VACCINE

Abstract: The present invention relates to novel proteins and to their production, from the prostase family. In particular, the invention pertains to a prostase protein or fragment thereof fused to an immunological fusion partner, such as NS1. Such antigens may be formulated to provide vaccines for the treatment of prostate tumours. Novel methods for purifying prostase protein and homologues are also provided.

Description: VACCINE The present invention relates to protein derivatives of a protein known as prostase, a prostate-specific serine protease, to methods for their purification and manufacture, and also to pharmaceutical compositions containing such derivatives, and to their use in medicine. In particular such derivatives find utility in cancer vaccine therapy, particularly prostate cancer vaccine therapy and diagnostic agents for prostate tumours. In particular the derivatives of the invention include fusion proteins comprising prostase linked to an immunological or an expression enhancer fusion partner. The present invention also provides methods for purifying the prostase derivatives and for formulating vaccines for immunotherapeutically treating prostate cancer patients and prostase-expressing tumours other than prostate tumours, prostatic hyperplasia, and prostate intraepithelilial neoplasia (PIN). Prostate cancer is the most common cancer among males, with an estimated incidence of 30% in men over the age of 50. Overwhelming clinical evidence shows that human prostate cancer has the propency to metastasise to bone, and the disease appears to progress inevitably from androgen dependent to androgen refractory status, leading to increased patient mortality (Abbas F., Scardino P. "The Natural History of Clinical Prostate Carcinoma." In Cancer (1997); 80:827-833). This prevalent disease is currently the second leading cause of cancer death among men in the US. Despite considerable research into therapies for the disease, prostate cancer remains difficult to treat. Currently, treatment is based on surgery and/or radiation therapy, but these methods are ineffective in a significant percentage of cases (Frydenberg M., Strieker P., Kaye K. "Prostate Cancer Diagnosis and Management "The Lancet (1997); 349:1681-1687). Several tumour-associated antigens are already known. Many of these antigens may be interesting targets for immunotherapy, but are either not fully tumour-specific or are closely related to normal proteins, and hence bear with them the risk of organ-specific auto-immunity, once targeted by a potent immune response. When an auto-immune response to non-crucial organs can be tolerated, auto-immunity to heart, intestine and other crucial organs could lead to unacceptable safety profiles. Some previously identified prostate specific proteins like prostate specific antigen (PSA) and prostatic acid phosphatase (PAP), prostate- specific membrane antigen (PSMA) and prostate stem cell antigen (PSCA) have limited therapeutic potential and moreover are not always correlated with the presence of prostate cancer or with the level of metastasis (Pound C, Partin A., Eisenberg M. et al. "Natural History of Progression after PSA Elevation following Radical Prostatectomy." In Jama (1999); 281 :1591-1597) (Bostwick D., Pacelli A., Blute M. et al. "Prostate Specific Membrane Antigen Expression in Prostatic Intraepithelial Neoplasia and Adenocarcinoma." In Cancer (1998); 82:2256-2261). The existence of tumour rejection mechanisms has been recognised since several decades. Tumour antigens, though encoded by the genome of the organism and thus theoretically not recognized by the immune system through the immune tolerance phenomenon, can occasionally induce immune responses detectable in cancer patients. This is evidenced by antibodies or T cell responses to antigens expressed by the tumour (Xue BH., Zhang Y., Sosman J. et al. "Induction of Human Cytotoxic T-Lymphocytes Specific for Prostate-Specific Antigen." In Prostate (1997); 30(2):73-78). When relatively weak anti-tumour effects can be observed through the administration of antibodies recognizing cell surface markers of tumour cells, induction of strong T cell responses to antigens expressed by tumour cells can lead to complete regression of established tumours in animal models (mainly murine). It is now recognised that the expression of tumour antigens by a cell is not sufficient for induction of an immune response to these antigens. Initiation of a tumour rejection response requires a series of immune amplification phenomena dependent on the intervention of antigen presenting cells, responsible for delivery of a series of activation signals. Prostase is a prostate-specific serine protease (trypsin-like), 254 amino acid- long, with a conserved serine protease catalytic triad H-D-S and a amino-terminal pre- propeptide sequence, indicating a potential secretory function (P. Nelson, Lu Gan, C. Ferguson, P. Moss, R. Gelinas, L. Hood & K. Wand, "Molecular cloning and characterisation of prostase, an androgen-regulated serine protease with prostate restricted expression, In Proc. Natl. Acad. Sci. USA (1999) 96, 31 14-31 19). A putative glycosylation site has been described. The predicted structure is very similar to other known serine proteases, showing that the mature polypeptide folds into a O 01/04143 single domain. The mature protein is 224 amino acids-long, with one A2 epitope shown to be naturally processed. Prostase nucleotide sequence and deduced polypeptide sequence and homologs are disclosed in Ferguson, et al. (Proc. Natl. Acad. Sci. USA 1999, 96, 3114-3119) and in International Patent Applications No. WO 98/12302 (and also the corresponding granted patent US 5,955,306), WO 98/20117 (and also the corresponding granted patents US 5,840,871 and US 5,786, 148) (prostate-specific kallikrein) and WO 00/04149 (P703P). The present invention provides prostase protein fusions based on prostase protein and fragments and homologues thereof ("derivatives"). Such derivatives are suitable for use in therapeutic vaccine formulations which are suitable for the treatment of a prostate tumours. Prostase fragments of the invention will be of at least about 10 consecutive amino acids, preferably about 20, more preferably about 50, more preferably about 100, more preferably about 150 contiguous amino acids selected from the amino acid sequences as shown in SEQ ID N°7 or SEQ ID N°8. More particularly fragments will retain some functional property, preferably a immunological activity, of the larger molecule set forth in SEQ ID N°7 or SEQ ID N°8, and are useful in the methods described herein (e.g. in vaccine compositions, in diagnostics, etc.). In particular the fragments will be able to generate an immune response, when suitable attached to a carrier, that will recognise the protein of SEQ ID N°7 or SEQ ID N°8. Prostase homologues 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 90% identity, yet more preferably at least 95% identity, most preferably at least 97-99% identity, to that of SEQ ID NO:7 or SEQ ID N°8 over the entire length of SEQ ID NO:7 or SEQ ID N°8. Such polypeptides