition, the DNA arrays are hybridised with mixed cDNA probes from normal tissues to deteirnine the level of expression ofthe candidate gene in these tissues.

Example 3 : DNA microarrays. DNA micro-arrays are used to examine mRNA expression profiles of large collections of genes in multiple samples. This information is used to complement the data obtained by real-time PCR and provides an independent measure of gene expression levels in tumours and normal tissues.

Examples of current technologies for production of DNA micro-arrays include 1) The Affymetrix "GeneChip" arrays in which oligonucleotides are synthesised on the surface ofthe chip by solid phase chemical synthesis using a photolithographic process 2) DNA spotting technology in which small volumes of a DNA solution are robotically deposited and then immobilised onto the surface of a solid phase (e.g. glass). In both instances, the chips are hybridised with cDNA or cRNA that has been extracted from the tissue of interest (e.g. normal tissue, tumour etc...) and labelled with radioactivity or with a fluorescent reporter molecule. The labelled material is hybridised to the chip and the amount of probe bound to each sequence on the chip is determined using a specialised scanner. The experiment can be set-up with a single fluorescent reporter (or radioactivity) or, alternatively, can be performed using two fluorescent reporters. In this latter case, each ofthe two samples is labelled with one ofthe reporter molecules. The two-labelled samples are then hybridised competitively to the sequences on the DNA chip. The ratio of the two fluorescent signals is determined for each sequence on the chip. This ratio is used to calculate the relative abundance ofthe transcript in the two samples. Detailed protocols are available from a number of sources including "DNA Microarrays: A practical approach. Schena M. Oxford University Press 1999" and the World Wide Web (http://cmgm.stanford.edu/pbrown/protocols/index.html), http://arrayit.com/DNA- Microarray-Protocols/) and specialised distributors (e.g. Affymetrix).

Affymetrix DNA microarrays (GeneChip Hu35K), gridded with human Unigene clusters- representing probesets, were hybridised with RNA transcribed into cDNA from normal colon tissue and colorectal tumour biopsies of different stages (namely Dukes' A, B, C and D stages). Raw results of hybridisation experiments were analysed using Affymetrix data processing methods. For each hybridisation, an absolute analysis was obtained.

Comparison analysis, that allows to compare hybridisations, and therefore tissue overall expression profiles, were also obtained. In comparison analysis, several parameters are offered to evaluate expression status of probesets. Difference Call parameter, as it gives a straightforward status of expression for a probeset, was selected to undertake the GeneChip analysis. There are 5 possible outcome for Difference Call : Either a probeset- related transcript level is increased (I), marginally increased (MI), decreased (D), marginally decreased (MD), or does not change (NC) between hybridisations or tissues.

Probesets showing al least one increase (I) Difference Call, in any ofthe three above- mentioned comparisons, were selected. Single sequences were assembled with identical and overlapping ESTs, leading to assemblies. For each assembly, a virtual expression profile was calculated, and only assemblies composed of at least 80 % of ESTs sequenced from tumour, foetal or reproductive tract tissues were kept.

Further analysis using the Self Organizing Method (SOM) method was done. The method groups probesets with a similar expression profile in a number of clusters. Expression profile of probesets is assessed with an Affymetrix calculation, the Fold Change value. The number of clusters to group probesets in is defined by the user, and was set to 24 in this case. A further analysis ofthe 24 clusters was performed : Relevant clusters to our concern are those where expression of probesets is low in normal colon and high or increasing throughout the Dukes' stages.

CASB88 is represented by probeset RC_AA010188_at. It was ranked first when combining the virtual expression profile, the "difference call" and "fold change" parameters, and the SOM analysis, meaning that this gene seems to be overexpressed in colorectal cancer.

The results showing the increased expression in various stages of colon cancer (Dukes A to Dukes D) compared to normal colon (N) are shown in table 2:

Table 1 : CASB88 expression profile on HU35K Affymetrix GeneChip.

*see text Example 4 : Real-time RT-PCR analysis of CASB88 on colorectal tumour biopsies and normal tissues.

Real-time RT-PCR (U. Gibson. 1996. Genome Research: 6,996) is used to compare mRNA transcript abundance ofthe candidate antigen in matched tumour and normal tissues ofthe same organ from multiple patients. In addition, mRNA levels ofthe candidate gene in a panel of normal tissues are also evaluated by this approach.

Total RNA from tumour and matched normal tissue is extracted from snap frozen biopsies using TriPure reagent (Boehringer). Total RNA from normal tissues is purchased from InVitrogen or is extracted from snap frozen biopsies using TriPure reagent (Boehringer). Poly-A+ mRNA is purified from total RNA after DNAase treatment using oligo-dT magnetic beads (Dynal). Quantification ofthe mRNA is performed by spectrofluorimetry (VersaFluor, BioRad) using Sybrll dye (Molecular Probes). Primers for real-time PCR amplification are designed with the Perkin-Elmer Primer Express software using default options for TaqMan amplification conditions.

Real-time reactions are assembled according to standard PCR protocols using 2 ng of purified mRNA for each reaction. Either Sybrll dye (Molecular Probes) is added at a final dilution of 1/75000 for real-time detection, or standard TaqMan detection is used. Amplification (40 cycles) and real-time detection is performed in a Perkin-Elmer Biosystems PE7700 system using conventional instrument settings. Ct values are calculated using the PE7700 Sequence Detector software. Several Ct values are obtained for each samples : for the patient samples, the tumour Ct (CtT) and the matched normal tissue Ct (CtN) values on the candidate present invention antigen and for the panel of normal tissue samples, a CtXY for each normal tissue XY is calculated. An another Ct (CtA) is also obtained on Actin gene, as an internal reference, for all ofthe samples.

.As the efficiency of PCR amplification under the prevailing experimental conditions is close to the theoretical amplification efficiency, 2^{(CtA"ctT/N/XY)} value is an estimate ofthe relative TAA transcript level ofthe sample, standardised with respect to Actin transcript level. A value of 1 thus suggests the candidate antigen and Actin have the same expression level. Real-time PCR reactions were performed on tumour colon and matching normal colon and a panel of normal tissues with 4 different primer pairs. Between 6 and 12 patient biopsies, and 12 and 23 commercial or biopsie normal tissue samples were used, depending on the primer pair. Both Sybr and TaqMan detection were undergone. CASB88 transcript levels are calculated as described above. The proportion of patients over-expressing the candidate antigen, as well as transcript over-expressions in tumour of colon (Tc) versus matched normal colon (Nc) and average of normal tissues (N) are also calculated from this data set (shown in Tc/Nc and Tc N columns, respectively). Normal tissues were further evaluated using a fifth primer pair (Primer Pair 5). Results are shown in Tables 2 & 3 and illustrated in Figures 1 to 11 (a list of used abbreviations for normal tissues is given in Table 4).

Table 2 : CASB88 RT-PCR expression profile on colorectal tumours and normal tissues using Sybr detection.

* A normal tissue has a high transcript level when it is higher than average Tc transcript level.

Table 3 : CASB88 RT-PCR expression profile on colorectal tumours and normal tissues using TaqMan detection.

* A normal tissue has a high transcript level when it is higher than average Tc transcript level.

Tables 2 & 3, Figures 1 to 11, clearly show that CASB88, while being barely expressed in normal adult tissues, apart from testis, is highly over-expressed in a majority of colorectal tumours, with an over-expression rate of at least more than ten fold. CASB88 tumour associated antigen is therefore a suitable vaccine candidate to treat cancer patients, more particulary patients suffering form colorectal cancer. Table 4 shows the tissue abbreviations used in Figures 1 to 11.

Table 4 : Normal Tissue Abbreviations.

Example 5 : Real time PCR analysis of CASB88 on lung tumour, preneoplasic lesions and normal tissue cDNAs libraries.

Presence of CASB88 transcript in lung tumours was assessed by PCR on three cDNA libraries made from lung tumours, preneoplasic lesions and a pool of normal tissues (see below, construction of cDNA libraries).

