HK1189598A - Consensus prostate antigens nucleic acid molecule encoding the same and vaccine and uses comprising the same - Google Patents

Description

Consensus prostate antigens, nucleic acid molecules encoding said antigens, vaccines comprising said nucleic acid molecules and uses thereof

Technical Field

The present invention relates to nucleic acid molecules encoding consensus prostate proteins and fragments thereof; an improved prostate cancer vaccine, an improved method for inducing an immune response against prostate cancer cells, an improved method for prophylactically and/or therapeutically immunizing an individual against prostate cancer.

Background

Prostate cancer is an important therapeutic immune target. The development of immunotherapies is complicated by the need to develop immunogens capable of inducing strong immune responses, including preferably CTL responses.

The direct administration of nucleic acid sequences to vaccinate against animal and human diseases has been studied and much effort has been focused on efficient and effective methods of nucleic acid delivery to produce the necessary expression of the desired antigen, resulting in an immunogenic response and ultimately the success of this technology.

DNA vaccines have a number of conceptual advantages over conventional vaccination methods such as live attenuated viruses and recombinant protein-based vaccines. DNA vaccines are safe, stable, easy to produce, and well tolerated in humans, with preclinical testing showing little evidence of plasmid integration [ Martin, T., et al, plasmid DNA malaria vaccine: potential for genome integration following intramuscular injection Hum Gene Ther,1999.10(5): pages 759-68; nichols, W.W., et al, potential DNA vaccine integration into the host genome Ann N Y Acadsi, 1995.772: pp 30-9 ]. Furthermore, DNA vaccines are well suited for repeated administration due to the fact that the efficacy of the vaccine is not affected by pre-existing antibody titers against the vector [ Chattergoon, m., j.boyer and d.b.weiner, gene immunization: a new era of vaccines and immunotherapeutics FASEB J,1997.11(10): pages 753-63. However, one major obstacle to clinical adoption of DNA vaccines has been the reduction in immunogenicity of the platform when transferred to larger animals [ Liu, m.a. and j.b.ulmer, human clinical trials of plasmid DNA vaccines Adv Genet,2005.55: pages 25-40 ]. Recent technological advances in the engineering of DNA vaccine immunogens such as codon optimization, RNA optimization and addition of immunoglobulin leader sequences have improved the expression and immunogenicity of DNA vaccines [ Andre, S., et al, enhanced immune responses elicited by DNA vaccination using synthetic gp120 sequences with optimized codon usage J Virol,1998.72(2): pp 1497-503; dell, L., et al, multiple effects of codon usage optimization on the expression and immunogenicity of DNA candidate vaccines encoding the Gag protein type 1 human immunodeficiency virus J Virol,2001.75(22): pp. 10991-1001; lacdy, D.J., et al, immunogenicity of novel DNA vaccines against avian influenza based on consensus sequences Vaccine,2007.25(16): pages 2984-9; frelin, L.et al, codon optimization and mRNA amplification effectively enhanced the immunogenicity of the hepatitis C virus non-structural 3/4A Gene Gene Ther,2004.11(6): pages 522-33 ].

Recent technological advances in plasmid delivery systems have improved the expression and immunogenicity of DNA vaccines, including techniques such as electroporation [ Hirao, l.a., et al, intradermal/subcutaneous immunization by electroporation has improved the delivery and potential of plasmid vaccines in pigs and macaques. Luckay, A., et al, plasmid DNA vaccine design and Effect of in vivo electroporation on vaccine-specific immune responses obtained in macaques J Virol,2007.81(10): pages 5257-69; ahlen, G.et al, in vivo electroporation enhanced the immunogenicity, protein expression, inflammation and infiltration of CD3+ T cells of hepatitis C virus nonstructural 3/4A DNA by increased local DNA uptake J Immunol,2007.179(7): pages 4741-53 ].

Furthermore, studies have shown that the use of a consensus immunogen may be able to increase the breadth of the cellular immune response (break) compared to the native antigen alone [ Yan, J., et al, enhanced cellular immune response elicited by an enveloped DAN vaccine based on an engineered HIV-1 subtype B consensus sequence Mol Ther,2007.15(2): pages 411-21; the construction and function of the ancestral tree center of the protein of Rolland, M., et al, human immunodeficiency virus type 1J Virol,2007.81(16): pages 8507-14 ]. However, breaking the immune tolerance of cancer antigens and generating autoimmunity should be considered as major obstacles to cancer vaccines.

There remains a need for nucleic acid constructs encoding prostate cancer antigens and compositions for inducing immune responses against prostate cancer antigens, thereby breaking immune tolerance. There remains an effective prophylactic and therapeutic treatment against prostate cancer that is cost effective.

Summary of the preferred embodiments

Aspects of the invention include nucleic acid molecules comprising a coding sequence encoding one or more proteins selected from the group consisting of: a) 2, SEQ ID NO; a protein having 98% homology to SEQ ID NO. 2, provided that amino acids 69, 78, 80, 82, 102, 110, 137, 139, 165, 189, 203, 220, 232 and 248 of SEQ ID NO. 2 are conserved; or an immunogenic fragment of SEQ ID NO. 2 comprising amino acids corresponding to at least 256 amino acid residues of SEQ ID NO. 2, provided that amino acids 69, 78, 80, 82, 102, 110, 137, 139, 165, 189, 203, 220, 232 and 248 of SEQ ID NO. 2 are conserved; b) 4, SEQ ID NO; a protein having 98% homology to SEQ ID NO.4, provided that amino acids 21, 86, 127, 129, 154, 156, 182, 195, 206, 218, 220, 237, 249, 255, 265, 271 and 275 of SEQ ID NO.4 are conserved; or an immunogenic fragment of SEQ ID NO.4 comprising amino acids corresponding to at least 274 amino acid residues of SEQ ID NO.4, provided that amino acids 21, 86, 127, 129, 154, 156, 182, 195, 206, 218, 220, 237, 249, 255, 265, 271, and 275 of SEQ ID NO.4 are conserved; c) 6, SEQ ID NO; a protein having 98% homology to SEQ ID NO.6, provided that amino acids 14, 15, 32, 47, 58, 79, 111, 157, 223, 320, 350, 475, 499, 569, 613, 624, 653, 660, 663, 733 and 734 of SEQ ID NO.6 are conserved; or an immunogenic fragment of SEQ ID No.6 comprising amino acids corresponding to at least 735 amino acid residues of SEQ ID No.6, provided that amino acids 14, 15, 32, 47, 58, 79, 111, 157, 223, 320, 350, 475, 499, 569, 613, 624, 653, 660, 663, 733 and 734 of SEQ ID No.6 are conserved; d) 8 in SEQ ID NO; a protein having at least 98% homology to SEQ ID No. 8, provided that amino acids 20, 30, 31, 48, 63, 74, 95, 127, 173, 239, 336, 366, 491, 515, 564, 585, 629, 640, 669, 676, 679, 749, and 750 of SEQ ID No. 8 are conserved; or an immunogenic fragment of SEQ ID NO. 8 comprising amino acids corresponding to at least 751 amino acid residue of SEQ ID NO. 8, provided that amino acids 20, 30, 31, 48, 63, 74, 95, 127, 173, 239, 336, 366, 491, 515, 564, 585, 629, 640, 669, 676, 679, 749 and 750 of SEQ ID NO. 8 are conserved; e) 10 in SEQ ID NO; a protein having 98% homology to SEQ ID NO 10; or an immunogenic fragment of SEQ ID NO. 10 comprising amino acids corresponding to at least 333 amino acid residues of SEQ ID NO. 10; f) 12 is SEQ ID NO; a protein having 98% homology to SEQ ID NO 12; or an immunogenic fragment of SEQ ID NO. 12 comprising amino acids corresponding to at least 349 amino acid residues of SEQ ID NO. 12; g) 14, SEQ ID NO; a protein having 98% homology to SEQ ID NO. 14 or an immunogenic fragment of SEQ ID NO. 14 comprising amino acids corresponding to at least 129 amino acid residues of SEQ ID NO. 14; or h) a signal peptide linked to amino acids 19-131 of SEQ ID NO 14; a protein having a signal peptide linked to an amino acid sequence having 98% homology with amino acids 19-131 of SEQ ID NO. 14; or a signal peptide having an immunogenic fragment linked to amino acids 19-131 of SEQ ID NO. 14, a protein comprising at least 110 amino acid residues of SEQ ID NO. 14 and linked to a fragment of the signal peptide. In some embodiments, the nucleic acid molecule is selected from the group consisting of nucleic acid molecules encoding proteins a), b), c) or d).