include those comprising the amino acid of SEQ ID NO:7 or SEQ ID N°8. The prostase antigen derivative or fragments and homologues thereof may in a preferred embodiment carry a mutation in the active site of the protein, to reduce substantially or preferably eliminate its protease biological activity. Preferred mutations involve replacing the Histidine and Aspartate catalytic residues of the serine protease. In a preferred embodiment, prostase contains a Histidine-Alanine O 01/04143 mutation at residue 71 of prostase sequence (Ferguson, et al. (Proc. Natl. Acad. Sci. USA 1999, 96, 3114-3119) which corresponds to amino acid 43 of P703PDE5 sequence (SEQ ID N°8). The mutated protein preferably has a significant decrease in the catalytic efficiency (expressed in enzymatic specific activity) of the protein as compared to the non-mutated one. Preferably the reduction in the catalytic efficiency is at least by a factor of 10 3 , more preferably at least by a factor of 10 6 . The protein which has undergone a histidine alanine mutation is hereafter referred to as * (star). In one embodiment, the present invention relates to fusions proteins, comprising the tumour-associated prostase or fragment or homologues thereof and a heterologous protein or part of a protein acting as a fusion partner. The protein and the fusion partner may be chemically conjugated, but are preferably expressed as recombinant fusion proteins in a heterologous expression system. In a preferred embodiment of the invention there is provided a prostase fusion protein or fragment or homologues thereof linked to an immunological fusion partner that may assist in providing T helper epitopes. Thus the fusion partner may act through a bystander helper effect linked to secretion of activation signals by a large number of T cells specific to the foreign protein or peptide, thereby enhancing the induction of immunity to the prostase component as compared to the non-fused protein. Preferably the heterologous partner is selected to be recognizable by T cells in a majority of humans. In another embodiment, the invention provides a prostase protein or fragment or homologues thereof linked to a fusion partner that acts as an expression enhancer. Thus the fusion partner may assist in aiding in the expression of prostase in a heterologous system, allowing increased levels to be produced in an expression system as compared to the native recombinant protein. Preferably the fusion partner will be both an immunological fusion partner and an expression enhancer partner. Accordingly, the present invention in the embodiment provides fusion proteins comprising the tumour-specific prostase or a fragment thereof linked to a fusion partner. Preferably the fusion partner is acting both as an immunological fusion partner and as an expression enhancer partner. Accordingly, in a preferred form of the invention, the fusion partner is the non- structural protein from influenzae virus, NS1 (hemagglutinin) or fragment thereof. Typically the N-terminal 81 amino acids are utilised, although different fragments may be used provided they include T-helper epitopes (C. Hackett, D. Horowitz, M. Wysocka & S. Dillon, 1992, J. Gen. Virology, 73, 1339-1343). When NSl is the immunological fusion partner it has the additional advantage in that it allows higher expression yields to be achieved. In particular, such fusions are expressed at higher yields than the native recombinant prostase proteins. In preferred embodiments, the prostase moiety within the fusion is selected from the group comprising SEQ ID NO: 5 (Millenium WO 98/12302), SEQ ID NO: 6 (Incyte WO 98/20117), SEQ ID NO: 7 (PNAS (1999) 96, 3114-3119), and SEQ ID NO: 8 (Corixa WO 00/04149, P703PDE5 sequence). Yet in a most preferred embodiment, the fusion protein comprises the N-terminal 81 amino acids of NSl non structural protein fused to the 5 to 226 carboxy-terminal amino acids from mutated prostase, as set forth in SEQ ID NO: 1 or SEQ ID N°3. The proteins of the present invention are expressed in an appropriate host cell, and preferably in E. coli. In a preferred embodiment the proteins are expressed with an affinity tag, such as for example, a histidine tail comprising between 5 to 9 and preferably six histidine residues, most preferably at least 4 histidine residues. These are advantageous in aiding purification through for example ion metal affinity chromatography (IMAC). The present invention also provides a nucleic acid encoding the proteins of the present invention. Such sequences can be inserted into a suitable expression vector and used for DNA/RNA vaccination or expressed in a suitable host. In additional embodiments, genetic constructs comprising one or more of the polynucleotides of the invention are introduced into cells in vivo. This may be achieved using any of a variety or well-known approaches. One of the preferred methods for in vivo delivery of one or more nucleic acid sequences involves the use of an expression vector such as a recombinant live viral or bacterial microorganism. Suitable viral expression vectors are for example poxviruses (e.g; vaccinia, fowlpox, canarypox), alphaviruses (Sindbis virus, Semliki Forest Virus, Venezuelian Equine Encephalitis Virus), adenoviruses, adeno-associated virus, picornaviruses (poliovirus, rhinovirus), and herpesviruses (varicella zoster virus, etc). Other preferred methods for in vivo delivery of one or more nucleic acid sequences involves the use of a bacterial expression vector, such as Listeria, Salmonella , Shigella and BCG. Inoculation and in vivo infection with this live vector will lead to in vivo expression of the antigen and induction of immune responses. These viruses and bactena can be virulent, or attenuated in vanous ways in order to obtain live vaccines. Such live vaccines also form part of the invention. A DNA sequence encoding the proteins of the present invention can be synthesized using standard DNA synthesis techniques, such as by enzymatic gation as descπbed by D.M. Roberts et al in Biochemistry 1985, 24, 5090-5098, by chemical synthesis, by in vitro enzymatic polymerization, or by PCR technology utilising for example a heat stable polymerase, or by a combination of these techniques. Enzymatic polymerisation of DNA may be earned out in vitro using a DNA polymerase such as DNA polymerase I (Klenow fragment) in an appropπate buffer containing the nucleoside tπphosphates dATP, dCTP, dGTP and dTTP as required at a temperature of 10°-37°C, generally in a volume of 50μl or less Enzymatic hgation of DNA fragments may be earned out using a DNA hgase such as T4 DNA gase in an appropnate buffer, such as 0.05M Tns (pH 7 4), 0.01M MgCl 2 , 0.01M dithiothreitol, ImM spermidme, ImM ATP and O.lmg/ml bovine serum albumin, at a temperature of 4°C to ambient, generally in a volume of 50ml or less. The chemical synthesis of the DNA polymer or fragments may be earned out by conventional phosphotnester, phosphite or phosphoramidite chemistry, using solid phase techniques such as those descnbed m 'Chemical and Enzymatic Synthesis of Gene Fragments - A Laboratory Manual' (ed. H.G. Gassen and A. Lang), Verlag Chemie, Weinheim (1982), or in other scientific publications, for example M.J. Gait, H.W.D Matthes, M. Singh, B.S Sproat. and R.C. Titmas, Nucleic Acids Research. 