Construction of cDNA libraries from lung preneoplasic lesions and normal tissues Total RNA is extracted from a pool of lung solid tumour, a pool of 30 preneoplasic biopsies (endoscopic sampling) and a pool of normal tissues (Tri-Pure, Roche). mRNA is purified on oligo-d(T) magnetic beads (Dynal). Then the same protocol was applied to all mRNA preparations :350 ng of mRNA were reverse-transcribed using Superscript II enzyme (Life Technologies). The second cDNA strand was synthesized using RNAseH- DNA polymerase I and was blunted with the Pfu DNA polymerase. T3 promoter was specifically ligated at the cDNA 5' end. cDNA was attached to streptavidin beads (Dynal) via its biotinylated 3' end. In vitro transcribed mRNA (using T3 RNA polymerase) was purified on oligo-d(T) magnetic beads (Dynal). cDNA library was constructed from 5 μg of purified RNA using the classical Stratagene protocol.

Real time PCR analysis

10⁶ particles of Lambda phages were directly used as template of PCR amplification for analysis. Real time PCR reactions, and standardization relative to Actin transcript, were undergone as described above (in example 3).

Example 6 : Northern-Southern blot analysis

Limited amounts of mixed tumour and matched normal tissue cDNA are amplified by Advantage PCR (see above). Messenger RNA from multiple normal tissues is also amplified using the same procedure. The amplified cDNA (1 μg) is elecfrophoresed on a 1.2% agarose gel and transferred onto a nylon membrane. The membrane is hybridised (AlkPhos Direct System) with a probe prepared using a fragment ofthe candidate TAA cDNA. Northern-Southern analysis provides information on transcript size, presence of splice variants and transcript abundance in tumour and normal tissues.

Example 7 : Northern Blot Analysis

Northern blots are produced according to standard protocols using 1 μg of poly A+ mRNA. Radioactive probes are prepared using the Ready-to-Go system (Pharmacia).

Example 8 : In silico detection ofthe full length cDNA sequence EST sequence databases are screened with experimentally obtained cDNA sequence fragments, using the BLAST algorithm (Altschul, S.F., Gish, W., Miller, ., Myers, E.W. & Lipman, D.J. (1990) "Basic local alignment search tool." J. Mol. Biol. 215:403- 41). The aim is to search for overlapping or longer identical EST sequences. Matched EST sequences are then assembled together, using the SeqMan software ofthe Lasergene package (DNASTAR). The consensus sequence ofthe resulting assembly is an EST- derived longer cDNA. This EST-derived cDNA is further analysed using the GeneMark software to find a potential open reading frame (ORF). The translated sequence ofthe ORF is compared with protein databases, using the Blast algorithm, to find homologues. If any, the homologous protein sequences are further used to complete the cDNA prediction by searching for genomic contig homologies using the Wise2 algorithm, leading to a genome-derived, virtual cDNA sequence. This virtual cDNA in finally assembled with EST-derived cDNA, and the new consensus cDNA undergoes a final check against ESTs to confirm the Wise2 prediction, and correct potential sequencing errors and frameshifts. The virtual cDNA is considered as a virtual full length cDNA once a full ORF (from start to stop codons), with clear protein homologies and coding potential.

CASB88 virtual full-length polynucleotide sequence is given in SEQ ID NO:5, and its deduced full length polypeptide in SEQ ID NO:6. CASB88 transcript virtual sequence was futher experimentally verified as described in Example 10.

Example 9 : Experimental Identification of the full length cDNA sequence

To experimentally confirm CASB88 virtual full length sequence, CASB88 transcript was PCR amplified on a colorectal carcinoma cell line cDNA : mRNA was extracted and purified from LS41 IN cell line (ATCC catalog number CRL-2159). cDNA was synthesised using the GeneRacer technology (InVitrogen) which ensures the amplification of only full-length transcripts. 250 ng of mRNA were treated with CIP. mRNA 5' ends were decapped with TAP (Tobacco Acid Pyrophosphatase) and were ligated to a specific RNA oligonucleotide. The ligated mRNA was reverse transcribed into cDNA using an oligod(T) tailed primer. Amplification of cDNA was performed using both GeneRacer flanking primers (Advantage, Clontech). Then, CASB88 was PCR generated using gene specific primers on amplified cDNA. PCR product was diluted and re-ran on an agarose gel to precisely check product size, cloned in pCR4-TOPO cloning vector (InVitrogen) following the manufacturer instructions, and sequenced.

CASB88 deduced polynucleotide sequence following that approach is disclosed in SEQ ID NO:l, and conceptually translated polypeptide in SEQ ID NO:2.

An alternative approach is a screening of a colorectal tumour cDNA library: Total RNA is extracted from colorectal solid tumour biopsies (Tri-Pure, Roche). mRNA is purified on oligo-d(T) magnetic beads (Dynal). 5 μg of polyA÷ mRNA was reverse-transcribed using Superscript II polymerase. Second strand was synthesised using RNAseH-DNA polymerase I and blunted with the Pfu DNA polymerase. EcoRI adapters were specifically ligated at the cDNA 5' end. cDNA was size-fractionated using a Sepharose CL-2B column and ligated into phage arms (Uni-ZAP lambda, Stratagene). About 1.5 xl 0⁶ independent phages are plated for each screening ofthe library. Phage plaques are transferred onto nylon filters and hybridised using a cDNA probe labelled with AlkPhos Direct. Positive phages are detected by chemiluminescence. Positive phages are excised from the agar plat, eluted in 500μl SM buffer and confirmed by gene-specific PCR. Eluted phages are converted to single strand Ml 3 bacteriophage by in vivo excision. The bacteriophages are then converted to double strand plasmid DNA by infection of E. coli : Phage DNA is in vitro packaged and amplified in E. coli. cDNA inserts are transfeπed into plasmids using the mass excision protocol (Stratagene). Infected bacteria are plated and submitted to a second round of screening with the cDNA probe. Plasmid DNA is purified from positive bacterial clones and sequenced on both strands.

When the full length gene cannot be obtained directly from the cDNA library, missing sequence is isolated using RACE technology (Marathon Kit, ClonTech.). This approach relies on reverse transcribing mRNA into double strand cDNA, ligating linkers onto the ends ofthe cDNA and amplifying the desired extremity ofthe cDNA using a gene- specific primer and one ofthe linker oligonucleotides. Marathon PCR products are cloned into a plasmid (pCRII-TOPO, InVitrogen) and sequenced.

Example 10 : Digital Northern A complementary approach to experimental antigen tissue expression characterisation is to explore the human "Expressed Sequence Tags" (ESTs) database. ESTs are small fragments of cDNA made from a collection of mRNA extracted from a particular tissue or cell line. Such database currently provides a massive amount of human ESTs (2 10⁶) from several hundreds of cDNA tissue libraries, including tumour tissues from various types and states of disease. By means of informatics tools (BLAST), a comparison search ofthe CASB88 sequence is performed in order to have further insight into tissue expression.

Ofthe 39 ESTs that can be fully aligned to CASB88 (ie that are 100% identical over their entire length), 30 were sequenced in tumour cDNA libraries, 4 were sequenced in fetal or reproductive tract tissue cDNA libraires, and only 5 were sequenced normal or unknown tissue cDNA libraries. This clearly confirms present invention transcript is over- expressed in tumour tissues while being barely expressed in normal tissues. Taken with other example results, this makes CASB88 antigen a suitable vaccine candidate to treat cancer patients. Example 11 :

11.1 Expression and purification of tumour-specific antigens

Expression in microbial hosts, or alternatively in vitro transcription/translation, is used to produce the antigen ofthe invention for vaccine purposes and to produce protein fragments or whole protein for rapid purification and generation of antibodies needed for characterisation ofthe naturally expressed protein by immunohistochemistry or for follow-up of purification.

Recombinant proteins may be expressed in two microbial hosts, E. coli and in yeast (such as Saccharomyces cerevisiae or Pichia pastoris). This allows the selection ofthe expression system with the best features for this particular antigen production. In general, the recombinant antigen will be expressed in E. coli and the reagent protein expressed in yeast.

The expression strategy first involves the design ofthe primary structure ofthe recombinant antigen. In general an expression fusion partner (EFP) is placed at the N terminal extremity to improve levels of expression that could also include a region useful for modulating the immunogenic properties ofthe antigen, an immune fusion partner

(IFP). In addition, an affinity fusion partner (AFP) useful for facilitating further purification is included at the C-terminal end. When the recombinant strains are available, the recombinant product is characterised by the evaluation ofthe level of expression and the prediction of further solubility ofthe protein by analysis ofthe behaviour in the crude extract.