In another aspect, the invention includes a method of treating a subject diagnosed with prostate cancer, comprising administering to the subject a nucleic acid molecule described herein.

In another aspect, a protein is provided, the protein selected from the group consisting of: a) 2, SEQ ID NO; a protein having 98% homology to SEQ ID NO. 2, provided that amino acids 69, 78, 80, 82, 102, 110, 137, 139, 165, 189, 203, 220, 232 and 248 of SEQ ID NO. 2 are conserved; or an immunogenic fragment of SEQ ID NO. 2 comprising an amino acid corresponding to at least 261 amino acid residues of SEQ ID NO. 2, provided that amino acids 69, 78, 80, 82, 102, 110, 137, 139, 165, 189, 203, 220, 232 and 248 of SEQ ID NO. 2 are conserved; b) 4, SEQ ID NO; a protein having 98% homology to SEQ ID NO.4, provided that amino acids 21, 86, 127, 129, 154, 156, 182, 195, 206, 218, 220, 237, 249, 255, 265, 271 and 275 of SEQ ID NO.4 are conserved; or an immunogenic fragment of SEQ ID NO.4 comprising amino acids corresponding to at least 274 amino acid residues of SEQ ID NO.4, provided that amino acids 21, 86, 127, 129, 154, 156, 182, 195, 206, 218, 220, 237, 249, 255, 265, 271, and 275 of SEQ ID NO.4 are conserved; c) 6, SEQ ID NO; a protein having 98% homology to SEQ ID No.6, provided that amino acids 14, 15, 32, 47, 58, 79, 111, 157, 223, 320, 350, 475, 499, 569, 613, 624, 653, 660, 663, 733 and 734 of SEQ ID No.6 are conserved; or an immunogenic fragment of SEQ ID No.6 comprising amino acids corresponding to at least 735 amino acid residues of SEQ ID No.6, provided that amino acids 14, 15, 32, 47, 58, 79, 111, 157, 223, 320, 350, 475, 499, 569, 613, 624, 653, 660, 663, 733 and 734 of SEQ ID No.6 are conserved; d) 8 in SEQ ID NO; a protein having at least 98% homology to SEQ ID No. 8, provided that amino acids 20, 30, 31, 48, 63, 74, 95, 127, 173, 239, 336, 366, 491, 515, 564, 585, 629, 640, 669, 676, 679, 749, and 750 of SEQ ID No. 8 are conserved; or an immunogenic fragment of SEQ ID NO. 8 comprising amino acids corresponding to at least 751 amino acid residue of SEQ ID NO. 8, provided that amino acids 20, 30, 31, 48, 63, 74, 95, 127, 173, 239, 336, 366, 491, 515, 564, 585, 629, 640, 669, 676, 679, 749 and 750 of SEQ ID NO. 8 are conserved; e) 10 in SEQ ID NO; a protein having 98% homology to SEQ ID NO 10; or an immunogenic fragment of SEQ ID NO. 10 comprising amino acids corresponding to at least 333 amino acid residues of SEQ ID NO. 10; f) 12 is SEQ ID NO; a protein having 98% homology to SEQ ID NO 12; or an immunogenic fragment of SEQ ID NO. 12 comprising amino acids corresponding to at least 349 amino acid residues of SEQ ID NO. 12; g) 14, SEQ ID NO; a protein having 98% homology to SEQ ID NO. 14; or an immunogenic fragment of SEQ ID NO. 14 comprising amino acids corresponding to at least 129 amino acid residues of SEQ ID NO. 14; or h) a signal peptide linked to amino acids 19-131 of SEQ ID NO 14; a protein having a signal peptide linked to an amino acid sequence having 98% homology with amino acids 19-131 of SEQ ID NO. 14; or a signal peptide having an immunogenic fragment linked to amino acids 19-131 of SEQ ID NO. 14, a protein comprising at least 110 amino acid residues of SEQ ID NO. 14 and linked to a fragment of the signal peptide. In some embodiments, the protein is selected from protein a), b), c) or d).

Some aspects of the invention include methods of treating a subject diagnosed with prostate cancer, comprising delivering to the subject a protein described herein.

Other aspects of the invention are pharmaceutical compositions comprising a nucleic acid molecule provided herein and a pharmaceutically acceptable excipient.

Brief description of the drawings

Figure 1 shows results from in vitro translation performed to confirm expression of PSA and PSMA antigens.

Figure 2A shows cellular immunogenicity data. The cellular immunogenicity of PSA antigens was determined by interferon-gamma ELISpot.

Figure 2B shows cellular immunogenicity data. The cellular immunogenicity of PSA antigens was determined by interferon-gamma ELISpot.

Figures 3A-C show CD4+ T cell responses characterized by flow cytometry by displaying graphs showing PSA-specific (left panel), PSMA-specific (middle panel), and total vaccine-specific (right panel) cytokine production: CD4+ T cells producing IFN γ (fig. 3A); (FIG. 3B) of IL-2 producing cells CD4+ T; and TNF α -producing CD4+ T cells% (fig. 3C).

Figures 4A-C show CD8+ T cell responses characterized by flow cytometry by displaying graphs showing PSA-specific (left panel), PSMA-specific (middle panel), and total vaccine-specific (right panel) cytokine production: CD8+ T cells producing IFN γ (fig. 4A); IL-2 producing CD8+ T cells% (FIG. 4B); and TNFa-producing CD8+ T cells (fig. 4C).

Figures 5A-B show ELISA data for PSA-specific antibodies one week after final immunization. (FIG. 5A) PSA IgG endpoint titers. (FIG. 5B) representative IgG titration curves.

Detailed Description

Provided herein are consensus sequences of prostate proteins and isolated nucleic acid molecules encoding them, in particular, the prostate antigen Prostate Specific Antigen (PSA), Prostate Specific Membrane Antigen (PSMA), six transmembrane epithelial antigen of prostate antigen (STEAP), and prostate specific stem cell antigen (PSCA).

The prostate cancer antigens described herein are consensus sequences derived from a pool of homologous antigens across multiple species, including the species targeted by the vaccine. The selected species from which the antigenic sequences are aligned to form a consensus sequence, e.g., homo sapiens (human), macaque (rhesus monkey), and cynomolgus monkey (cynomolgus monkey), should be selected based on proximity of species on the phylogenetic tree (phylogenetic tree). The consensus antigen is not identical but has close identity to the native prostate antigen, and its sequence shares at least 85%, preferably 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. Such described consensus cancer antigens are capable of breaking tolerance (or eliciting autoimmunity) of the targeted species and generating an effective immune response against prostate cancer antigens. Provided herein are methods of producing DNA vaccines based on consensus cancer antigens.