1982, 10, 6243; B.S. Sproat, and W. Bannwarth, Tetrahedron Letters, 1983, 24, 5771 , M.D Matteucci and M.H. Caruthers, Tetrahedron Letters, 1980, 21, 719; M.D Matteucci and M.H. Caruthers, Journal of the Amencan Chemical Society, 1981, 103, 3185, S P. Adams et al, Journal of the Amencan Chemical Society, 1983, 105, 661 , N.D. Sinha, J Biemat, J. McMannus, and H. Koester, Nucleic Acids Research, 1984, 12, 4539, and H W.D. Matthes et al , EMBO Journal, 1984, 3, 801. In a further embodiment of the invention is provided a method of producing a protein as descnbed herein The process of the invention may be performed by conventional recombinant techniques such as descnbed in Maniatis et al , Molecular Cloning - A Laboratory Manual, Cold Spnng Harbor, 1982-1989. In particular, the process of the invention may preferably comprise the steps of: i) preparing a replicable or integrating expression vector capable, in a host cell, of expressing a DNA polymer comprising a nucleotide sequence that encodes the protein or an immunogenic derivative thereof; ii) transforming a host cell with said vector; ii) culturing said transformed host cell under conditions permitting expression of said DNA polymer to produce said protein; and iv) recovering said protein. The term 'transforming' is used herein to mean the introduction of foreign DNA into a host cell. This can be achieved for example by transformation, transfection or infection with an appropriate plasmid or viral vector using e.g. conventional techniques as described in Genetic Engineering; Eds. S.M. Kingsman and A.J. Kingsman; Blackwell Scientific Publications; Oxford, England, 1988. The term 'transformed' or 'transformant' will hereafter apply to the resulting host cell containing and expressing the foreign gene of interest. Preferably recombinant antigens of the invention are expressed in unicellular hosts, most preferably in bacterial systems, most preferably in E. coli. Preferably the recombinant strategy includes cloning a gene construct encoding a NSl fusion protein, the gene construct comprising from 5' to 3' a DNA sequence encoding NSl joined to a DNA sequence encoding the protein of interest, into an expression vector to form a DNA fragment encoding a NS 1 - carboxyl-terminal P703P fusion protein. An affinity polyhistidine tail may be engineered at the carboxy- terminus of the fusion protein allowing for simplified purification through affinity chromatography. The expression vectors are novel and also form part of the invention. The replicable expression vectors may be prepared in accordance with the invention, by cleaving a vector compatible with the host cell to provide a linear DNA segment having an intact replicon, and combining said linear segment with one or more DNA molecules which, together with said linear segment encode the desired product, such as the DNA polymer encoding the protein of the invention, or derivative thereof, under ligating conditions. Thus, the hybnd DNA may be pre-formed or formed dunng the construction of the vector, as desired. The choice of vector will be determined in part by the host cell, which may be prokaryotic or eukaryotic but are preferably E. coh, yeast or CHO cells. Suitable vectors include plasmids, bacteπophages, cosmids and recombinant viruses. Expression and cloning vectors preferably contain a selectable marker such that only the host cells expressing the marker will survive under selective conditions. Selection genes include but are not limited to the one encoding protein that confer a resistance to ampicil n, tetracyclin or kanamycin. Expression vectors also contain control sequences which are compatible with the designated host. For example, expression control sequences for E. coh, and more generally for prokaryotes, include promoters and nbosome binding sites. Promoter sequences may be naturally occumng, such as the β-lactamase (penicilhnase) (Weissman 1981, In Interferon 3 (ed. L. Gresser), lactose (lac) (Chang et al. Nature, 1977, 198: 1056) and tryptophan (tip) (Goeddel et al. Nucl. Acids Res. 1980, 8, 4057) and lambda-denved P L promoter system. In addition, synthetic promoters which do not occur m nature also function as bacterial promoters. This is the case for example for the tac synthetic hybnd promoter which is denved from sequences of the trp and lac promoters (De Boer et al., Proc. Natl Acad Sci. USA 1983, 80, 21-26). These systems are particularly suitable with E. coli. Yeast compatible vectors also carry markers that allow the selection of successful transformants by conferring prototrophy to auxotrophic mutants or resistance to heavy metals on wild-type strains. Control sequences for yeast vectors include promoters for glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg. 1968, 7, 149), PHO5 gene encoding acid phosphatase, CUPl gene, ARG3 gene, GAL genes promoters and synthetic promoter sequences.. Other control elements useful in yeast expression are terminators and leader sequences. The leader sequence is particularly useful since it typically encodes a signal peptide compπsed of hydrophobic amino acids which direct the secretion of the protein from the cell. Suitable signal sequences can be encoded by genes for secreted yeast proteins such as the yeast invertase gene and the a-factor gene, acid phosphatase, killer toxin, the a-mating factor gene and recently the heterologous mulinase signal sequence denved from INUIA gene of Kluyveromyces marxianus.. Suitable vectors have been developed for expression in Pichia pastons and Saccharomyces cerevisiae. A vanety of P pastoris expression vectors are available based on vanous mducible or constitutive promoters ( Cereghmo and Cregg, FEMS Microbiol. Rev. 2000,24 45-66). For the production of cytosohc and secreted proteιns,the most commonly used P. pastons vectors contain the very strong and tightly regulated alcohol oxidase (AOX1) promoter The vectors also contain the P pastoris histidinol dehydrogenase (HIS4) gene for selection in hιs4 hosts. Secretion of foreign protein require the presence of a signal sequence and the S cerevisiae prepro alpha mating factor leader sequence has been widly and successfully used in Pichia expression system. Expression vectors are integrated into the P. pastons genome to maximize the stability of expression strains. As in S cerevisiae, cleavage of a P.pastoris expression vector within a sequence shared by the host genome (AOX1 or HIS4) stimulates homologous recombination events that efficiently target integration of the vector to that genomic locus. In general, a recombinant strain that contains multiple integrated copies of an expression cassette can yield more heterologous protein than single-copy strain. The most effective way to obtain high copy number transformants requires the transformation of Pichia recipient strain by the sphaeroplast technique (Cregg et all 1985, Mol.Cell.Biol. 5: 3376-3385) . The preparation of the replicable expression