After growth on appropriate culture medium and induction ofthe recombinant protein expression, total extracts are analysed by SDS-PAGE. The recombinant proteins are visualised in stained gels and identified by Western blot analysis using specific antibodies.

A comparative evaluation ofthe different versions ofthe expressed antigen will allow the selection ofthe most promising candidate that is to be used for further purification and immunological evaluation.

The purification scheme follows a classical approach based on the presence of an His affinity tail in the recombinant protein. In a typical experiment the disrupted cells are filtered and the acellular extracts loaded onto an Ion Metal Affinity Chromatography (IMAC; Ni++NTA from Qiagen) that will specifically retain the recombinant protein. The retained proteins are eluted by 0-500 mM Imidazole gradient (possibly in presence of a detergent) in a phosphate buffer.

11.2 Antibody production and immunohistochemistry

Small amounts of relatively purified protein can be used to generate immunological tools in order to a) detect the expression by immunohistochemistry in normal or cancer tissue sections; b) detect the expression, and to follow the protein during the purification process (ELISA/ Western Blot); or c) characterise/ quantify the purified protein (ELISA).

11.2.1 Polyclonal antibodies: Immunisation

Rabbits are immunised , intramuscularly (LM.) , 3 times at 3 weeks intervals with lOOμg of protein, formulated in the adjuvant 3D-MPL/QS21. Three weeks after each immunisation a blood sample is taken and the antibody titer estimated in the serum by ELISA using the protein as coating antigen following a standard protocol.

ELISA

96 well microplates (maxisorb Nunc) are coated with 5μg of protein overnight at 4°C. After Ihour saturation at 37°C with PBS NCS 1%, serial dilution ofthe rabbit sera is added for IH 30 at 37°C (starting at 1/10). After 3 washings in PBS Tween, anti rabbit biotinylated anti serum (Amersham ) is added (1/5000). Plates are washed and peroxydase coupled streptavidin (1/5000) is added for 30 min at 37°C. After washing, 50μl TMB (BioRad) is added for 7 min and the reaction then stopped with H2SO4 0.2M. The OD can be measured at 450 nm and midpoint dilutions calculated by SoftmaxPro.

11.2.2 Monoclonal antibodies: Immunisation

5 BALB/c mice are immunised 3 times at 3 week intervals with 5 μg of purified protein. Bleedings are performed 14 days post II and 1 week post 3. The sera are tested by Elisa on purified protein used as coated antigen. Based on these results (midpoint dilution > 10000 ) one mouse is selected for fusion

Fusion/ HATselection Spleen cells are fused with the SP2/0 myeloma according to a standard protocol using PEG 40% and DMSO 5%. Cells are then seeded in 96 well plates 2.5 xl04 - 105 cells/well and resistant clones will be selected in HAT medium. The supernatant of these hybridomas will be tested for their content in specific antibodies and when positive, will be submitted to 2 cycles of limited dilution . After 2 rounds of screening, 3 hybridomas will be chosen for ascitis production.

11.2.3 Immunohistochemistry

When antibodies are available, immuno staining is performed on normal or cancer tissue sections, in order to determine : 0 the level of expression of the antigen of the invention in cancer relative to normal tissue or

0 the proportion of cancer of a certain type expressing the antigen

0 if other cancer types also express the antigen

0 the proportion of cells expressing the antigen in a cancer tissue

Tissue sample preparation

After dissection, the tissue sample is mounted on a cork disk in OCT compound and rapidly frozen in isopentane previously super cooled in liquid nitrogen (-160°C). The block will then be conserved at -70°C until use. 7-10μm sections will be realised in a cryostat chamber (-20, -30°C).

Staining

Tissue sections are dried for 5 min at room Temperature (RT), fixed in acetone for lOmin at RT, dried again, and saturated with PBS 0.5% BSA 5% serum. After 30 min at RT either a direct or indirect staining is performed using antigen specific antibodies. A direct staining leads to a better specificity but a less intense staining whilst an indirect staining leads to a more intense but less specific staining. 11.3 Analysis of human cellular immune responses to the antigen of the invention

Immunological relevance of CASB88 can be assesed by in vitro priming of human T cells. All T cell lymphocytes, T cell lines and dendritic cells are derived from PBMCs (peripheral blood mononuclear cells) of healthy donors or cancer patients (preferred donors are from HLA-A2 subtype).

Epitopes binding of HLA alleles prediction: The HLA Class I binding peptide sequences (nonamers, decamers) are predicted by the Parker's algorithm [Parker K, et al. 1994] (http:^imas.dcrt.nih.gov/molbio/hla_bind/), and the Rammensee method [Rammensee, et al. 1997] [Rammensee, et al. 1995] (http://sy eithi.bmi-heidelberg.com Scripts/MHCServer.dll/EpPredict.hτm). Only predicted epitopes that score above a cut-off of 100 and 20 for the Parker algorithm and the Rammensee method, respectively, are selected.

The HLA Class II binding peptide sequences (nonamers) are predicted using the Tepitope algorithm [Sturniolo, et al. 1999].

CD8+ T-cell response:

Two strategies to raise the CD8+ T cell lines are used: a peptide-based approach and a whole gene-based approach. Both approaches require the full-length cDNA of interest in the correct reading frame to be cloned in an appropriate delivery system and to be used to predict the sequence ofthe HLA binding peptides.

Peptide-based approach:

For this approach, an HLA-A2.1/Kb transgenic mouse model is used for screening of the HLA-A2J peptides. Briefly, transgenic mice are immunized with adjuvanted HLA-A2 peptides, those able to induce a CD 8 response (as defined by an efficient lysis or g-IFN production on peptide-pulsed target cells) are further analyzed in the human system.

Human dendritic cells (cultured according to [Romani et al. 1994]) will be pulsed with the selected peptides and used to stimulate CD8+-sorted T cells (by Facs). After several weekly stimulation, the CD8+ lines are first tested on peptide-pulsed autologous BLCL (EBV-B transformed cell lines). To verify the proper in vivo processing of the peptide, the CD8+ lines are then tested on cDNA-transfected tumour cells (HLA-A2 transfected LnCaP, Skov3 or CAMA tumour cells).

Whole gene-based approach :

CD8+ T cell lines are primed and stimulated with either gene-gun transfected dendritic cells, retrovirally transduced B7J -transfected fibroblasts, recombinant pox virus [Kim et al. 1997] or adeno virus [Butterfield et al. 1998] infected dendritic cells. Virus infected cells are very efficient to present antigenic peptides since the antigen is expressed at high level but can only be used once to avoid the over-growth of viral T cells lines.

After alternated stimulation, the CD8+ lines are tested on cDNA-transfected tumour cells as indicated above. Peptide specificity and identity is determined to confirm the immunological validation of antigen ofthe present invention.

CD4+ T-cell response:

Similarly, the CD4+ T-cell immune response can also be assessed. Generation of specific CD4+ T cells is made using dendritic cells loaded with recombinant purified protein or peptides to stimulate the T-cells.

References :

Butterfield LH, Jilani SM, Chakraborty NG, Bui LA, Ribas A, Dissette VB, Lau R, Gamradt SC, Glaspy JA, McBride WH, Mukherji B, Economou JS. (1998) Generation of melanoma-specific cytotoxic T lymphocytes by dendritic cells transduced with a MART- 1 adeno virus. J Immunol. 161: 5607-13.

Kim CJ, Prevette T, Cormier J, Overwijk W, Roden M, Restifo NP, Rosenberg SA, Marincola FM. (1997) Dendritic cells infected with poxviruses encoding MART-1/Melan A sensitize T lymphocytes in vitro. J Immunother.20:216-286. Parker K, Bednarek M, Coligan J (1994). Scheme for ranking potential HLA-A2 binding peptides based on independent binding of individual peptide side-chains. J. Immunol. 152:163.

Rammensee, Bachmann, Stevanovic (1997). MHC ligands and peptide motifs. Landes Bioscience.

Rammensee, Friede, Stevanovic (1995). MHC ligands and peptide motifs: 1st listing, Immunogenetics 41, 178-228.

Romani N, Gruner S, Brang D, Kampgen E, Lenz A, Trockenbacher B, Konwalinka G, Fritsch PO, Steinman RM, Schuler G. (1994) Proliferating dendritic cell progenitors in human blood. JExp Med. 180:83-93.