Aspects of the invention include nucleic acid molecules comprising a coding sequence encoding one or more proteins selected from the group consisting of: a) 2, SEQ ID NO; a protein having 98% homology to SEQ ID NO. 2, provided that amino acids 69, 78, 80, 82, 102, 110, 137, 139, 165, 189, 203, 220, 232 and 248 of SEQ ID NO. 2 are conserved; or an immunogenic fragment of SEQ ID NO. 2 comprising amino acids corresponding to at least 256 amino acid residues of SEQ ID NO. 2, provided that amino acids 69, 78, 80, 82, 102, 110, 137, 139, 165, 189, 203, 220, 232 and 248 of SEQ ID NO. 2 are conserved; b) 4, SEQ ID NO; a protein having 98% homology to SEQ ID NO.4, provided that amino acids 21, 86, 127, 129, 154, 156, 182, 195, 206, 218, 220, 237, 249, 255, 265, 271 and 275 of SEQ ID NO.4 are conserved; or an immunogenic fragment of SEQ ID NO.4 comprising amino acids corresponding to at least 274 amino acid residues of SEQ ID NO.4, provided that amino acids 21, 86, 127, 129, 154, 156, 182, 195, 206, 218, 220, 237, 249, 255, 265, 271, and 275 of SEQ ID NO.4 are conserved; c) 6, SEQ ID NO; a protein having 98% homology to SEQ ID NO.6, provided that amino acids 14, 15, 32, 47, 58, 79, 111, 157, 223, 320, 350, 475, 499, 569, 613, 624, 653, 660, 663, 733 and 734 of SEQ ID NO.6 are conserved; or an immunogenic fragment of SEQ ID No.6 comprising amino acids corresponding to at least 735 amino acid residues of SEQ ID No.6, provided that amino acids 14, 15, 32, 47, 58, 79, 111, 157, 223, 320, 350, 475, 499, 569, 613, 624, 653, 660, 663, 733 and 734 of SEQ ID No.6 are conserved; d) 8 in SEQ ID NO; a protein having at least 98% homology to SEQ ID No. 8, provided that amino acids 20, 30, 31, 48, 63, 74, 95, 127, 173, 239, 336, 366, 491, 515, 564, 585, 629, 640, 669, 676, 679, 749, and 750 of SEQ ID No. 8 are conserved; or an immunogenic fragment of SEQ ID NO. 8 comprising amino acids corresponding to at least 751 amino acid residue of SEQ ID NO. 8, provided that amino acids 20, 30, 31, 48, 63, 74, 95, 127, 173, 239, 336, 366, 491, 515, 564, 585, 629, 640, 669, 676, 679, 749 and 750 of SEQ ID NO. 8 are conserved; e) 10 in SEQ ID NO; a protein having 98% homology to SEQ ID NO 10; or an immunogenic fragment of SEQ ID NO. 10 comprising amino acids corresponding to at least 333 amino acid residues of SEQ ID NO. 10; f) 12 is SEQ ID NO; a protein having 98% homology to SEQ ID NO 12; or an immunogenic fragment of SEQ ID NO. 12 comprising amino acids corresponding to at least 349 amino acid residues of SEQ ID NO. 12; g) 14, SEQ ID NO; a protein having 98% homology to SEQ ID NO. 14; or an immunogenic fragment of SEQ ID NO. 14 comprising amino acids corresponding to at least 129 amino acid residues of SEQ ID NO. 14; or an immunogenic fragment of SEQ ID NO. 14 comprising at least 129 amino acid residues of SEQ ID NO. 14; or h) a signal peptide linked to amino acids 19-131 of SEQ ID NO. 14; a protein having a signal peptide linked to an amino acid sequence having 98% homology with amino acids 19-131 of SEQ ID NO. 14; or a signal peptide having an immunogenic fragment linked to amino acids 19-131 of SEQ ID NO. 14, a protein comprising at least 110 amino acid residues of SEQ ID NO. 14 and linked to a fragment of the signal peptide. Two consensus protein sequences for PSA are disclosed: PSA consensus antigen sequence 1(SEQ ID NO:2) and PSA consensus antigen sequence 2(SEQ ID NO: 4). Two consensus protein sequences for PSMA are disclosed: PSMA consensus antigen sequence 1(SEQ ID NO:6) and PSMA consensus antigen sequence 2(SEQ ID NO: 8). Two consensus protein sequences for STEAP (also referred to herein as STEAP1) are disclosed: STEAP consensus antigen sequence 1(SEQ ID NO:10) and STEAP consensus antigen sequence 2(SEQ ID NO: 12). A consensus protein sequence for PSCA is disclosed: PSCA consensus antigen sequence (SEQ ID NO: 14). SEQ ID NO 14 includes the IgE signal peptide. In some embodiments, the PSCA consensus antigen may comprise amino acids 19-131 of SEQ ID NO. 14 linked to a signal sequence other than the IgE signal of SEQ ID NO. 14. In some embodiments, the nucleic acid molecule is selected from the group consisting of nucleic acid molecules encoding the above-described proteins a), b), c) or d). In other embodiments, the nucleic acid molecule is a nucleic acid molecule encoding one or more proteins selected from the group consisting of: at least one selected from the group consisting of nucleic acid molecules encoding proteins a) or b), and at least one selected from the group consisting of nucleic acid molecules encoding proteins c) or d).

The nucleic acid molecule may also be a molecule encoding one or more proteins selected from the group consisting of: 2, SEQ ID NO; 4, SEQ ID NO; 6, SEQ ID NO; 8 in SEQ ID NO; SEQ ID NO. 10; 12 is SEQ ID NO; or SEQ ID NO 14; preferably, SEQ ID NO 2; 4, SEQ ID NO; 6 or 8 SEQ ID NO. In some embodiments, the nucleic acid molecule may be a nucleic acid molecule encoding one or more proteins selected from the group consisting of: at least one selected from SEQ ID NO. 2 or SEQ ID NO.4, and at least one selected from SEQ ID NO.6 or SEQ ID NO. 8.

In another aspect, a protein is provided, the protein selected from the group consisting of: a) 2, SEQ ID NO; a protein having 98% homology to SEQ ID NO. 2, provided that amino acids 69, 78, 80, 82, 102, 110, 137, 139, 165, 189, 203, 220, 232 and 248 of SEQ ID NO. 2 are conserved; or an immunogenic fragment of SEQ ID NO. 2 comprising amino acids corresponding to at least 256 amino acid residues of SEQ ID NO. 2, provided that amino acids 69, 78, 80, 82, 102, 110, 137, 139, 165, 189, 203, 220, 232 and 248 of SEQ ID NO. 2 are conserved; b) 4, SEQ ID NO; a protein having 98% homology to SEQ ID NO.4, provided that amino acids 21, 86, 127, 129, 154, 156, 182, 195, 206, 218, 220, 237, 249, 255, 265, 271 and 275 of SEQ ID NO.4 are conserved; or an immunogenic fragment of SEQ ID NO.4 comprising amino acids corresponding to at least 274 amino acid residues of SEQ ID NO.4, provided that amino acids 21, 86, 127, 129, 154, 156, 182, 195, 206, 218, 220, 237, 249, 255, 265, 271, and 275 of SEQ ID NO.4 are conserved; c) 6, SEQ ID NO; a protein having 98% homology to SEQ ID No.6, provided that amino acids 14, 15, 32, 47, 58, 79, 111, 157, 223, 320, 350, 475, 499, 569, 613, 624, 653, 660, 663, 733 and 734 of SEQ ID No.6 are conserved; or an immunogenic fragment of SEQ ID No.6 comprising amino acids corresponding to at least 735 amino acid residues of SEQ ID No.6, provided that amino acids 14, 15, 32, 47, 58, 79, 111, 157, 223, 320, 350, 475, 499, 569, 613, 624, 653, 660, 663, 733 and 734 of SEQ ID No.6 are conserved; d) 8 in SEQ ID NO; a protein having at least 98% homology to SEQ ID No. 8, provided that amino acids 20, 30, 31, 48, 63, 74, 95, 127, 173, 239, 336, 366, 491, 515, 564, 585, 629, 640, 669, 676, 679, 749, and 750 of SEQ ID No. 8 are conserved; or an immunogenic fragment of SEQ ID NO. 8 comprising amino acids corresponding to at least 751 amino acid residue of SEQ ID NO. 8, provided that amino acids 20, 30, 31, 48, 63, 74, 95, 127, 173, 239, 336, 366, 491, 515, 564, 585, 629, 640, 669, 676, 679, 749 and 750 of SEQ ID NO. 8 are conserved; e) 10 in SEQ ID NO; a protein having 98% homology to SEQ ID NO 10; or an immunogenic fragment of SEQ ID NO. 10 comprising amino acids corresponding to at least 333 amino acid residues of SEQ ID NO. 10; f) 12 is SEQ ID NO; a protein having 98% homology to SEQ ID NO 12; or an immunogenic fragment of SEQ ID NO. 12 comprising amino acids corresponding to at least 349 amino acid residues of SEQ ID NO. 12; g) 14, SEQ ID NO; a protein having 98% homology to SEQ ID NO. 14; or an immunogenic fragment of SEQ ID NO. 14 comprising amino acids corresponding to at least 129 amino acid residues of SEQ ID NO. 14; or h) a signal peptide linked to amino acids 19-131 of SEQ ID NO 14; a protein having a signal peptide linked to an amino acid sequence having 98% homology with amino acids 19-131 of SEQ ID NO. 14; or a signal peptide having an immunogenic fragment linked to amino acids 19-131 of SEQ ID NO. 14, a protein comprising at least 110 amino acid residues of SEQ ID NO. 14 and linked to a fragment of the signal peptide. In some embodiments, the protein is selected from the group consisting of: proteins a), b), c) or d). In other embodiments, the protein encodes one or more proteins selected from the group consisting of: at least one selected from the group consisting of proteins a) or b), and at least one selected from the group consisting of proteins c) or d).