vector may be earned out conventionally with appropπate enzymes for restπction, polymeπsation and Hgation of the DNA, by procedures descnbed in, for example, Maniatis et al. cited above. The recombinant host cell is prepared, in accordance with the invention, by transforming a host cell with a replicable expression vector of the invention under transforming conditions. Suitable transforming conditions are conventional and are descnbed in, for example, Maniatis et al. cited above, or "DNA Cloning" Vol. II, D.M Glover ed., IRL Press Ltd, 1985. The choice of transforming conditions depends upon the choice of the host cell to be transformed. For example, in vivo transformation using a live viral vector as the transforming agent for the polynucleotides of the invention is descnbed above Bactenal transformation of a host such as £ coh may be done by direct uptake of the polynucleotides (which may be expression vectors containing the desired sequence) after the host has been treated with a solution of CaCl? (Cohen et al , Proc. Nat. Acad Sci., 1973, 69, 2110) or with a solution compnsing a mixture of rubidium chlonde (RbCl), MnCl 2 , potassium acetate and glycerol, and then with 3-[N-morphohno]- propane-sulphonic acid, RbCl and glycerol. Transformation of lower eukaryotic organisms such as yeast cells in culture by direct uptake may be earned out by using the method of Hinnen et al (Proc Natl. Acad. Sci. 1978, 75 • 1929-1933). Mammalian cells in culture may be transformed using the calcium phosphate co-precipitation of the vector DNA onto the cells (Graham & Van der Eb, Virology 1978, 52, 546). Other methods for introduction of polynucleotides into mammalian cells include dextran mediated transfection, polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotιde(s) into hposomes, and direct micro-injection of the polynucleotides into nuclei. The invention also extends to a host cell transformed with a nucleic acid encoding the protein of the invention or a replicable expression vector of the invention. Cultunng the transformed host cell under conditions permitting expression of the DNA polymer is earned out conventionally, as descnbed in, for example, Maniatis et al. and "DNA Cloning" cited above. Thus, preferably the cell is supplied with nutnent and cultured at a temperature below 50°C, preferably between 25°C and 35°C, most preferably at 30°C. The incubation time may vary from a few minutes to a few hours, according to the proportion of the polypeptide in the bacteπal cell, as assessed by SDS-PAGE or Western blot. The product may be recovered by conventional methods according to the host cell and according to the localisation of the expression product (intracellular or secreted into the culture medium or into the cell peπplasm) Thus, where the host cell is bactenal, such as E coh it may, for example, be lysed physically, chemically or enzymatically and the protein product isolated from the resulting lysate. Where the host cell is mammalian, the product may generally be isolated from the nutnent medium or from cell free extracts Where the host cell is a yeast such as Saccharomyces cerevisiae or Pichia pastoris, the product may generally be isolated from from lysed cells or from the culture medium, and then further punfied using conventional techniques. The specificity of the expression system may be assessed by western blot using an antibody directed against the polypeptide of interest. Conventional protein isolation techniques include selective precipitation, adsorption chromatography, and affinity chromatography including a monoclonal antibody affinity column. When the proteins of the present invention are expressed with a histidine tail (His tag), they can easily be punfied by affinity chromatography using an ion metal affinity chromatography column (IMAC) column. In a preferred embodiment of the invention the proteins of the present invention is provided with an affinity tag, such as a polyhistidine tail. In such cases the protein after the blocking step is preferably subjected to affinity chromatography For those proteins with a polyhistidine tail, immobilised metal ion affinity chromatography (IMAC) may be performed The metal ion, may be any suitable ion for example zinc, nickel, iron, magnesium or copper, but is preferably zinc or nickel Preferably the IMAC buffer contains detergent, preferably a non-ionic detergent such as Tween 80, or a zwittenonic detergent such as Empigen BB, as this may result in lower levels of endotoxin in the final product. Further chromatographic steps include for example a Q-Sepharose step that may be operated either before of after the IMAC column. Preferably the pH is in the range of 7.5 to 10, more preferably from 7.5 to 9.5, optimally between 8 and 9, ideally 8.5. The proteins of the present invention are provided either soluble a liquid form or in a lyophihsed form, which is the preferred form. It is generally expected that each human dose will compose 1 to 1000 μg of protein, and preferably 30-300 μg- The present invention also provides pharmaceutical composition compπsmg a protein of the present invention in a pharmaceutically acceptable excipient. A preferred vaccine composition compnses at least NS1-P703P* (SEQ ID N°l) or NS1-P703P (SEQ ID N°3). Said protein has, preferably, blocked thiol groups and is highly punfied, e g. has less than 5% host cell contamination. Such vaccine may optionally contain one or more other tumour-associated antigen and denvatives. For example, suitable other associated antigen include PAP-1, PSA (prostate specific antigen), PSMA (prostate-specific membrane antigen), PSCA (Prostate Stem Cell Antigen), STEAP. Vaccine preparation is generally descnbed in Vaccine Design ("The subunit and adjuvant approach" (eds. Powell M F. & Newman M.J). (1995) Plenum Press New York) Encapsulation within posomes is descnbed by Fullerton, US Patent 4,235,877. The proteins of the present invention are preferably adjuvanted in the vaccine formulation of the invention. Suitable adjuvants are commercially available such 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 (SmithKline Beecham, 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 or interleukin-2, -7, or -12, may also be used as adjuvants. In the formulations of the invention it is preferred that the adjuvant composition induces an immune response predominantly of the TH1 type. High levels of Thl-type cytokines (e.g., IFN-γ, TNFα, IL-2 and IL-12) tend to favour the induction of cell mediated immune responses to an administered antigen. 