Sturniolo T, Bono E, Ding J, Raddrizzani L, Tuereci O, Sahin U, Braxenthaler M,

Gallazzi F, Protti MP, Sinigaglia F, Hammer J. (1999) Generation of tissue-specific and promiscuous HLA ligand databases using DNA microarrays and virtual HLA class II matrices. Nat Biotechnol 17: 555-61.

Results

A- Prediction of Class I epitopes using the Parker method.

B8 Nonamer 75 RPRGRPRKL 160.000 SEQ ID NOJ l

NB : Score is an estimate of half-time of disassociation of a molecule containing this subsequence.

B- Prediction of Class I epitopes using the Rammensee method.

C- Prediction of Class II epitopes using the Tepitope method.

SEQUENCE INFORMATION SEQ ID NO:l

GAAAGGGTGACTGGCGGCGGGCGCCGCGGTCGGGCTGGCTGCCGGGCAGCatggaggagctgagcagcgtgg gcgagcaggtcttcgccgccgagtgcatcctgagcaagcggctccgcaagggcaagctggagtacctggtca agtggcgcggctggtcctccaaacataacagctgggagccggaggagaacatcctggacccgaggctgctcc tggccttccagaagaaggaacatgagaaggaggtgcagaaccggaagagaggcaagaggccgagaggccggc caaggaagctcactgccatgtcctcctgcagccggcgctccaagctcaaggaacccgatgctccctccaaat ccaagtccagcagttcctcctcttcctccacgtca cctcctcttcctcagatgaagaggatgacagtgact taga gctaagaggggtccccggggccgcgagacccacccagtgccgcagaagaaggcccagatcctggtgg ccaaacccgagctgaaggatcccatccggaagaagcggggacgaaagcccctgcccccagagcaaaaggcaa cccgaagacccgtgagcctggccaaggtgc gaagaccgcccggaaggatctgggggccccggccagcaagc tgccccctccactcagcgcccccgttgcaggcctggcagctctgaaggcccacgccaaggaggcctgtggcg gccccagtgccatggccaccccagggaacctggccagcctaatgaagggcatggccagtagccccggccggg gtggcatcagctggcggagctccatcgtgcactacatgaaccggatgacccagagccaggcccaggctgcca gcaggttggcgctgaaggcccaggccaccaacaagtgcggcctcgggctggacctgaaggtgaggacgcaga aaggggagctgggaatgagccctccaggaagcaaaatcccgaaggcccccagcggtggggctgtggagcaga aagtggggaacacagggggccccccgcacacccatggtgccagcagggtgcctgctgggtgcccaggccccc agccagcacccacccaggagctgagcctccaggtcttggacttgcagagtgccaagaatggcatgcccgggg tgggtctccttgcccgccacgccaccgccaccaagggtgtcccggccaccaacccagcccctgggaagggca ctgggagtggcctcattggggccagcggggccaccatgcccaccgacacaagcaaaagtgagaagctggctt ccagagcagtggcgccacccacccctgccagcaagagggactgtgtcaagggcagtgctacccccagtgggc aggagaaccgcacagcccccggagaagcccgcaaggcggccacactgccagagatgagcgcaggtgaggaga gtagcagctcggactccgaccccgaccccgcctcgccgcccagcactggacagaacccatcagtgtccgttc agaccagccaggactggaagcccacccgcagcctcatcgagcacgtatttgtcaccgacgtcactgccaacc catcaccgtcacagtgaaggagtctcccaccagcgtgggcttcttcaacctgaggcattac gaAGCCCCG GCGCCACCAGCTGCGCGGTCTTACTCCCCTTCCCTGCCTATGGTGTCGCTTGGCTAAGTGACTCCCAGCCCA AGCCCCCTCAAGAGTCTGGGTCGGGGGAGGAGGAAGTGGGTGGCCTCCTTGATGGGCAGGCTGGAAGGGACT TTTCCCGCACCCCACTTTTGTCCCAGGACATAGGGCAGGGGGCCTCACTGCCTTGTTGGTCTCCACCTTGTT CCTACCTCTGCAGGCCTCTTTGCTCTCCCCTCTTGCCTCAAGGAAACCCGGTGGCACCTGTGGCTCCAGGTG ACTGTCTTGAACAGAGCGGGCTTCTTCATGGCTGCGTTGTTGCTGAGTTTGAACTGCTCCTCCCTGGCCTGC GTGACTGAATCACAGCTTTGGTCCCTGTCTTGCAGGGACTGAGGTGTCAGGAGGGGACTTCTGGCCCACCTT GCCTTCAGCCCTGGAGTGGGCAGAGAGTATTGTGGGGAGGCATGGCCAGTGGGACTAGTGTTCCCTCCATCT GGCCACAGCTTTTGGGAGATGGGGTGGGCAGGGGTGGTCCTGGCTGGCATTGCCTGAGCCGGCAGTGATGAA GTGGGGAGCTTGCCCTTGACAGGTGGGGGCTGGCTGGGGCCTTAATGTGAAAAGACAGTGGCAGGCAGCTGG AGTAGAGCGAGCCCAGCAGCCCTAAAAGGCTGCCTTCATGGCCATCTAGCCCCAGTTCAGGGCAGCATCCAT AGCCCACAAGCCAGCGTGGGTGGGGCGGGGGTGGTCCCACAGCTGGGTTCCACCTGAAGAGCCTCCGTGCCT CGGAGCAGGAGAGGCAGGCTATGGCTGCCACCCTCCCTCCTGCCTGTGTCCCAGTGAGAACTGACCTGAGTC CCCTTCCAAACCCAGACCCACCTCCTGCCCCAGGCCCACTGAAGCATGTTCCATTTCTAAAAAGCCCAGAGT TCAGTGTGTCCCAAGGAAAACCCAAAGTGGAGGTGCTCAGGTCCAGGGGAGTCCAGTGGGCAGGACCCTTGG CAGGCAAGCCCCTCCCTTCACTCCCAGGACCTACCTTCTGCTAGTAAAGGACTGGCTTCATTCTAATTATGG CCCACAGACTGCCCCGGAGACCTGGAGGACAGCAGTGCTGGCACTTGGGTGTCCATGGGCCCGTCTGCCGGC TCTGCCTGTGCTGCAAGTGTTGGCCGTGGGTCCAGCCAACAACTCCCTACGTCCTGTGTGGGGCCCTGCCCA AGTGGATGAGGCATTCCTTGAGGAGTATCATTTTCCCTGACAATCCCCATCACCTTTAGGGGTTCCCTGCTT GGCTCCTTTCCAGCTGAAAAACTAGACCTGTGCCATTGGGGAAGCTGGACAAAGTCTAGGGGGCCCGCCTGG TAGAGGGTCCCGGGAAGCTGGATCTGTCAGCCTCGGCCCTGAGGCCCCTGTTAACTCAAGACTGTGAGCTGC CTCTAGGTGGTCACGTCTGGGAGCTAGCTTGTATGGCTTCTGACCAGTATCAGGATTTCTGTTCTGAGAGCA GCGTGGGCAGCAAGGCAGGGCAGCCCAGAGGTGGCAGCGGCAGGCAATCTGGTCACTAGGTCTTTGTGATGC CAAAAATAAAAGAGGGTGGGGTGGGTGCTTTCTGTTCCTCTGATTGGATGGAGTCCGCCAGCAGGCATGGGG CTACATTCCAGTGCCTGACTATAGGGAGGCACTCCTGATTCCATGGAGCAGCCCGGACTTTGAGAATGGGCT CTGGTTTGCGGGGGGCAGGCGTACCAGACTGCAAGACCCCCCAGTACCTCACCGTGCCAAATAGGAAGAGGT GGCCTTGGTGTAGCCAAATGGATCTTTTTAACAGTGTGCCTTTGGGGAGGGACCCATGTCCATGGCTTCGTT GAGGGCCATCCATATGCCAGCTGGGGGCCAGCCCACAGTGGCCATATTGGCTGCAGCAGGAATGGTGCCCAC CTCGGCGAATTGAAGGGCTAAGAGTCCCAGATAGCTAGGCCAGAGCTGGAAGCAGACAGTAAGGGGAAGAGC TGCTCCCACAGGAGAGGGAGAGATTCCAGCTCACTGCGCAGCCTGGGAGGAGGCGTGGATCCTGGCACGCTG AGCCTCAGGCACCAGCCTCCCTGTGCTCGACAGCAAAGTCTTGACTCCTTCCTGCTGAGCACTGTGCTACCT TCACTGCTCCAAAGCCAGACTAACAGCTCTCCAAGCCCTTGGGGTGACTCGGCTTCCAGGAGCTGTTGGAGA AATGAGGATGTCTGTCCCTGTCTGCCTGGGCAGGCCAGATTCCTCCCCAGCAGCCGGGTCTCTCCAGACCCT GATTCGGTGCCTTTCTGTTTACCAGCTACTTCAATCCCAAAGTTTGAATCTGCAGATACCTTACTCCCAGCC ACTTTGCCTTCTTACTGTGTTGTGTGTTTTTCCTGGTGCTTCAAGAGCGTGTGCAGGGCAAGTGCCGTCACT GGGAACTGCACCAGATGCTCAGACTTGGTTGTCTTATGTTTACCAATAAATAAAAGTAGACTTTTTCTATTT TTATTTGCTGCTATTTGTGTGTGTGTTTGTGTTTGTGTAGCTAGGTATCTGGCACTTCTGACGATGCATTGT TGCTTTTTTCCCGAAGGTCCCGCAGGAACTGTGGCAATGGTGTGTGTGTGAAATGGTGTGTTAACCGCGTTT TGTTTGCTCCTGTATTGAATAGGAAGCAGTGGCCAGTCTGTCTTCCTTAGAGATGTTAGCATATTTTTATAT GTATATATTTTGTACCAAAAAAGAGTGTTCCTTGTTTTGGTTACACTCGAAATTCTGACCTAGCTGGAGAGG GCTCTGGGCCGAGAGCTTTCACTAAGGGGAGACTTCAGGGGAGGATCAAGCTTTGAACCAAAGCCAATCACT GGCTTGATTTGTGTTTTTTAATTAAA-^AAAAAATCATTCATGTATGCCACTTCTAAAAAAAAAAAAAAAAAA AAAAAAA