The protein may also be selected from SEQ ID NO 2; 4, SEQ ID NO; 6, SEQ ID NO; 8 in SEQ ID NO; 10 in SEQ ID NO; 12 or 14 SEQ ID NO; preferably, SEQ ID NO 2; 4, SEQ ID NO; the protein of SEQ ID NO 6 or SEQ ID NO 8. In some embodiments, the protein may be a protein selected from the group consisting of: at least one selected from SEQ ID NO. 2 or SEQ ID NO.4, and at least one selected from SEQ ID NO.6 or SEQ ID NO. 8.

Nucleic acid coding sequences have been generated to enhance and optimize expression. The codons used in such nucleic acid molecules are selected to produce RNA with reduced secondary structure formation due to intramolecular hybridization. Nucleic acid sequences encoding PSA consensus antigen sequence 1(SEQ ID NO:1) and PSA consensus antigen sequence 2(SEQ ID NO:3) are disclosed. Similarly, the nucleic acid coding sequences for PSMA consensus antigen sequence 1(SEQ ID NO:5 or nucleotides 1-2250 of SEQ ID NO: 5) and PSMA consensus antigen sequence 2(SEQ ID NO:7 or nucleotides 1-2301 of SEQ ID NO: 7) as well as STEAP consensus antigen sequence 1(SEQ ID NO:9), STEAP consensus antigen sequence 2(SEQ ID NO:11) and PSCA consensus antigen sequence (SEQ ID NO:13) are provided. Also provided are nucleic acid sequences having 98% homology to SEQ ID NO.1 and encoding PSA consensus antigen sequence 1(SEQ ID NO. 2) or a protein having up to 98% homology to SEQ ID NO. 2 (preferably comprising amino acids 69, 78, 80, 82, 102, 110, 137, 139, 165, 189, 203, 220, 232 and 248 of SEQ ID NO. 2), and nucleic acid sequences having 98% homology to SEQ ID NO. 3 and encoding PSA consensus antigen sequence 2(SEQ ID NO. 4) or a protein having up to 98% homology to SEQ ID NO.4 (preferably comprising amino acids 21, 86, 127, 129, 154, 156, 182, 195, 206, 218, 220, 237, 249, 255, 265, 271 and 275 of SEQ ID NO. 4). Likewise, a nucleic acid sequence having at least 98% homology to nucleotide 2250 of SEQ ID NO.5 and encoding PSMA consensus antigen sequence 1(SEQ ID NO:6) or a protein having up to 98% homology to SEQ ID NO:6 (preferably comprising amino acids 14, 15, 32, 47, 58, 79, 111, 157, 223, 320, 350, 475, 499, 569, 613, 624, 653, 660, 663, 733 and 734) or having 98% homology to nucleotide 2301 of SEQ ID NO.7 and encoding PSMA consensus antigen sequence 1(SEQ ID NO:8) or a protein having up to 98% homology to SEQ ID NO:8 (preferably comprising amino acids 20, 30, 31, 48, 63, 74, 95, 127, 173, 239, 336, 366, 491, 515, 564, 585, 629, 640, 669, 239, 336, 366, 491, 515, 564, 585, 629, 669, and 734), 676. 679, 749 and 750), and nucleotides having 98% homology to SEQ ID NO9 and encoding STEAP consensus antigen sequence 1(SEQ ID NO:10) or a protein having up to 98% homology to SEQ ID NO:10, nucleotides having 98% homology to SEQ ID NO:11 and encoding STEAP consensus antigen sequence 2(SEQ ID NO:12) or a protein having up to 98% homology to SEQ ID NO:12, and nucleotides having 98% homology to SEQ ID NO:13 and encoding PSCA consensus antigen sequence (SEQ ID NO:14) or a protein having up to 98% homology to SEQ ID NO: 14. In some embodiments, the nucleic acid molecule encodes a protein comprising an IgE signal peptide (e.g., SEQ ID NO:3 encoding SEQ ID NO:4, nucleotides 1-2301 of SEQ ID NO:7 encoding SEQ ID NO:8, SEQ ID NO:11 encoding SEQ ID NO:12, and SEQ ID NO:13 encoding SEQ ID NO: 14).

Compositions comprising nucleic acid molecules comprising coding sequences for the isolated nucleic acid molecules provided herein can be used to induce an immune response against a prostate protein (when administered into an animal). Compositions comprising one or more of such nucleic acid sequences can be used as vaccines or vaccine components to prophylactically or therapeutically elicit immunity to prostate cancer. Likewise, compositions comprising the consensus protein can be used to induce an immune response against prostate proteins when administered into an animal. Combinations of compositions comprising nucleic acid molecules comprising coding sequences of the isolated nucleic acid molecules provided herein can be used to induce an immune response against prostate proteins, and can be used together as a vaccine or vaccine component to prophylactically or therapeutically elicit immunity against prostate cancer. Likewise, compositions comprising the consensus protein can be used to induce an immune response against prostate proteins when administered into an animal. Compositions comprising one or more of such consensus proteins are useful as vaccines or vaccine components to prophylactically or therapeutically elicit immunity against prostate cancer.

Vaccines comprising the nucleic acid sequences provided herein are provided. In some embodiments, vaccines are provided that comprise a nucleic acid sequence encoding one or more consensus prostate antigens selected from the group consisting of: consensus PSA antigen 1, consensus PSA antigen 2, consensus PSMA antigen 1, consensus PSMA antigen 2, consensus STEAP antigen 1, consensus STEAP antigen 2, and consensus PSCA. Methods of inducing an immune response using nucleic acid sequences encoding one or more prostate antigens selected from the group consisting of: consensus PSA antigen 1, consensus PSA antigen 2, consensus PSMA antigen 1, consensus PSMA antigen 2, consensus STEAP antigen 1, consensus STEAP antigen 2, and consensus PSCA.

Vaccines comprising one or more of consensus PSA antigen 1, consensus PSA antigen 2, consensus PSMA antigen 1, consensus PSMA antigen 2, consensus STEAP antigen 1, consensus STEAP antigen 2, and consensus PSCA are also provided. Also provided are methods of inducing an immune response using one or more of consensus PSA antigen 1, consensus PSA antigen 2, consensus PSMA antigen 1, consensus PSMA antigen 2, consensus STEAP antigen 1, consensus STEAP antigen 2, and consensus PSCA.

Methods of protecting an individual from prostate cancer or treating an individual who has been identified as having prostate cancer are provided. The method comprises the following steps: administering to the individual an effective amount of one or more nucleic acid molecules comprising one or more of the nucleic acid molecules provided herein. In some methods, delivery of the nucleic acid molecule is facilitated by electroporation of the targeted tissue or tissue receiving the nucleic acid molecule. The nucleic acid sequence is expressed in cells of the individual, inducing an immune response against the prostate protein encoded by the nucleic acid sequence.

1. Definition of

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used in the specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the content clearly dictates otherwise.