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. Accordingly, suitable adjuvants for use in eliciting a predominantly Thl-type response include, for example a combination of monophosphoryl lipid A, preferably 3-de-O-acylated monophosphoryl lipid A (3D-MPL) together with an aluminium salt. Other known adjuvants which preferentially induce a TH1 type immune response include CpG containing oligonucleotides. The oligonucleotides are characterised in that the CpG dinucleotide is unmethylated. Such oligonucleotides are well known and are described in, for example WO 96/02555. Immunostimulatory DNA sequences are also described, for example, by Sato et al., Science 273:352, 1996. Another preferred adjuvant is a saponin, preferably QS21 (Aquila Biopharmaceuticals Inc., Framingham, MA), which may be used alone or in combination with other adjuvants. For example, an enhanced system involves the combination of a monophosphoryl lipid A and saponin derivative, such as the combination of QS21 and 3D-MPL 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. A particularly potent adjuvant formulation involving QS21, 3D-MPL and tocopherol in an oil-in-water emulsion is described in WO 95/17210. A particularly potent adjuvant formulation involving QS21 3D-MPL & tocopherol in an oil in water emulsion is described in WO 95/17210 and is a preferred formulation. Other preferred adjuvants include Montanide ISA 720 (Seppic, France), SAF (Chiron, California, United States), ISCOMS (CSL), MF-59 (Chiron), Detox (Ribi, Hamilton, MT), RC-529 (Corixa, Hamilton, MT) and other aminoalkyl glucosaminide 4-phosphates (AGPs). Other preferred adjuvants include adjuvant molecules of the general formula (I): HO(CH 2 CH 2 O) n -A-R Wherein, n is 1-50, A is a bond or -C(O)-, R is Cι. 50 alkyl or Phenyl Cι. 50 alkyl. One embodiment of the 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 C 1 . 50 , preferably C - C 20 alkyl and most preferably Cι 2 alkyl, and A is a bond. The concentration of the 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 (12 th 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. Accordingly in one embodiment of the present invention there is provided a vaccine comprising a protein of the present invention, more preferably a NS1-P703P* adjuvanted with a monophosphoryl lipid A or derivative thereof, QS21 and tocopherol in an oil in water emulsion. Preferably the vaccine additionally comprises a saponin, more preferably QS21. Another particular suitable adjuvant formulation including CpG and a saponin is described in WO 00/09159 and is a preferred formulation. Most preferably the saponin in that particular formulation is QS21. Preferably the formulation additionally comprises an oil in water emulsion and tocopherol. Any of a variety of delivery vehicles may be employed within pharmaceutical compositions and vaccines to facilitate production of an antigen-specific immune response that targets tumour cells. Delivery vehicles include 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 of the T cell response, to have anti-tumour 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 peri-tumoural tissues, and may be autologous, allogeneic, syngeneic or xenogeneic cells. Certain preferred embodiments of the present invention use dendritic cells or progenitors thereof as antigen-presenting cells. Dendritic cells are highly potent APCs (Banchereau and Steinman, Nature 592:245-251, 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. 50:501-529, 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, peritumoral 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, lipopolysaccharide 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 CD11) and costimulatory molecules (e.g., CD40, CD80, CD86 and 4-1BB). APCs may generally be transfected with a polynucleotide encoding prostase tumour protein (or derivative thereof) such that the prostase tumour polypeptide, or an immunogenic portion thereof, is expressed on the cell surface. Such transfection may take place ex vivo, and a composition or vaccine 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 prostase rumour polypeptide, DNA (naked or within a plasmid vector) or RNA; or with antigen-expressing recombinant bacterium or viruses (e.g., vaccinia, fowlpox, adenovirus or lentivirus vectors). Vaccines and pharmaceutical compositions may be presented in unit-dose or multi-dose containers, such as sealed ampoules or vials. Such containers are preferably hermetically sealed to preserve sterility of the formulation until use. In general, formulations may be stored as suspensions, solutions or emulsions in oily or aqueous vehicles. Alternatively, a vaccine or pharmaceutical composition may be stored in a freeze-dried condition requiring only the addition of a sterile liquid carrier immediately prior to use. The present invention also provides a method for producing a vaccine formulation comprising mixing a protein of the present invention together with a pharmaceutically acceptable excipient, such as 3D-MPL. Another aspect of the invention is the use of a protein or nucleic acid as claimed herein for the manufacture of a vaccine for immunotherapeutically treating a patient suffering from prostate cancer or other prostase-associated tumours. FIGURES LEGENDS Figure 1 : Design of the fusion protein NSl- p703* -His expressed in E. coli Figure 2: Primary structure of the fusion protein NS 1 - p703 * -His expressed in E. coli (SEQ ID N°1). Figure 3: Coding sequence of NSl ι -81 -P703P*-His (SEQ ID N°2). Figure 4: Cloning strategy to produce NSl-P703P*-His in E. coli Figure 5: Plasmid map of RIT 14952 Figure 6: E. coli NSl-P703P*-His fermentation process Figure 7: E. coli NSl-P703P*-His purification process Figure 8: Immunogenicity of NS1-P703P* adjuvanted with SBAS2 Figure 9: Primary structure of the fusion protein NSl- p703 -His expressed in E. coli (SEQ ID N°3). Figure 10: Coding sequence of NSl ι -8] -P703P-His (SEQ ID N°4). The invention will be further described by reference to the following examples: EXAMPLE I: Preparation of the recombinant E. coli strain expressing the fusion protein NS1- P703P*-3-His 1. - Protein design The expression strategy followed for this candidate included the design of the most appropriate primary structure for the recombinant protein that could have the best expectation for both, good level of expression and easy purification process. Although the chance that a recombinant protein could keep its protease biological activity when formulated for vaccination is really very low, the mutation of the active side was made in order to reduce substantially or preferably eliminate its proteolytical biological activity. Accordingly, the His residue at position 43 of SEQ ID N° 8, has been mutated into an Ala residue. The design of the fusion protein NSl- p703* -His to be expressed in E. coli is described in figure 1. This fusion contains the N-terminal (81 amino acid) of non structural protein of Influenzae virus, followed by the non processed amino acid sequence of prostate antigen (amino acids 5->226 of p703pde5 sequence described in SEQ ID N°8 containing the mutation His- Ala of the 43 residue of the protease active site followed by the His tail. The Histidine tail was added to prostase to enable versatile purification of the fusion and processed protein. The length of the fusion is 313 aminoacids. The primary structure of the resulting protein has the sequence described in figure 2. The coding sequence corresponding to the above protein is illustrated in figure 3 and was subsequently placed under the control of λpL promoter in a E. coli expression plasmid. 