SEQ ID NO:2

MEELSSVGEQVFAAECILSKRLRKGKLEYLVKWRG SSKHNS EPEENILDPRLLLAFQKKEHEKEVQNRKR GKRPRGRPRKLTAMSSCSRRSKLKEPDAPSKSKSSSSSSSSTSSSSSSDEEDDSDLDAKRGPRGRETHPVPQ KKAQILVAKPELKDPIRKKRGRKPLPPEQKATRRPVSLAKVLKTARKDLGAPASKLPPPLSAPVAGLAALKA HAKEACGGPSAi-ATPGNLASLMKGMASSPGRGGIS RSSIVHYMNRMTQSQAQAASRLALKAQATNKCGLGL DLKVRTQ GELGMSPPGSKIPKAPSGGAVEQKVGNTGGPPHTHGASRVPAGCPGPQPAPTQELSLQVLDLQS AKNGMPGVGLLARHATATKGVPATNPAPGKGTGSGLIGASGATMPTDTSKSEKLASRAVAPPTPASKRDCVK GSATPSGQENRTAPGEARKAATLPEMSAGEESSSSDSDPDPASPPSTGQNPSVSVQTSQDWKPTRSLIEHVF VTDVTANLITVTVKESPTSVGFFNLRHY SEQ ID NO:3

MEELSSVGEQVFAAECILSKRLRKGKLEYLVKWRGWSSKHNSWEPEENILDPRLLLAFQKKEHEKEVQNRKR GKRPRGRPRKHTATSSCSRRSKLKEPDAPSKSKSSSSSSSSTSSSSSSDEEEDDSDLDSKRGPRGRETHPVP QKKAQILVAKPELKDPIRKKRGRKPLPPEQAARRPVSLAKVLKTTRKDLGTSAAKLPPPLSAPVAGLAALK AHTKEACGGPST- TPENLASLMKGMAGSPSRGGI QSSIVHYMNR SQSQVQAASRLALKAQATNKCGLGL DLKVRTQKGGELGGSPAGGKVPKAPGGGAAEQQRGNHSGSPGAQLAPTQELSLQVLDLQSVKNGVPGVGLLA RHAPAKAIPATNPATGKGPGSGPTGANMTNAPTDNNKGEKLTCKATALPAPSVKRDTVKSVAASGGQEGHTA PGEGR PPALSELSTGEENSSSDSDPDSTSLPSAAQNLSVAIQTSQD PTRSLIEHVFVTDVTANLITVTV KESPTSVGFFNLRHY

SEQ ID NO:4

CCGGCGCGCCTATTGGCCCGGCGGCTGCGGGTAGAGCAGCGCGGGCGACTCCGGGGCCCGTGCGCGGGGCGG GGCGCGGCGGGGGCGGCGCTTTGTGTGCAGCAGTGAGCCGGGGTCTGCGGGGGCCGGCCGGCGGCGGGCGGC GGCATGGAGGAGCTGAGCAGCGTGGGCGAGCAGGTCTTCGCCGCCGAGTGCATCCTGAGCAAGCGGCTCCGC AAGGGCAAGCTGGAGTACCTGGTCAAGTGGCGCGGCTGGTCCTCCAAACACAACAGCTGGGAGCCAGAAGAG AACATTTTGGACCCGAGGCTGCTCCTAGCCTTCCAGAAGAAGGAACATGAGAAGGAGGTTCAGAACCGGAAG AGAGGCAAGAGACCCAGGGGCAGGCCGAGGAAACACACAGCCACATCCTCCTGCAGCCGGCGCTCCAAGCTC AAGGAACCAGATGCGCCATCCAAATCCAAATCCAGCAGTTCGTCCTCTTCCTCCACATCTTCCTCCTCTTCC TCGGACGAAGAGGAAGACGACAGCGACCTAGACTCCAAGAGAGGCCCCCGGGGCCGTGAAACCCATCCAGTG CCTCAGAAAAAAGCCCAGATCCTGGTAGCCAAGCCAGAGCTGAAGGATCCCATTAGAAAGAAACGGGGACGC AAGCCTCTACCCCCAGAACAGAAGGCAGCTCGGAGACCCGTCAGCCTGGCCAAGGTGCTAAAGACCACCAGG AAGGATCTGGGGACCTCAGCCGCCAAGCTGCCCCCTCCACTCAGCGCTCCGGTGGCAGGCCTGGCTGCCCTG AAGGCCCACACCAAAGAGGCCTGTGGTGGCCCCAGCACTATGGCGACCCCAGAGAACCTGGCCAGTCTGATG AAAGGCATGGCCGGGAGCCCCAGCAGAGGCGGCATCTGGCAGAGCTCCATCGTACACTACATGAACCGCATG AGCCAGAGTCAGGTTCAGGCTGCCAGCCGACTGGCACTCAAGGCCCAGGCCACCAACAAGTGCGGTCTCGGG CTAGACCTGAAAGTGAGGACGCAGAAGGGGGGTGAGCTAGGGGGGAGCCCCGCAGGAGGCAAGGTCCCGAAG GCCCCCGGTGGCGGAGCTGCAGAGCAGCAGAGAGGGAACCATTCGGGGAGCCCAGGTGCTCAGCTGGCACCC ACTCAGGAGTTGAGCCTTCAGGTCCTTGACTTGCAAAGCGTCAAGAACGGTGTGCCTGGTGTGGGCCTGCTT GCTCGCCATGCCCCAGCCAAGGCTATTCCTGCTACCAACCCAGCCACAGGGAAAGGTCCTGGGAGCGGCCCC ACAGGAGCAAACATGACCAACGCTCCCACAGACAACAACAAAGGGGAAAAGCTGACTTGCAAGGCAACGGCT CTGCCTGCCCCTTCCGTCAAGCGGGACACCGTTAAAAGCGTCGCTGCCTCCGGCGGGCAGGAGGGCCACACA GCCCCGGGAGAAGGCCGAAAGCCACCTGCGCTGTCTGAGCTGAGCACGGGAGAGGAGAATAGTAGCTCTGAC TCGGACCCTGACTCGACCTCGCTTCCCA