The recitation of numerical ranges herein explicitly includes each intervening number with the same degree of accuracy. For example, for the range of 6-9, the numbers 7 and 8 are included in addition to 6 and 9, and for the range of 6.0-7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 and 7.0 are explicitly included.

a. Adjuvant

As used herein, "adjuvant" means any molecule added to the DNA plasmid vaccines described herein to enhance the immunogenicity of the antigen encoded by the DNA plasmid and encoding nucleic acid sequences described below.

b. Antibodies

As used herein, "antibody" means an antibody or fragment, fragment or derivative thereof of the class IgG, IgM, IgA, IgD, or IgE, including Fab, F (ab')2, Fd, and single chain antibodies, diabodies (diabodies), bispecific antibodies, bifunctional antibodies, and derivatives thereof. The antibody may be an antibody isolated from a serum sample of a mammal, a polyclonal antibody, an affinity purified antibody, or a mixture thereof, which antibody exhibits sufficient binding specificity to the desired epitope or a sequence derived therefrom.

c. Coding sequence

As used herein, "coding sequence" or "coding nucleic acid" means a nucleic acid (RNA or DNA molecule) comprising a nucleotide sequence that encodes a protein. The coding sequence may also include initiation and termination signals operably linked to regulatory elements capable of directing expression in the cells of the individual or mammal to which the nucleic acid is administered, including promoters and polyadenylation signals.

d. Complementary to each other

As used herein, "complementary" or "complementary" means a nucleic acid that can undergo Watson-Crick (e.g., A-T/U and C-G) or Hoogsteen base pairing (Watson-base pairing) between nucleotides or nucleotide analogs of the nucleic acid molecule.

e. Consensus or consensus sequences

As used herein, "consensus" or "consensus sequence" means a polypeptide sequence based on an analysis of an alignment of multiple subtypes of a particular prostate antigen. Nucleic acid sequences encoding the consensus polypeptide sequence may be prepared. Vaccines comprising proteins containing consensus sequences and/or nucleic acid molecules encoding such proteins can be used to induce broad immunity against specific prostate antigens.

f. Electroporation

As used interchangeably herein, "electroporation," "electroporation," or "Electrokinetic (EP)" means inducing microscopic pathways (pores) in a biological membrane using transmembrane electric field pulses; their presence allows biomolecules such as plasmids, oligonucleotides, siRNA, drugs, ions and water to pass from one side of the cell membrane to the other.

g. Fragments

As used herein with respect to nucleic acid sequences, "fragment" means a nucleic acid sequence or portion thereof that encodes a polypeptide that is capable of eliciting an immune response in a mammal that is cross-reactive with full-length prostate antigen. The fragment may be a DNA fragment selected from at least one of various nucleotide sequences encoding consensus amino acid sequences and constructs comprising such sequences. The DNA fragment may comprise the coding sequence of an immunoglobulin leader sequence, such as an IgE or IgG sequence. The DNA fragment may encode a protein fragment as shown below.

By "fragment" with respect to a polypeptide sequence is meant a polypeptide capable of eliciting an immune response in a mammal that cross-reacts with prostate antigens (including, for example, PSA, PSMA, STEAP, and PSCA).

The human PSA sequence is about 261 amino acids. A fragment of PSA consensus antigen 1 may comprise at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, preferably 98% or 99%, of SEQ ID No. 2, as long as the fragment comprises one or more of amino acids 69, 78, 80, 82, 102, 110, 137, 139, 165, 189, 203, 220, 232, and 248. Fragments of PSA consensus antigen 1 may comprise 255, 256, 257, 258, 259 or 260 amino acids of SEQ ID NO. 2, but preferably 256 amino acids or more. A fragment of PSA consensus antigen 2 may comprise at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, preferably 98% or 99%, of SEQ ID No.4, as long as the fragment comprises one or more of amino acids 21, 86, 127, 129, 154, 156, 182, 195, 206, 218, 220, 237, 249, 255, 265, 271, and 275. All such fragments of PSA consensus antigen 2 may also optionally exclude amino acids 1-17. In some embodiments, fragments of PSA consensus antigen 2 may optionally include one or more of amino acids 1-17, and one or more of amino acids from amino acid 18 to amino acid 278, and fragments of PSA consensus antigen 2 may also include 255, 256, 257, 258, 259, or 260 amino acids, but preferably 274 amino acids or more, of SEQ ID No. 4.

The human PSMA sequence is about 749-750 amino acids. A fragment of PSMA consensus antigen 1 may comprise at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, preferably 98% or 99%, of SEQ ID No.6, as long as the fragment comprises one or more of amino acids 14, 15, 32, 47, 58, 79, 111, 157, 223, 320, 350, 475, 499, 569, 613, 624, 653, 660, 663, 733, and 734. Fragments of PSMA consensus antigen 1 may comprise 745, 746, 747, 748, or 749 amino acids, but preferably 735 amino acids or more, of SEQ ID NO 6. A fragment of PSMA consensus antigen 2 may comprise at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, preferably 98% or 99%, of SEQ ID No. 8, provided that the fragment comprises one or more of amino acids 20, 30, 31, 48, 63, 74, 95, 127, 173, 239, 336, 366, 491, 515, 564, 585, 629, 640, 669, 676, 679, 749, and 750. All such fragments of PSA consensus antigen 2 may also optionally exclude amino acids 1-16. In some embodiments, fragments of PSA consensus antigen 2 may optionally comprise one or more of amino acids 1-16 and one or more of amino acids from amino acid 17 to amino acid 766, and fragments of PSMA consensus antigen 2 may further comprise 760, 761, 762, 763, 764, or 765 amino acids of SEQ ID NO:8, but preferably 751 amino acids or more.

The human STEAP sequence is about 339 amino acids. The consensus STEAP sequence may comprise an amino acid sequence of an immunoglobulin leader such as IgE or IgG. Consensus STEAP antigen 2 contained an 18 amino acid leader sequence at position 1 instead of methionine. A fragment of PSMA consensus antigen 2 may comprise the leader sequence and at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, preferably 98% or 99% of amino acids 18-356 of SEQ ID NO 12. A fragment of PSMA consensus antigen 1 may comprise amino acids 1-350, 1-351, 1-352, 1-353, 1-354, or 1-355 of SEQ ID NO 12.

The human PSCA sequence is about 114 amino acids. The consensus STEAP sequence may comprise an amino acid sequence of an immunoglobulin leader such as IgE or IgG. The consensus PSCA antigen contained an 18 amino acid leader sequence at position 1 in place of methionine. A fragment of the PSCA consensus antigen may comprise the leader sequence and at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, preferably 98% or 99%, of amino acids 18-131 of SEQ ID NO. 14. A fragment of PSMA consensus antigen 1 may comprise amino acids 1-125, 1-126, 1-127, 1-128, 1-129, or 1-130 of SEQ ID NO. 14.

h. Gene construct

As used herein, the term "genetic construct" refers to a DNA or RNA molecule comprising a nucleotide sequence encoding a protein. The coding sequence includes initiation and termination signals operably linked to regulatory elements capable of directing expression in the cells of the individual to which the nucleic acid molecule is administered, including promoters and polyadenylation signals. As used herein, the term "expressible form" refers to a genetic construct that comprises the necessary regulatory elements operatively linked to a coding sequence encoding a protein (so that the coding sequence can be expressed when present in the cells of an individual).

i. Homology of

Homology and phylogenetic maps (phylogenetic maps) for multiple sequence alignments were generated using ClustalW (the general purpose multiple sequence alignment program for DNA or proteins).

j. Are identical

As used herein, "identical" or "identity," in the context of two or more nucleic acid or polypeptide sequences, means that the sequences have a specified percentage of residues that are the same over a specified region. The percentages can be calculated as follows: optimally aligning the two sequences, comparing the two sequences over a specified region, determining the number of positions in the two sequences at which the identical residue is present to produce the number of matched positions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying the result by 100 to produce the percentage of sequence identity. In the case where two sequences are of different lengths or are aligned to produce one or more staggered ends and the specified region of comparison includes only a single sequence, the residues of the single sequence are included in the denominator in the calculation rather than the numerator. When comparing DNA to RNA, thymine (T) and uracil (U) can be considered equivalent. Identity can be calculated manually or using computer sequence algorithms such as BLAST or BLAST 2.0.

k. Immune response

As used herein, "immune response" means the activation of the host immune system, e.g., a mammal's immune system, in response to the introduction of an antigen, e.g., a prostate consensus antigen. The immune response may be in the form of a cellular or humoral response or both.

Nucleic acids

As used herein, "nucleic acid" or "oligonucleotide" or "polynucleotide" means at least two nucleotides covalently linked together. The description of the single strand also defines the sequence of the complementary strand. Thus, nucleic acids also include the complementary strand of the single strand described. Many variants of a nucleic acid can be used for the same purpose as a given nucleic acid. Thus, nucleic acids also include substantially identical nucleic acids and their complements. The single strand provides a probe that can hybridize to a target sequence under stringent hybridization conditions. Thus, nucleic acids also include probes that hybridize under stringent hybridization conditions.