2. - The E. Coli expression system For the production of NS 1 the DNA encoding the 81 amino-terminal residues of NS l (non-structural protein from influenzae virus) has been cloned into the expression vector pMG 81. This plasmid utilises signals from lambda phage DNA to drive the transcription and translation of inserted foreign genes. The vector contains the lambda PL promoter PL, operator OL and two utilisation sites (NutL and NutR) to relieve transcriptional polarity effects when N protein is provided (Gross et al., 1985. Mol. & Cell. Biol. 5: 1015). Vectors containing the PL promoter, are introduced into an E. coli lysogenic host to stabilise the plasmid DNA. Lysogenic host strains contain replication-defective lambda phage DNA integrated into the genome (Shatzman et al., 1983; In Experimental Manipulation of Gene Expression. Inouya (ed) pp 1-14. Academic Press NY). The lambda phage DNA directs the synthesis of the cl repressor protein which binds to the OL repressor of the vector and prevents binding of RNA polymerase to the PL promoter and thereby transcription of the inserted gene. The cl gene of the expression strain AR58 contains a temperature sensitive mutation so that PL directed transcription can be regulated by temperature shift, i.e. an increase in culture temperature inactivates the repressor and synthesis of the foreign protein is initiated. This expression system allows controlled synthesis of foreign proteins especially of those that may be toxic to the cell (Shimataka & Rosenberg, 1981. Nature 292: 128). 3. - The E. Coli strain AR58: The AR58 lysogenic E. coli strain used for the production of the NS1 -P703P*- His protein is a derivative of the standard NTH E.coli K12 strain N99 (F- su- galK2, lacZ- thr-). It contains a defective lysogenic lambda phage (galE::TN10, 1 Kil- cI857 DHl). The Kil- phenotype prevents the shut off of host macromolecular synthesis. The cI857 mutation confers a temperature sensitive lesion to the cl repressor. The DHl deletion removes the lambda phage right operon and the hosts bio, uvr3, and chlA loci. The AR58 strain was generated by transduction of N99 with a P lambda phage stock previously grown on an SA500 derivative (galE::TN10, 1 Kil- cI857 DHl). The introduction of the defective lysogen into N99 was selected with tetracycline by virtue of the presence of a TN10 transposon coding for tetracyclin resistance in the adjacent galE gene. N99 and SA500 are E.coli K12 strains derived from Dr. Martin Rosenberg's laboratory at the National Institutes of Health. 4. - Construction of the vector designed to express the recombinant protein NS1- P703P*-His The starting materials were: 1) A cDNA plasmid received from CORIXA p703pde5 (WO 00/04149), where the putative signal sequence and a piece of the pro-peptide of P703P is missing (see fig 1) and containing the coding sequence for prostase antigen; 2) Vector pRIT 14901 containing the long version of the PL promoter; and 3) Plasmid PMG81 containing the 81aa of NSi coding region from Influenzae virus. The cloning strategy outlined in figure 4 included the following steps: a) PCR amplification of the p703 sequence with Ncol and Spel restriction sites The template for the PCR reaction was the cDNA plasmid received from CORLXA, the oligonucleotide sense canl39 : 5'GCG CCC ATG GTT GGG GAG GAC TGC AGC CCG 3', and the oligonucleotide antisense can 134 : 5'GGG ACT AGT ACT GGC CTG GAC GGT TTT CTC 3'; b) Insertion of the amplified sequences into the commercial vector Litmus 28 (biolabs), leading to the intermediate plasmid pRIT 14949; c) Directed His -> Ala mutagenesis of residue 43 of the p703 sequence contained in the plasmid pRIT 14949, using the sense oligonucleotide can 140 : 5 'CTG TCA GCC GCA GCG TGT TTC CAG 3 'and the antisense oligonucleotide canl41 : 5' CTG GAA AC A CGC TGC GGC TGA CAG 3', leading to the obtention of plasmid pRIT 14950; d) Isolation of the Ncol - Spel fragment from the plasmid pRIT 14950; e) From pMG81 plasmid, purification of NS l fragment (81aa) after digestion of the restriction sites BamHI - Ncol; f) Ligation of both fragments were ligated to the expression plasmid pRIT 14901 (pr PL long); g) Selection and charactensation of the E. coh AR58 strain transformants containing the plasmid pRIT 14952 (see figure 5) expressing the NSl- p703 mutated -His fusion protein The recombinant strain thus produces the NS1-P703P* His-tailed fusion protein of 313 amino acid residues long (see Figure 2), with the amino acids sequence descnbed in ID Nol and the coding sequence is descnbed in ID No2. EXAMPLE II: Preparation of the recombinant NSl-P703P*-3-His fusion protein 1. - Growth and induction of bacterial strain B1225 - Expression of NS1-P703P*- 3-His Cells of AR58 transformed with plasmid pRIT14952 (strain B1225) were grown in a 2 L flask containing 400 ml FEC015AA medium supplemented with kanamycin sulphate (lOOmg/L). After a 16h of incubation at 30°C and at 200 rpm, a small sample was removed from this flask for microscopic examination. 50 ml of this pre-culture was transferred into a 20-L fermentor containing 8.7 L of FEC012AF medium supplemented with kanamycin sulphate (50mg/L). The pH was adjusted to and maintained at 6.8 by addition of NH4OH (25 % v/v), and the temperature was maintained at 30°C. The aeration rate was kept constant at 20 L/min and the pO2 was regulated at 20% of saturation by feedback control of the agitation speed. The head pressure was maintained at 0.5 bar. This fed-batch fermentation process is based on glycerol as a carbon source. The feed solution was added at an initial rate of 0.04ml/mιn, and increased exponentially dunng the first 30 hours to limit the growth rate in order to be able to keep a minimum pO2 level of 20%. After 30 hours, the temperature of the fermentor was rapidly increased to 39.5°C in order to induce the intracellular expression of the antigen NSl-P703P*-Hιs. The feeding rate was maintained constant at 1.28 ml/mm during the whole induction phase (18h). Samples of broth were taken during both growth and induction phases in order to monitor bacterial growth and antigen expression. Microbiological identification and purity tests were also realised on these materials. At the end of fermentation, the biomass reached an optical density of about 130, corresponding to a dry cell weight of about 50g/L. The final volume was approximately 10.5 L. The cells containing the antigen were directly separated from the culture medium by centrifugation at 5000g for 1 h at 4°C and the pellet was stored in plastic bags at -70°C. 2. - Extraction of the protein: Recombinant NSl-P703P*-His protein, expressed in E. coli as inclusion bodies, was purified from cell homogenate using different steps (see figure 6). Briefly, frozen concentrated cells from fermentation harvest were thawed to +4°C before being resuspended in disruption buffer (phosphate 20 mM - NaCI 2M - EDTA 5 mM pH 7.5) to a final optical density (OD650) of 120. Two passes through a high- pressure homogeniser (1000 bars) disrupted the cells. EXAMPLE πi: Purification of fusion Protein NSl-P703P*-His a) Introduction As said above, the recombinant protein, NSl-P703P*-His is produced in E. coli in the form of inclusion bodies. A