SEQ ED NO:5

GGTCGGGCTGGCTGCCGGGCAGCatggaggagctgagcagcgtgggcgagcaggtcttcgccgccgagtgca tcctgagcaagcggctccgcaagggcaagctggagtacctggtcaagtggcgcggctggtcctccaaacata acagctgggagccggaggagaacatcctggacccgaggctge cctggccttccagaagaaggaacatgaga aggaggtgcagaaccggaagagaggcaagaggccgagaggccggccaaggaagctcactgccatgtcctcct gcagccggcgctccaagctcaaggaacccga gc ccctccaaatccaagtccagcagttcctcctcttcct ccacg ca cctcctcttcctcagatgaagaggatgacagtgacttaga gctaagaggggtccccggggcc gcgagacccacccagtgccgcagaagaaggcccagatcctggtggccaaacccgagctgaaggatcccatcc ggaagaagcggggacgaaagcccctgcccccagagcaaaaggcaacccgaagacccgtgagcctggccaagg gctgaagaccgcccggaaggatctgggggccccggccagcaagctgccccctccactcagcgcccccgttg caggcctggcagctctgaaggcccacgccaaggaggcctgtggcggccccagtgccatggccaccccagaga acctggccagcctaatgaagggcatggccagtagccccggccggggtggcatcagctggcagagctccatcg tgcactacatgaaccggatgacccagagccaggcccaggctgccagcaggttggcgctgaaggcccaggcca ccaacaagtgcggcctcgggctggacctgaaggtgaggacgcagaaaggggagctgggaatgagccctccag gaagcaaaatcccgaaggcccccagcggtggggctgtggagcagaaagtggggaacacagggggccccccgc acacccatggtgccagcagggtgcctgc gggtgcccaggcccccagccagcacccacccaggagctgagcc tccaggtcttggacttgcagagtgtcaagaatggcatgcccggggtgggtctccttgcccgccacgccaccg ccaccaagggtgtcccggccaccaacccagcccctgggaagggcactgggagtggcctca tggggccagcg gggccaccatgcccaccgacacaagcaaaagtgagaagctagcttccagagcagtggcgccacccacccctg ccagcaagagggactgtgtcaagggcagtgctacccccagtgggcaggagagccgcacagcccccggagaag cccgcaaggcggccacactgccagagatgagcgcaggtgaggagagtagcagctcggactccgaccccgact ccgcctcgccgcccagcactggacagaacccgtcagtgtccgttcagaccagccaggactggaagcccaccc gcagcctcatcgagcacg a tgtcaccgacgtcactgccaacctcatcaccgtcacagtgaaggag ctc ccaccagcgtgggcttcttcaacctgaggcattactgaAGCCCCGGCGCCACCAGCTGCGCGGTCTTACTCC CCTTCCCTGCCTATGGTGTCGCTTGGCTAAGTGACTCCCAGCCCAAGCCCCCTCAAGAGTCTGGGTCGGGGG AGGAGGAAGTGGGTGGCCTCCTTGATGGGCAGGCTTGGAAGGGACTTTTCCCGCACCCCACTTTTGTCCCAG GGACATAGGGCAGGGGGCCTCACTGCCTTGTTGGTCTCCACCTTGTTCCTACCTCTGCAGGCCTCTTTGCTC TCCCCTCTTGCCTCAAGGAAACCCGGTGGCACCTGTGGCTCCAGGTGACTGTCTTGAACAGAGCGGGCTTCT TCATGGCTGCGTTGTTGCTGAGTTTGAACTGCTCCTCCCTGGCCTGCGTGACTGAATCACAGCTTTGGTCCC TGTCTTGCAGGGACTGAGGTGTCAGGAGGGGACTTCTGGCCCACCTTGCCTTCAGCCCTGGAGTGGGCAGAG AGTATTGTGGGGAGGCATGGCCAGTGGGACTAGTGTTCCCTCCATCTGGCCACAGCTTTTGGGAGATGGGGT GGGCAGGGGTGGTCCTGGCTGGCATTGCCTGAGCCGGCAGTGATGAAGTGGGGAGCTTGCCCTTGACAGGTG GGGGCTGGCTGGGGCCTTAATGTGAAAAGACAGTGGCAGGCAGCTGGAGTAGAGCGAGCCCAGCAGCCCTAA AAGGCTGCCTTCATGGCCATCTAGCCCCAGTTCAGGGCAGCATCCATAGCCCACAAGCCAGCGTGGGTGGGG CGGGGGTGGTCCCACAGCTGGGTTCCACCTGAAGAGCCTCCGTGCCTCGGAGCAGGAGAGGCAGGCTATGGC TGCCACCCTCCCTCCTGCCTGTGTCCCAGTGAGAACTGACCTGAGTCCCCTTCCAAACCCAGACCCACCTCC TGCCCCAGGCCCACTGAAGCATGTTCCATTTCTAAAAAGCCCAGAGTTCAGTGTGTCCCAAGGAAAACCCAA AGTGGAGGTGCTCAGGTCCAGGGGAGTCCAGTGGGCAGGACCCTTGGCAGGCAAGCCCCTCCCTTCACTCCC AGGACCTACCTTCTGCTAGTAAAGGACTGGCTTCATTCTAATTATGGCCCACAGACTGCCCCGGAGACCTGG AGGACAGCAGTGCTGGCACTTGGGTGTCCATGGGCCCGTCTGCCGGCTCTGCCTGTGCTGCAAGTGTTGGCC GTGGGTCCAGCCAACAACTCCCTACGTCCTGTGTGGGGCCCTGCCCAAGTGGATGAGGCATTCCTTGAGGAG TATCATTTTCCCTGACAATCCCCATCACCTTTAGGGGTTCCCTGCTTGGCTCCTTTCCAGCTGAAAAACTAG ACCTGTGCCATTGGGGAAGCTGGACAAAGTCTAGGGGGCCCGCCTGGTAGAGGGTCCCGGGAAGCTGGATCT GTCAGCCTCGGCCCTGAGGCCCCTGTTAACTCAAGACTGTGAGCTGCCTCTAGGTGGTCACGTCTGGGAGCT AGCTTGTATGGCTTCTGACCAGTATCAGGATTTCTGTTCTGAGAGCAGCGTGGGCAGCAAGGCAGGGCAGCC CAGAGGTGGCAGCGGCAGGCAATCTGGTCACTAGGTCTTTGTGATGCCAAAAATAAAAGAGGGTGGGGTGGG TGCTTTCTGTTCCTCTGATTGGATGGAGTCCGCCAGCAGGCATGGGGCTACATTCCAGTGCCTGACTATAGG GAGGCACTCCTGATTCCATGGAGCAGCCCGGACTTTGAGAATGGGCTCTGGTTTGCGGGGGGCAGGCGTACC AGACTGCAAGACCCCCCAGTACCTCACCGTGCCAAATAGGAAGAGGTGGCCTTGGTGTAGCCAAATGGATCT TTTTAACAGTGTGCCTTTGGGGAGGGACCCATGTCCATGGCTTCGTTGAGGGCCATCCATATGCCAGCTGGG GGCCAGCCCACAGTGGCCATATTGGCTGCAGCAGGAATGGTGCCCACCTCGGCGAATTGAAGGGCTAAGAGT CCCAGATAGCTAGGCCAGAGCTGGAAGCAGACAGTAAGGGGAAGAGCTGCTCCCACAGGAGAGGGAGAGATT CCAGCTCACTGCGCAGCCTGGGAGGAGGCGTGGATCCTGGCACGCTGAGCCTCAGGCACCAGCCTCCCTGTG CTCGACAGCAAAGTCTTGACTCCTTCCTGCTGAGCACTGTGCTACCTTCACTGCTCCAAAGCCAGACTAACA GCTCTCCAAGCCCTTGGGGTGACTCGGCTTCCAGGAGCTGTTGGAGAAATGAGGATGTCTGTCCCTGTCTGC CTGGGCAGGCCAGATTCCTCCCCAGCAGCCGGGTCTCTCCAGACCCTGATTCGGTGCCTTTCTGTTTACCAG CTACTTCAATCCCAAAGTTTGAATCTGCAGATACCTTACTCCCAGCCACTTTGCCTTCTTACTGTGTTGTGT GTTTTTCCTGGTGCTTCAAGAGCGTGTGCAGGGCAAGTGCCGTCACTGGGAACTGCACCAGATGCTCAGACT TGGTTGTCTTATGTTTACCAATAAATAAAAGTAGACTTTTTCTATTTTTATTTGCTGCTATTTGTGTGTGTG TTTGTGTTTGTGTAGCTAGGTATCTGGCACTTCTGACGATGCATTGTTGCTTTTTTCCCGAAGGTCCCGCAG GAACTGTGGCAATGGTGTGTGTGTGAAATGGTGTGTTAACCGCGTTTTGTTTGCTCCTGTATTGAATAGGAA GCAGTGGCCAGTCTGTCTTCCTTAGAGATGTTAGCATATTTTTATATGTATATATTTTGTACCAAAAAAGAG TGTTCCTTGTTTTGGTTACACTCGAAATTCTGACCTAGCTGGAGAGGGCTCTGGGCCGAGAGCTTTCACTAA GGGGAGACTTCAGGGGAGGATCAAGCTTTGAACCAAAGCCAATCACTGGCTTGATTTGTGTTTTTTAATTAA AAAAAAAATCATTCATGTATGCCACTTCTAAAAAAAAAAAAAAAAAAAAAAAAA