The nucleic acid may be single-stranded or double-stranded, or may comprise portions of both double-stranded and single-stranded sequences. The nucleic acid can be DNA (genomic DNA and cDNA), RNA, or hybrids, wherein the nucleic acid can comprise a combination of deoxyribonucleotides and ribonucleotides, as well as a combination of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine. Nucleic acids can be obtained by chemical synthesis or by recombinant methods.

m. operatively connected

As used herein, "operably linked" means that expression of a gene is under the control of a promoter spatially linked thereto. The promoter may be located 5 '(upstream) or 3' (downstream) of the gene under its control. The distance between a promoter and a gene may be about the same as the distance between the promoter and the gene it controls in the gene from which the promoter is derived. As is known in the art, this change in distance can be modulated without loss of promoter function.

n. promoter

As used herein, "promoter" means a molecule of synthetic or natural origin that is capable of conferring, activating or enhancing the expression of a nucleic acid in a cell. The promoter may include one or more specific transcriptional regulatory sequences to further enhance expression of the gene and/or alter its spatial and/or temporal expression. Promoters may also include distal enhancer or repressor elements, which may be located as much as several thousand base pairs away from the transcription initiation site. Promoters may be derived from sources including viruses, bacteria, fungi, plants, insects, and animals. Promoters can regulate the expression of a gene module (gene component) constitutively or differentially, for the cell, tissue or organ in which expression occurs or for the developmental stage at which expression occurs, or in response to external stimuli such as physiological stress, pathogens, metal ions or inducers. Representative examples of promoters include the phage T7 promoter, the phage T3 promoter, the SP6 promoter, the lac operator-promoter, the tac promoter, the SV40 late promoter, the SV40 early promoter, the RSV-LTR promoter, the CMV IE promoter, the SV40 early promoter or the SV40 early promoter, and the CMV IE promoter.

Stringent hybridization conditions

As used herein, "stringent hybridization conditions" means conditions under which a first nucleic acid sequence (e.g., a probe) will hybridize to a second nucleic acid sequence (e.g., a target), e.g., in a complex mixture of nucleic acids. Stringent conditions are sequence dependent and will be different in different circumstances. Stringent conditions can be selected to determine the thermal melting point (T) of a particular sequence at ionic strength pH_m) About 5-10 ℃. T is_mCan be (at defined ionic strength, pH and nucleic acid concentration) 50% of the temperature at which a probe complementary to the target hybridizes to the target sequence at equilibrium (at T when the target sequence is present in excess, at_mNext, 50% of the probes are occupied in equilibrium). Stringent conditions can be conditions in which the salt concentration is less than about 1.0M sodium ion, e.g., a sodium ion concentration (or other salt) of about 0.01-1.0M, at ph7.0 to 8.3, and the temperature is at least about 30 ℃ (e.g., about 10-50 nucleotides for short probes) and at least about 60 ℃ (e.g., greater than about 50 nucleotides for long probes). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal can be at least 2 to 10 times background hybridization. Exemplary stringent hybridization conditions include the following:50% formamide, 5 XSSC and 1% SDS at 42 ℃ or 5 XSSC, 1% SDS at 65 ℃ and washed in 0.2 XSSC and 0.1% SDS at 65 ℃.

Substantially complementary to

As used herein, "substantially complementary" means that the first sequence is at least 60%, 65%, 70%, 75%, 80%, 85%, 70%, 80%, 85%, 80%, 270, 360, 450, 540, 630, 720, 810, 900, 990, 1080, 1170, 1260, 1350, 1440, 1530, 1620, 1710, 1800, 1890, 1980, 2070 or a region of more nucleotides or amino acids of the complementary sequence of the second sequence over 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 180, 270, 360, 450, 540, 630, 720, 810, 900, 990, 1080, 1170, 1260, 1350, 1440, 1530, 1620, 1710, 1800, 1890, 1980, 2070 or the two sequences hybridize under stringent hybridization conditions.

q. substantially the same

As used herein, "substantially identical" means that a first sequence is at least 60%, 65%, 70%, 75%, 80%, 85, 90, 95, 100, 180, 270, 360, 450, 540, 630, 720, 810, 900, 990, 1080, 1170, 1260, 1350, 1440, 1530, 1620, 1710, 1800, 1890, 1980, 2070 or more nucleotides or amino acids of a region of 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 180, 270, 360, 450, 540, 630, 720, 810, 900, 990, 1080, 1170, 1260, 1350, 1440, 1530, 1620, 1710, 1800, 1890, 1980, 2070 or, in the case that the first sequence is substantially complementary to a complementary sequence of a second sequence in the case of a nucleic acid.

Subtype or serotype

As used herein, "subtype" or "serotype" are used interchangeably and, when referring to a prostate cancer antigen, means a genetic variant of the prostate cancer antigen (genetic variant) such that one subtype (or variant) is recognized by the immune system separate from a different subtype.

s. variants

As used herein, "variant" with respect to a nucleic acid means (i) a portion or fragment of a reference nucleic acid sequence; (ii) a complement of a reference nucleotide sequence or portion thereof; (iii) a nucleic acid that is substantially identical to a reference nucleic acid or a complement thereof; or (iv) a nucleic acid that hybridizes under stringent conditions to a reference nucleic acid, its complement, or a sequence substantially identical thereto.

"variant" refers to a peptide or polypeptide that differs in amino acid sequence by insertion, deletion, or conservative substitution of amino acids, but retains at least one biological activity. A variant may also mean a protein whose amino acid sequence is substantially identical to that of the reference protein, which retains at least one biological activity. Conservative substitutions of amino acids, i.e., substitutions of amino acids with different amino acids having similar properties (e.g., hydrophilicity, degree and distribution of charged regions) are generally recognized in the art as including minor variations. These small changes can be identified in part by considering the hydropathic index of amino acids, as understood in the art. Kyte et al, J.mol.biol.157:105-132 (1982). The hydropathic index of an amino acid is based on consideration of its hydrophobicity and charge. It is known in the art that amino acids with similar hydropathic indices can be substituted and still retain protein function. In one aspect, amino acids having a hydropathic index of ± 2 are substituted. The hydrophilicity of amino acids can also be used to reveal substitutions that can produce proteins that retain biological function. Consideration of the hydrophilicity of amino acids in the context of a peptide allows the calculation of the maximum local average hydrophilicity of the peptide, useful measures relating to antigenicity and immunogenicity have been reported. U.S. Pat. No.4,554,101, incorporated herein by reference in its entirety. Substitution of amino acids with similar hydrophilicity values can result in peptides that retain biological activity, such as immunogenicity, as is understood in the art. Amino acids having hydrophilicity values within ± 2 can be substituted for each other. The hydrophobicity index and hydrophilicity value of an amino acid are affected by the particular side chain of that amino acid. Consistent with this observation, it is understood that amino acid substitutions that are compatible with biological function depend on the relative similarity of the amino acids, particularly the relative similarity of the side chains of the amino acids, as shown by hydrophobicity, hydrophilicity, charge, size, and other properties.

t. vector

As used herein, "vector" means a nucleic acid sequence comprising an origin of replication. The vector may be a vector, a bacteriophage, a bacterial artificial chromosome, or a yeast artificial chromosome. The vector may be a DNA or RNA vector. The vector may be an autonomously replicating extrachromosomal vector, preferably a DNA plasmid.

2. Consensus prostate antigen

Provided herein are consensus antigens capable of eliciting an immune response in a mammal against a prostate antigen. The consensus antigens may comprise epitopes that make them particularly effective immunogens against which prostate cancer cells can be induced. The consensus prostate antigen may comprise the full-length translation product, a variant thereof, a fragment thereof, or a combination thereof.

Seven different consensus prostate antigens have been designed. Two of the consensus prostate antigens are consensus PSA antigen 1(SEQ ID NO:2) and consensus PSA antigen 2(SEQ ID NO: 4). Two of the consensus prostate antigens were consensus PSMA antigen 1(SEQ ID NO:6) and consensus PSMA antigen 2(SEQ ID NO: 8). Two of the consensus prostate antigens are consensus STEAP antigen 1(SEQ ID NO:10) and consensus STEAP antigen 2(SEQ ID NO: 12). One of the consensus prostate antigens is the consensus PSCA antigen (SEQ ID NO: 14). The protein may comprise a sequence homologous to a prostate antigen, a fragment of a prostate antigen, and a protein having a sequence homologous to a fragment of a prostate antigen.