major issue for the set-up of the purification method was the oxidation of the recombinant protein with itself or with host cell contaminants, likely through covalent binding with disulphide bonds. The process as developed aimed at reducing the massive oxidation phenomenon in order to have a highly purified product together with an acceptable global yield, while preserving the product ability to mount an effective immune response against the antigen of interest. a) Description of the process The broken cell suspension was treated on a Pallsep VMF (Vibrating Membrane Filtration) system (Pall-Filtron) equipped with 0.45 μm membrane. The "pellet fraction" was first washed by diafiltration with 20 mM phosphate buffer pH 7.5 containing 0.5% Empigen BB detergent. The washed material was then solubilised in the same buffer containing 4M guanidine hydrochoride and 20 mM glutathion. The product was recovered through 0.45 μm filter and the permeate was treated with 200 mM iodoacetamide to prevent oxidative re-coupling. The carboxyami dated fraction was subjected to IMAC (Nickel-Chelating- Sepharose FF, Pharmacia). The column was first equilibrated with 20 mM Tris buffer pH 7.5 containing 4M urea, 0.5% Tween 80, and 20 mM imidazole. After the sample loading, the column was washed with the same buffer. The protein was then eluted in the previous buffer with 400 mM Imidazole. Before continuing the anion exchange chromatography, the conductance of the IMAC-eluate was reduced to below 5 mS/cm with 20 mM Tris buffer pH 8.5 containing 4M urea and 0.5-1.0 % Tween 80. The packed bed support (Q-Sepharose FF, Pharmacia) was equilibrated with the dilution buffer. After the sample loading and a washing step with the equilibration buffer, the protein was eluted with the same buffer containing 250 mM NaCI. The Q-Sepharose FF-eluate was then diafiltered against the appropriate storage buffer (20 mM Tris buffer pH 8.0) in a tangential flow filtration unit equipped with a 10 Kd cut-off membrane (Omega, Filton). Ultrafiltration retentate containing NSl-P703P*-His was sterile filtered through 0.22 μm membrane. The global purification yield was very high: around 3-4 g of purified material / L of homogenate (DO 120). EXAMPLE IV: Vaccine preparation using NSl-P703P*-His protein 1. - Vaccine preparation using NSl-P703P*-His protein: The vaccine used in these experiments is produced from a recombinant DNA, encoding a NSl-P703P*-His, expressed in E. coli from the strain AR58, either adjuvanted or not. As an adjuvant, the formulation comprises a mixture of 3 de -O- acylated monophosphoryl lipid A (3D-MPL) and QS21 in an oil/water emulsion. The adjuvant system SBAS2 has been previously described WO 95/17210. 3D-MPL: is an immunostimulant derived from the lipopolysaccharide (LPS) of the Gram-negative bacterium Salmonella minnesota. MPL has been deacylated and is lacking a phosphate group on the lipid A moiety. This chemical treatment dramatically reduces toxicity while preserving the immunostimulant properties (Ribi, 1986). Ribi Immunochemistry produces and supplies MPL to SB-Biologicals. Experiments performed at Smith Kline Beecham Biologicals have shown that 3D-MPL combined with various vehicles strongly enhances both the humoral and a TH1 type of cellular immunity. QS21: is a natural saponin molecule extracted from the bark of the South American tree Quillaja saponaria Molina. A purification technique developed to separate the individual saponines from the crude extracts of the bark, permitted the isolation of the particular saponin, QS21, which is a triterpene glycoside demonstrating stronger adjuvant activity and lower toxicity as compared with the parent component. QS21 has been shown to activate MHC class I restricted CTLs to several subunit Ags, as well as to stimulate Ag specific lymphocytic proliferation (Kensil, 1992). Aquila (formally Cambridge Biotech Corporation) produces and supplies QS21 to SB-Biologicals. Experiments performed at SmithKline Beecham Biologicals have demonstrated a clear synergistic effect of combinations of MPL and QS21 in the induction of both humoral and TH1 type cellular immune responses. The oil/water emulsion is composed an organic phase made of of 2 oils (a tocopherol and squalene), and an aqueous phase of PBS containing Tween 80 as emulsifier. The emulsion comprised 5% squalene 5% tocopherol 0.4%) Tween 80 and had an average particle size of 180 nm and is known as SB62 (see WO 95/17210). Experiments performed at SmithKline Beecham Biologicals have proven that the adjunction of this O/W emulsion to 3D-MPL/QS21 (SBAS2) further increases the immunostimulant properties of the latter against various subunit antigens. 2. - Preparation of emulsion SB62 (2 fold concentrate): Tween 80 is dissolved in phosphate buffered saline (PBS) to give a 2% solution in the PBS. To provide 100 ml two fold concentrate emulsion 5g of DL alpha tocopherol and 5ml of squalene are vortexed to mix thoroughly. 90ml of PBS/Tween solution is added and mixed thoroughly. The resulting emulsion is then passed through a syringe and finally microfluidised by using an Ml 1 OS microfluidics machine. The resulting oil droplets have a size of approximately 180 nm. 3. - Lyophilisation of NSl-P703P*-His: In practice, all compounds are placed in solution and sterilisation is achieved by filtration on a 0.2μm membrane. Formulations were performed the day of freeze- drying. The sequence of formulation was: H 2 O + sucrose 15.75% + Tris 100 mM pH8 + Tween80 10%- s miπ + NSl-P703p The volumes of all compounds are adjusted to have in final: 250-50-10 μg NS l-P703P*-His, in Tris 10 mM, tween 8O 0.2%, 3.15% sucrose. The vial was overfilled with by 1.25 x (reconstitution with 625 μl diluant, injection of 500μl). Using the Lyovac GT6 lyophilisation apparatus purchased from Steris (Germany), the lyophilisation cycle was performed during 3 days as follows: +35 12 h -28°C 6 h 6 h -32°C 4 h -52°C 30 h 4. - Preparation of NSl-P703P*-His QS21/3D MPL oil in water (SBAS2) formulation: The adjuvant is formulated as a combination of MPL and QS21, in an oil/water emulsion. 1) Formulation composition (injection volume: lOOμl); group 1 has received P703P*-His (20 μg) formulated in a combination of MPL and QS21, in an oil/water emulsion. Group 2 has received NSl-P703P*-His (25 μg) in a combination of MPL and QS21, in an oil/water emulsion. 