SEQ ID NO:6

MEELSSVGEQVFAAECILSKRLRKGKLEYLVK RG SSKHNSWEPEENILDPRLLLAFQKKEHEKEVQNRKR GKRPRGRPRKLTAMSSCSRRSKLKEPDAPSKSKSSSSSSSSTSSSSSSDEEDDSDLDAKRGPRGRETHPVPQ KKAQILVAKPELKDPIRKKRGRKPLPPEQKATRRPVSLAKVLKTARKDLGAPASKLPPPLSAPVAGLAALKA HAKEACGGPSAMATPENLASLMKGMASSPGRGGIS QSSIVHYMNRMTQSQAQAASRLALKAQATNKCGLGL DLKVRTQKGELGMSPPGSKIPKAPSGGAVEQKVGNTGGPPHTHGASRVPAGCPGPQPAPTQELSLQVLDLQS VKNGMPGVGLLARHATATKGVPATNPAPGKGTGSGLIGASGATMPTDTSKSEKLASRAVAPPTPASKRDCVK GSATPSGQESRTAPGEARKAATLPEMSAGEESSSSDSDPDSASPPSTGQNPSVSVQTSQDWKPTRSLIEHVF VTDVTANLITVTVKESPTSVGFFNLRHY

SEQ ID NO:7

SLIEHVFVT SEQ BD NO:8 KLPPPLSAPV SEQ NO:9

FVTDVTANL

SEQ T-D NO: 10

KVLKTARKDL

SEQ NO:ll

RPRGRPRKL SEQ JD NO:12

KESPTSVGFF

SEQ ID NO: 13

APVAGLAAL

SEQ D3 NO:14

KVRTQKGEL

SEQ NO:15

APASKLPPPL

SEQ ID NO: 16

GPQPAPTQEL SEQ D3 NO: 17

APGKGTGSGL

SEQ ID NO:18

NILDPRLLL

SEQ TD NO: 19

ILDPRLLLA

SEQ ID NO:20

QILVAKPEL SEQ TD NO:21 SLAKVLKTA

SEQ BD NO:22

DLGAPASKL

SEQ TD NO:23

PLSAPVAGL

SEQ ID NO:24

ATPGNLASL SEQ ID NO:25

GMSPPGSKI

SEQ DD NO:26

ELSLQVLDL

SEQ ID NO:27

GLLARHATA

SEQ DD NO:28

TANLITVTV

SEQ LD NO:29

RLRKGKLEYL SEQ JED NO:30

SAPVAGLAAL

SEQ LD NO:31

MATPGNLASL

SEQ ID NO:32

SLQVLDLQSA

SEQ ID NO:33

GLIGASGATM SEQ LD NO:34

LIEHVFVTDV SEQ ID NO:35

VTANLITVTV

SEQ LD NO:36

RLRKGKLEY

SEQ ID NO:37

KRLRKGKLEY

SEQ ID NO:38

SWEPEENILD SEQ ID NO:39

ILDPRLLLAF

SEQ ID NO:40

VTDVTANLIT

SEQ TD NO.-41

CILSKRLRK

SEQ ID NO:42

KGKLEYLVK

SEQ ID NO:43

RLLLAFQKK

SEQ ID NO:44

EVQNRKRGK SEQ LD NO:45

ILVAKPELK SEQ ID NO:46

ELKDPIRKK SEQ ID NO:47

PIRKKRGRK

5

SEQ DD NO:48

TRRPVSLAK

10 SEQ ID NO:49

PVSLAKVLK

SEQ ID NO:50

15

KDLGAPASK

SEQ LD NO:51

20 PVAGLAALK

SEQ LD NO:52

RLALKAQAT

9J^

SEQ ID NO:53

ALKAQATNK 30 SEQ ID NO:54

QVLDLQSAK

SEQ ID NO:55

35

KLASRAVAP

SEQ LD NO:56

0 RTAPGEARK

SEQ DD NO:57

SVQTSQD K 5 SEQ ID NO:58

ANLITVTVK 0 SEQ LD NO:59 ILSKRLRKGK

SEQ LD NO:60

RKGKLEYLVK SEQ LD NO:61

LVK RG SSK SEQ ID NO:62

KLTAMSSCSR SEQD3NO:63

KLKEPDAPSK

SEQ LD NO:64

QILVAKPELK

SEQ D NO:65

PLPPEQKATR

SEQ LD NO:66

ATRRPVSLAK

SEQLDNO:67

RKDLGAPASK

SEQ LD NO:68

APVAGLAALK

SEQ ID NO:69

GLAALKAHAK

SEQ LD NO:70

RLALKAQATN SEQ LD NO:71

GLDLKVRTQK

SEQ LD NO:72 ELGMSPPGSK

SEQ LD NO:73

KVGNTGGPPH

SEQ LD NO:74

LLARHATATK SEQ LD NO:75

AVAPPTPASK

SEQ LD NO:76

SLIEHVFVTD SEQ LD NO:77

VPQKKAQIL

SEQ LD NO:78

KPLPPEQKA

SEQ LD NO:79

RPVSLAKVL

SEQ LD NO:80

IPKAPSGGA SEQ LD NO:81

APGEARKAA SEQ LD NO:82

RPRGRPRKLT

SEQ LD NO:83

GPRGRETHPV

SEQ LD NO:84

PPLSAPVAGL SEQ LD NO.-85

QPAPTQELSL SEQ LD NO:86

APGEARKAAT

SEQ LD NO:87

SPTSVGFFNL

SEQ LD NO:88

SKRLRKGKL

SEQ LD NO:89

LRKGKLEYL

SEQ LD NO:90

RSKLKEPDA

SEQ LD NO:91

GPRGRETHP

SEQ LD NO:92

RKKRGRKPL

SEQ LD NO:93

KATRRPVSL

SEQ LD NO:94

VLKTARKDL SEQ LD NO:95

DLKVRTQKG

SEQ LD NO:96

GEARKAATL

SEQ LD NO:97

WRSSIVHYM SEQ LD NO.-98

LVKWRGWSS SEQ LD NO:99

YMNRMTQSQ

SEQ LD NO: 100

YLVKWRGWS

SEQ LD NO:101

VHYMNRMTQ

SEQ LD NO: 102

LGLDLKVRT

Claims

Claims

1. An isolated polypeptide comprising an amino acid sequence which has at least 85% identity to the amino acid sequence of SEQ ID NO:2 over the entire length of of SEQ ID NO:2 respectively.

2. An isolated polypeptide as claimed in claim 1 in which the amino acid sequence has at least 95% identity to SEQ ID NO:2.

3. The polypeptide as claimed in claim 1 comprising the amino acid sequence of SEQ ID NO:2.

4. The isolated polypeptide of SEQ ID NO:2.

5. A polypeptide comprising an immunogenic fragment of a polypeptide as claimed in any one of claims 1 to 4 (if necessary when coupled to a carrier) which is capable of raising an immune response which recognises the polypeptide of SEQ ID NO:2.

6. An immunogenic fragment of SEQ ID NO:2 wherein the fragment comprises a sequence of one or more of SEQ ID NO:7 to SEQ ID NO:102.