Consensus PSA antigen 1(SEQ ID NO:2) has about 91% homology with the human PSA sequence, about 95% homology with cynomolgus PSA and about 96% homology with cynomolgus PSA. Consensus PSA antigen 1 differs from the human PSA sequence by amino acids 69, 78, 80, 82, 102, 110, 137, 139, 165, 189, 203, 220, 232, and 248 of SEQ ID NO 2.

Consensus PSA antigen 2(SEQ ID NO:4) has about 90-91% homology to the human PSA sequence, about 95% homology to cynomolgus PSA and about 95% homology to cynomolgus PSA. Consensus PSA antigen 2 has a leader sequence at its N-terminus. Consensus PSA antigen 2 also differs from the human PSA sequence by amino acids 21, 86, 127, 129, 154, 156, 182, 195, 206, 218, 220, 237, 249, 255, 265, 271, and 275 of SEQ ID NO. 4.

Consensus PSMA antigen 1(SEQ ID NO:6) has about 96% homology with the human PSMA sequence and about 94% homology with cynomolgus PSMA. Consensus PSMA antigen 1 differs from the human PSMA sequence by amino acids 14, 15, 32, 47, 58, 79, 111, 157, 223, 320, 350, 475, 499, 569, 613, 624, 653, 660, 663, 733, and 734 of SEQ ID NO 6.

Consensus PSMA antigen 2(SEQ ID NO:8) has about 96% homology with the human PSA sequence and about 94% homology with cynomolgus PSA. Consensus PSMA antigen 2 contained a leader sequence at its N-terminus. Consensus PSMA antigen 2 also differs from the human PSA sequence by amino acids 20, 30, 31, 48, 63, 74, 95, 127, 173, 239, 336, 366, 491, 515, 564, 585, 629, 640, 669, 676, 679, 749, and 750 of SEQ ID NO. 8.

Consensus STEAP antigen 1(SEQ ID NO:10) has about 94% homology with some human STEAP sequences and about 99% homology with other human STEAP sequences. Consensus STEAP antigen 1(SEQ ID NO:10) also has about 94% homology to cynomolgus PSMA.

Consensus STEAP antigen 2(SEQ ID NO:12) has about 88% homology with the human STEAP sequence and about 94% homology with other human STEAP sequences. Consensus STEAP antigen 2(SEQ ID NO:12) also has about 94% homology to cynomolgus PSMA. Consensus STEAP antigen 2 contains a leader sequence at its N-terminus.

The consensus PSCA antigen (SEQ ID NO:14) has about 87% homology with human PSCA. The consensus PSCA antigen (SEQ ID NO:14) differs from human PSCA by including a leader sequence at its N-terminus.

The protein may have a sequence with 98% homology to PSA consensus antigen sequence 1(SEQ ID NO:2), PSA consensus antigen sequence 2(SEQ ID NO:4), PSMA consensus antigen sequence 1(SEQ ID NO:6), PSMA consensus antigen sequence 2(SEQ ID NO:8), STEAP consensus antigen sequence 1(SEQ ID NO:10), STEAP consensus antigen sequence 2(SEQ ID NO:12), or PSCA consensus antigen sequence (SEQ ID NO: 14).

The protein may have a sequence with 99% homology to PSA consensus antigen sequence 1(SEQ ID NO:2), PSA consensus antigen sequence 2(SEQ ID NO:4), PSMA consensus antigen sequence 1(SEQ ID NO:6), PSMA consensus antigen sequence 2(SEQ ID NO:8), STEAP consensus antigen sequence 1(SEQ ID NO:10), STEAP consensus antigen sequence 2(SEQ ID NO:12), or PSCA consensus antigen sequence (SEQ ID NO: 14).

As noted above, some embodiments comprise a leader sequence on the N-terminus. In some embodiments, the leader sequence is the IgE leader sequence of SEQ ID NO 16. In some embodiments of the protein sequences provided herein, SEQ ID NO 16 is removed therefrom. Likewise, in some embodiments of the nucleic acid sequences provided herein, SEQ ID NO:15 (which encodes SEQ ID NO:16) is removed therefrom.

Thus, some embodiments relate to proteins comprising a signal peptide linked to SEQ ID NO 2, 6 or 10 in place of the N-terminal methionine shown in the claims (the coding sequence for the signal peptide typically includes a start codon encoding the N-terminal methionine). Some embodiments relate to proteins comprising a signal peptide linked to amino acids 19-131 of SEQ ID NO. 14. Some embodiments relate to proteins comprising a signal peptide linked to a protein having 98% homology to SEQ ID No. 2, provided that amino acids 69, 78, 80, 82, 102, 110, 137, 139, 165, 189, 203, 220, 232, and 248 of SEQ ID No. 2 are conserved. Some embodiments relate to proteins comprising a signal peptide linked to a protein having 98% homology to SEQ ID No.6, provided that amino acids 14, 15, 32, 47, 58, 79, 111, 157, 223, 320, 350, 475, 499, 569, 613, 624, 653, 660, 663, 733 and 734 of SEQ ID No.6 are conserved. Some embodiments relate to a protein comprising a signal peptide linked to a protein having 98% homology to SEQ ID NO 10, which in each case is linked at its N-terminus, in place of the N-terminal methionine indicated in the claims (the coding sequence for the signal peptide typically includes an initiation codon encoding the N-terminal methionine). Some embodiments relate to proteins comprising a signal peptide linked to a protein having 98% homology to amino acids 19-131 of SEQ ID NO. 14. Some embodiments relate to proteins comprising a signal peptide linked to an immunogenic fragment of SEQ ID NO:2 (comprising amino acids corresponding to at least 256 amino acid residues of SEQ ID NO:2) provided that amino acids 69, 78, 80, 82, 102, 110, 137, 139, 165, 189, 203, 220, 232, and 248 of SEQ ID NO:2 are conserved. Some embodiments relate to proteins comprising a signal peptide linked to an immunogenic fragment of SEQ ID No.6 (comprising amino acids corresponding to at least 735 amino acid residues of SEQ ID No. 6) provided that amino acids 14, 15, 32, 47, 58, 79, 111, 157, 223, 320, 350, 475, 499, 569, 613, 624, 653, 660, 663, 733 and 734 of SEQ ID No.6 are conserved. Some embodiments relate to proteins comprising a signal peptide linked to an immunogenic fragment of SEQ ID NO:10 (comprising amino acids corresponding to at least 333 amino acid residues of SEQ ID NO: 10). Some embodiments relate to a protein comprising a signal peptide of the protein linked to a signal peptide having an immunogenic fragment linked to amino acids 19-131 of SEQ ID No. 14, a fragment comprising at least 110 amino acid residues of SEQ ID No. 14.

3. Gene sequences, constructs and plasmids

Nucleic acid molecules encoding the consensus amino acid sequence are generated to optimize stability and expression in humans. Codon usage is determined based on, among other things, minimizing intramolecular interactions and the formation of secondary structures and using codons that result in increased expression. The vaccine may comprise one or more nucleic acid sequences encoding one or more forms of a consensus form of immunogenic protein selected from the group of sequences produced to optimize stability and expression in humans. Generating a nucleic acid sequence comprising the coding sequence for the IgE leader sequence at the 5' end of the optimized consensus coding nucleic acid sequence, which encodes a protein having the IgE leader sequence at the N-terminus of the consensus amino acid sequence. In some embodiments, the nucleic acid sequence encoding the IgE leader sequence is SEQ ID NO 15.