2) Components 3) Formulations The formulations were prepared extemporaneously on the day of injection. The formulations containing 3D-MPL and QS21 in an oil/water emulsion (SBAS2B formulations - Groups 2 and 3) were performed as follows: P703p (20μg) (group 2) and NSl-P703P*-His (25 μg) (group 3) were diluted in 10-fold concentrated PBS pH 6.8 and H 2 O before consecutive addition of SB62 (50μl), MPL (20μg), QS21 (20μg) and 1 μg/ml thiomersal as preservative at 5 min intervals. All incubations were carried out at room temperature with agitation. The non-adjuvanted formulations (Groups 4 and 5) were performed as follows: P703p (20μg) (group 4) and NSl-P703P*-His (25μg) (group 5) were diluted in 1.5 M NaCI and H 2 O before addition of 1 μg/ml thiomersal as preservative at 5 min intervals. All incubations were carried out at room temperature with agitation. The final vaccine is obtained after reconstitution of the lyophilised NS1- P703P*-His preparation with the adjuvant or with PBS alone. The adjuvant controls without antigen were prepared by replacing the protein by PBS. EXAMPLE V: Immunogenicity using NSl-P703P*-His protein 1. - Immunogenicity of NSl-P703P*-His in mice The aim of the experiment was to characterise the immune response induced in mice by vaccination with the purified recombinant mutated NSl-p703*-His molecule produced in E coli, in the presence or the absence of an adjuvant. a) - Immunization protocol: Groups of 10 immunocompetent Balb/c mice, 6 to 8 weeks old mice, were vaccinated twice, intramuscularly, at 2 weeks interval with 25 μg of mutated NS1- P703 formulated or not in SB AS02B (50μl SB62 / lOμg MPL / lOμg QS21). 14 days after the second injection, blood was taken and the sera were tested for the presence of anti-P703 antibodies. b) - Total IeG Antibody response: The anti P703 antibody response has been assessed in the sera of the mice 14 days after the latest vaccination. This has been done by ELISA using NS1- P703P* as coating antigen. E coli extracts were used to check for the possible presence of antibodies against host contaminants. c) - Results: The results show that 1) a higher immune response (IgGl) is induced by NSl - P703P* as evidenced in the sera of mice injected with the NS1-P703P* protein alone as compared to the sera of normal control mice; 2) high antibody titers are found in animals receiving the NS1-P703P* molecule formulated in the AS02B adjuvant. The isotypic profile of the NSl-p703p specific IgG response has also been measured. As shown in Figure 8, IgGl were detected when mice received the NS1- P703P* protein alone, however the isotypic profile was pushed towards a THl response (more IgG2a) by the presence of the AS02 adjuvant. EXAMPLE VI: 1. - NSl-P703P-His In an analogous fashion NS l-P703P-His was prepared. The amino acid and DNA sequences are depicted in SEQUENCE ID Nos. 3 and 4. Briefly, the strategy to express a NSl-P703P-His fusion protein in E.coli included the following steps: a) As a starting materiel, the same starting material as described in Example I (amino acids 5->226 of p703pde5 sequence described in SEQ ID N°8); b) PCR amplification to flank the p703 unmutated sequence cloning restriction sites; c) Inseπion in a PMG81 vector (promoter pL long) containing the NSl gene; d) Transformation of the recipient strain AR58 or AR120 e) Selection of recombinant strain. The resulting protein can be purified in an analogous manner to the NS1- P703P*-His mutated protein. The primary structure of the resulting protein has the sequence described in figure 9. The coding sequence corresponding to the above protein is illustrated in figure 10. REFERENCES: - Abbas F., Scardino P. "The Natural History of Clinical Prostate Carcinoma." Cancer 80:827-833 (1997) - Bostwick D., Pacelli A., Blute M. et al. "Prostate Specific Membrane Antigen "Expression in Prostatic Intraepithelial Neoplasia and Adenocarcinoma." Cancer 82:2256-2261 (1998) Frydenberg M., Strieker P., Kaye K. "Prostate Cancer Diagnosis and Management." The Lancet 349:1681-1687 (1997) - C. Hackett, D. Horowitz, M. Wysocka & S. Dillon, J. Gen. Virology, 73, 1339- 1343 (1992) - Kensil C.R., Soltysik S., Patel U., et al. in: Channock R.M. , Ginsburg H.S., Brown F., et al., (eds.), Vaccines 92, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), 36-40: (1992) - Nelson P., Lu Gan, Ferguson C, Moss P., Gelinas R., Hood L. & Wand K, "Molecular cloning and characterisation of prostase, an androgen-regulated serin protease with prostate restricted expression", Proc. Natl. Acad. Sci. USA 96, 3114-31 19 (1999) - Pound C, Partin A., Eisenberg M. et al. "Natural History of Progression after PSA Elevation following Radical Prostatectomy." Jama 281 : 1591-1597 (1999) Ribi E., et al. in: Levine L., Bonventre P.F., Morello J., et al. (eds)., American Society for Microbiology, Washington DC, Microbiology 1986, 9-13; (1986) Xue BH., Zhang Y., Sosman J. et al. "Induction of Human Cytotoxic T- Lymphocytes Specific for Prostate-Specific Antigen." Prostate 30(2):73-78 (1997)

Claims: CLAIMS: 1. A prostase protein or a derivative thereof linked to a fusion partner. 2. A protein as claimed in claim 1 wherein the prostase antigen is selected from the group consisting of SEQ ID N° 5, SEQ ID N° 6, SEQ ID N° 7, and SEQ ID N° 8. 3. A protein as claimed in claim 1 or 2 wherein the fusion partner is an immunological fusion partner or an expression enhancer fusion partner. 4. A protein as claimed in claim 1 to 3 wherein the fusion partner is NS 1 protein from influenza or a fragment thereof. 5. An antigen as claimed in any of claims 1 to 4 wherein the fusion protein further comprises an affinity tag. 6. A nucleic acid sequence encoding a protein as claimed in any of claims 1 to 5. 7. An expression vector containing a nucleic acid of claim 6. 8. A host cell transformed with a nucleic acid sequence of claim 6 or with a vector as claimed in claim 7. 9. A vaccine containing a protein as claimed in any of claims 1 to 5 or a nucleic acid as claimed in claim 6. 10. A vaccine as claimed in claim 9 additionally comprising an adjuvant, and/or immunostimulatory cytokine or chemokine. 11. A vaccine as claimed in claim 9 or 10 wherein the protein is presented in an oil in water or a water in oil emulsion vehicle. 12. A vaccine as claimed in claim 10 or 1 1 wherein the adjuvant comprises 3D-MPL, QS21, a CpG oligonucleotide or a polyethylene ether or ester. 13. A vaccine as claimed in any of claims 9 to 12 additionally comprising one or more other antigens. 14. A vaccine as claimed herein for use in medicine. 15. Use of a protein or nucleic acid as claimed herein for the manufacture of a vaccine for immunotherapeutically treating a patient suffering from prostate cancer or other prostase-associated tumours. 16. A process for the production of a protein as claimed in any of claims 1 to 5 comprising transforming a host cell with a nucleic acid sequence of claim 6, expressing said sequence and isolating the desired product. 17. A process for the production of a vaccine, comprising the steps of purifying a prostase protein or a derivative thereof, by the process of claim 16 and admixing the resulting protein as claimed herein with a suitable adjuvant, diluent or other pharmaceutically acceptable excipient. 18. A method of treating patients susceptible to or suffering from prostate-cancer comprising administering to said patients a pharmaceutically active amount of the vaccine according to claims 9 to 14.

Inventors: CABEZON SILVA TERESA, DILLON DAVIN CLIFFORD, CABEZON SILVA, TERESA, DILLON, DAVIN, CLIFFORD
Applicants: CABEZON SILVA TERESA, CORIXA CORP, DILLON DAVIN CLIFFORD, SMITHKLINE BEECHAM BIOLOG, CABEZON SILVA, TERESA, CORIXA CORPORATION, DILLON, DAVIN, CLIFFORD, SMITHKLINE BEECHAM BIOLOGICALS S.A.
Application year: 2000
Main classification: C07K14
IPC codes: A61K39, A61K48, C07K14, C12N1, C12N5, C12N9, C12N15