7. A polypeptide as claimed in any of claims 1 to 6 wherein said polypeptide is part of a larger fusion protein.

8. A polypeptide as claimed in any of claims 1 to 6 chemically conjugated to a carrier protein.

9. An isolated polynucleotide encoding a polypeptide as claimed in any of claims 1 to 6.

10. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide that has at least 85% identity to the amino acid sequence of SEQ ID NO:2, over the entire length of SEQ ID NO:2; or a nucleotide sequence complementary to said isolated polynucleotide.

11. An isolated polynucleotide comprising a nucleotide sequence that has at least 85% identity to a nucleotide sequence encoding a polypeptide of SEQ ID NO:2, over the entire coding region; or a nucleotide sequence complementary to said isolated polynucleotide.

12. An isolated polynucleotide which comprises a nucleotide sequence which has at least 70% identity to that of SEQ ID NO: 1 over the entire length of SEQ ID NO: 1 ; or a nucleotide sequence complementary to said isolated polynucleotide.

13. The is lated polynucleotide as defined in any one of claims 9 to 12 in which the identity is at least 95%.

14. An isolated polynucleotide selected from:

(a) a polynucleotide comprising a nucleotide sequence encoding the polypeptide of SEQ ID NO:2;

(b) the coding region ofthe polynucleotide of SEQ ID NOT; and

(c) a polynucleotide obtainable by screening an appropriate human library under stringent hybridization conditions with a labeled probe having the sequence of SEQ ID NO: 1 or a fragment thereof said polynucleotide encoding a protein which is capable of rasing an immune response, (if necessary when coupled to a carrier) which recognises the polypeptide of sequence ID N°2; or a nucleotide sequence complementary to said isolated polynucleotide.

15. An expression vector comprising an isolated polynucleotide according to any one of claims 9 to 14.

16. A recombinant live microorganism comprising an isolated polynucleotide according to any one of claims 9 to 14.

17. A host cell comprising the expression vector of claim 15 or the isolated polynucleotide of claims 9 to 14.

18. A process for producing a polypeptide of claims 1 to 8 comprising culturing a host cell of claim 17 under conditions sufficient for the production of said polypeptide and recovering the polypeptide from the culture medium.

19. A vaccine comprising an effective amount ofthe polypeptide of any one of claims 1 to 8 and a pharmaceutically acceptable carrier.

20. A vaccine comprising an effective amount ofthe polynucleotide of any one of claims 9 to 14 and a pharmaceutically effective carrier.

21. A vaccine comprising an effective amount of antigen presenting cells, modified by in vitro loading with a polypeptide of any one of claims 1 to 8, or genetically modified in vitro to express a polypeptide of claims 1 to 8 and a pharmaceutically effective carrier.

22. A vaccine as claimed in any one of claims 19 to 21 which additionally comprises a TH-1 inducing adjuvant.

23. A vaccine as claimed in claim 22 in which the TH-1 inducing adjuvant is selected from the group of adjuvants comprising: 3D-MPL, QS21, a mixture of QS21 and cholesterol, and a CpG oligonucleotide or a mixture of two or more said adjuvants.

24. An antibody immunospecific for the polypeptide or immunological fragment as claimed in any one of claims 1 to 6.

25. A method for screening to identify compounds which stimulate or which inhibit the function ofthe polypeptide of any one of claims 1 to 6 which comprises a method selected from the group consisting of:

(a) measuring the binding of a candidate compound to the said polypeptide (or to the cells or membranes bearing the polypeptide) or a fusion protein thereof by means of a label directly or indirectly associated with the candidate compound;

(b) measuring the binding of a candidate compound to the said polypeptide (or to the cells or membranes bearing the polypeptide) or a fusion protein thereof in the presence of a labeled competitior; (c) testing whether the candidate compound results in a signal generated by activation or inhibition ofthe said polypeptide, using detection systems appropriate to the cells or cell membranes bearing the polypeptide;

(d) mixing a candidate compound with a solution containing a polypeptide of any one of claims 1 to 8, to form a mixture, measuring activity ofthe polypeptide in the mixture, and comparing the activity ofthe mixture to a standard; or

(e) detecting the effect of a candidate compound on the production of mRNA encoding said polypeptide and said polypeptide in cells, using for instance, an ELISA assay.

26. A method for the treatment of a subject by immunoprophylaxis or therapy comprising in vitro induction of immune responses to a molecule of any one of claims 1 to 6, using in vitro incubation ofthe polypeptide of any one of claims 1 to 8 or the polynucleotide of any one of claims 9 to 14 with cells from the immune system of a mammal, and reinfusing these activated immune cells to the mammal for the treatment of disease.

27. A method as claimed in claim 26 wherein the treatment is for Crohn's disease, Colitis ulcerosa, colorectal cancer, lung cancer and preneoplasic lesions, breast, brain, uterus, muscle, eye and germ cell cancers, Wilm's tumour, retinoblastoma, rabdomyosarcoma, leimyosarcoma and synovial sarcoma.

28. An agonist or antagonist to the polypeptide of claims 1 to 6.

29. A compound which is:

(a) an agonist or antagonist to the polypeptide of claims 1 to 6; (b) isolated polynucleotide of claims 9 to 14; or

(c) a nucleic acid molecule that modulates the expression ofthe nucleotide sequence encoding the polypeptide of any one of claims 1 to 6; for use in therapy.

30. A process for diagnosing a disease or a susceptibility to a disease in a subject related to expression or activity of a polypeptide of any one of claims 1 to 6 in a subject comprising analyzing for the presence or amount of said polypeptide in a sample derived from said subject.

31. A process for diagnosing a disease or a susceptibility to a disease in a subject related to expression or activity of a polynucleotide of any one of claims 9 to 14 in a subject comprising analyzing for the presence or amount of said polynucleotide in a sample derived from said subject.

32. A process according to claim 30 or 31 wherein said disease is Crohn's disease, Colitis ulcerosa, colorectal cancer, lung cancer and preneoplasic lesions, breast, brain, uterus, muscle, eye and germ cell cancers, Wilm's tumour, retinoblastoma, rabdomyosarcoma, leimyosarcoma and synovial sarcoma.

33. An isolated polynucleotide selected form the group consisting of:

(a) an isolated polynucleotide comprising a nucleotide sequence which has at least 70% identity to SEQ ID NO:5 over the entire length of SEQ ID NO:5; (b) an isolated polynucleotide comprising the polynucleotide of SEQ ID NO:5;

(c) the polynucleotide of SEQ ID NO:5; or

(d) an isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide which has at least 85% identity to the amino acid sequence of SEQ ID NO:6, over the entire length of SEQ ID NO:6.

34. A polypeptide selected from the group consisting of:

(a) a polypeptide which comprises an amino acid sequence which has at least 85% identity to that of SEQ ID NO:6 over the entire length of SEQ ID NO:6;

(b) a polypeptide in which the amino acid sequence has at least 85% identity to the amino acid sequence of SEQ ID NO:6 over the entire length of SEQ ID NO:6;

(c) a polypeptide which comprises the amino acid of SEQ ID NO:6;

(d) a polypeptide which is the polypeptide of SEQ ID NO:6; or

(e) a polypeptide which is encoded by a polynucleotide comprising the sequence contained in SEQ ID NO:5.

35. A live vaccine composition comprising an expression vector according to claim 15 or recombinant live micro-organism according to claim 16.

36. Use of a polynucleotide as claimed in any one of claims 9 to 14 for the manufacture of a medicament in the treatment of cancer.

37. Use of a polynucleotide as claimed in any one of claims 9 to 14 for the manufacture of a medicament in the treatment of Crohn's disease, Colitis ulcerosa, colorectal cancer, lung cancer and preneoplasic lesions, breast, brain, uterus, muscle, eye and germ cell cancers, Wilm's tumour, retinoblastoma, rabdomyosarcoma, leimyosarcoma and synovial sarcoma.

38. Use of a polypeptide as claimed in any one of claims 1 to 8 for the manufacture of a medicament in the treatment of cancer.

39. Use of a polypeptide as claimed in any one of claims 1 to 8 for the manufacture of a medicament in the treatment of Crohn's disease and Colitis ulcerosa, colorectal cancer, lung cancer and preneoplasic lesions, breast, brain, uterus, muscle, eye and germ cell cancers, Wilm's tumour, retinoblastoma, rabdomyosarcoma, leimyosarcoma and synovial sarcoma.