Nucleic acid sequences are provided that encode PSA consensus antigen sequence 1 (protein sequence SEQ ID NO: 2; nucleic acid sequence SEQ ID NO:1), PSA consensus antigen sequence 2 (protein sequence SEQ ID NO: 4; nucleic acid sequence SEQ ID NO:3), PSMA consensus antigen sequence 1 (protein sequence SEQ ID NO: 6; nucleic acid sequence having nucleotides 1-2250 of SEQ ID NO: 5), PSMA consensus antigen sequence 2 (protein sequence SEQ ID NO: 8; nucleic acid sequence having nucleotides 1-2301 of SEQ ID NO: 7), STEAP consensus antigen sequence 1 (protein sequence SEQ ID NO: 10; nucleic acid sequence SEQ ID NO:9), STEAP consensus antigen sequence 2 (protein sequence SEQ ID NO: 12; nucleic acid sequence SEQ ID NO:11) or PSCA consensus antigen sequence (protein sequence SEQ ID NO: 14; nucleic acid sequence SEQ ID NO: 13). In addition to the PSMA encoding nucleotides, the nucleic acid sequence SEQ ID NO:5 encoding the PSMA consensus antigen sequence 1 also contained an additional 9 codons (27 nucleotides) just before the stop codon, encoding the HA tag (SEQ ID NO:32), not shown in SEQ ID NO: 6. The HA tag is a peptide sequence corresponding to an influenza epitope (influenza epitope) for detection of protein expression (performed using commercially available anti-HA tag antibodies), among others. SEQ ID NO 5 encodes SEQ ID NO 6 and an additional 9 amino acids sequence SEQ ID NO 32 linked at its N-terminus to the C-terminus of SEQ ID NO 6. In some embodiments, the PSMA-1 consensus antigen is encoded by SEQ ID NO 5 and comprises a protein having the amino acid sequence of SEQ ID NO 6 linked at its C-terminus to the N-terminus of SEQ ID NO 32. In some embodiments, the PSMA-1 consensus antigen is encoded by nucleotides 1-2250 of SEQ ID NO.5 and comprises a protein having the amino acid sequence of SEQ ID NO. 6. The coding sequence having nucleotides 1-2250 of SEQ ID NO.5 has one or more stop codons at its 3' end. In addition to the nucleotides encoding the IgE signal linked to PSMA, the nucleic acid sequence SEQ ID NO 7 encoding the PSMA consensus antigen sequence 2 comprises the protein and an additional 9 codons (27 nucleotides) just before the stop codon, said 9 codons encoding the HA tag (SEQ ID NO:32), not shown in SEQ ID NO: 8. SEQ ID NO 7 encodes SEQ ID NO 8 and an additional 9 amino acids sequence SEQ ID NO 32 linked at its N-terminus to the C-terminus of SEQ ID NO 8. In some embodiments, the PSMA-2 consensus antigen is encoded by SEQ ID NO 7 and comprises a protein having the amino acid sequence of SEQ ID NO 8 linked at its C-terminus to the N-terminus of SEQ ID NO 32. In some embodiments, the PSMA-2 consensus antigen is encoded by nucleotides 1-2301 of SEQ ID NO.7 and comprises a protein having the amino acid sequence of SEQ ID NO. 8. The coding sequence having nucleotides 1-2301 of SEQ ID NO.7 has one or more stop codons at its 3' end.

An isolated nucleic acid molecule can encode a protein having a sequence with 98% homology to PSA consensus antigen sequence 1(SEQ ID NO:2) as long as amino acids 69, 78, 80, 82, 102, 110, 137, 139, 165, 189, 203, 220, 232, and 248 of SEQ ID NO:2 are conserved, a protein having a sequence with 98% homology to PSA consensus antigen sequence 2(SEQ ID NO:4) as long as amino acids 21, 86, 127, 129, 154, 156, 182, 195, 206, 218, 220, 237, 249, 255, 265, 271, and 275 of SEQ ID NO:4 are conserved, a protein having a sequence with 98% homology to PSMA consensus antigen sequence 1(SEQ ID NO:6) as long as amino acids 14, 15, 32, 47, 58, 79, 111, 157, 223, 320, 350, 613, 499, 569, 613, 624, 653, 475, and 220 of SEQ ID NO:6 are conserved 660. 663, 733 and 734 are conserved, proteins having a sequence with 98% homology to PSMA consensus antigen sequence 2(SEQ ID NO:8), as long as amino acids 20, 30, 31, 48, 63, 74, 95, 127, 173, 239, 336, 366, 491, 515, 564, 585, 629, 640, 669, 676, 679, 749 and 750 of SEQ ID NO:8 are conserved, proteins having a sequence with 98% homology to STEAP consensus antigen sequence 1(SEQ ID NO:10), STEAP consensus antigen sequence 2(SEQ ID NO:12) or to PSCA consensus antigen sequence (SEQ ID NO: 14).

An isolated nucleic acid molecule can encode a protein having a sequence with 99% homology to PSA consensus antigen sequence 1(SEQ ID NO:2) as long as amino acids 69, 78, 80, 82, 102, 110, 137, 139, 165, 189, 203, 220, 232, and 248 of SEQ ID NO:2 are conserved, a protein having a sequence with 99% homology to PSA consensus antigen sequence 2(SEQ ID NO:4) as long as amino acids 21, 86, 127, 129, 154, 156, 182, 195, 206, 218, 220, 237, 249, 255, 265, 271, and 275 of SEQ ID NO:4 are conserved, a protein having a sequence with 99% homology to PSMA consensus antigen sequence 1(SEQ ID NO:6) as long as amino acids 14, 15, 32, 47, 58, 79, 111, 157, 223, 320, 350, 613, 499, 569, 613, 624, 653, 475, and 220 of SEQ ID NO:6 are conserved 660. 663, 733 and 734 are conserved, proteins having a sequence with 99% homology to PSMA consensus antigen sequence 2(SEQ ID NO:8), as long as amino acids 20, 30, 31, 48, 63, 74, 95, 127, 173, 239, 336, 366, 491, 515, 564, 585, 629, 640, 669, 676, 679, 749 and 750 of SEQ ID NO:8 are conserved, proteins having a sequence with 99% homology to STEAP consensus antigen sequence 1(SEQ ID NO:10), STEAP consensus antigen sequence 2(SEQ ID NO:12) or to PSCA consensus antigen sequence (SEQ ID NO: 14).

An isolated nucleic acid molecule can encode a protein having a sequence with 98% homology to a sequence encoding PSA consensus antigen sequence 1(SEQ ID NO:1), PSA consensus antigen sequence 2(SEQ ID NO:3), PSMA consensus antigen sequence 1(SEQ ID NO:5 or preferably nucleotides 1-2250 of SEQ ID NO: 5), PSMA consensus antigen sequence 2(SEQ ID NO:7 or preferably nucleotides 1-2301 of SEQ ID NO: 7), STEAP consensus antigen sequence 1(SEQ ID NO:9), STEAP consensus antigen sequence 2(SEQ ID NO:11), or PSCA consensus antigen sequence (SEQ ID NO: 13).

An isolated nucleic acid molecule can encode a protein having a sequence with 99% homology to a sequence encoding PSA consensus antigen sequence 1(SEQ ID NO:1), PSA consensus antigen sequence 2(SEQ ID NO:3), PSMA consensus antigen sequence 1(SEQ ID NO:5 or preferably nucleotides 1-2250 of SEQ ID NO: 5), PSMA consensus antigen sequence 2(SEQ ID NO:7 or preferably nucleotides 1-2301 of SEQ ID NO: 7), STEAP consensus antigen sequence 1(SEQ ID NO:9), STEAP consensus antigen sequence 2(SEQ ID NO:11), or PSCA consensus antigen sequence (SEQ ID NO: 13).

An isolated nucleic acid molecule can encode a protein comprising a leader sequence at the N-terminus. In some embodiments, the nucleic acid molecule can encode the IgE leader sequence of SEQ ID NO 16. In some embodiments, an isolated nucleic acid molecule can encode a protein comprising a signal peptide linked to SEQ ID NO:2, SEQ ID NO:6, or SEQ ID NO:10 in place of the N-terminal methionine shown in the claims (the coding sequence for the signal peptide typically includes an initiation codon encoding the N-terminal methionine). In some embodiments, the isolated nucleic acid molecule can encode a protein comprising a signal peptide linked to amino acids 19-131 of SEQ ID NO. 14. In some embodiments, the isolated nucleic acid molecule can encode a protein comprising a signal peptide linked to a protein having 98% homology to SEQ ID No. 2, so long as amino acids 69, 78, 80, 82, 102, 110, 137, 139, 165, 189, 203, 220, 232, and 248 of SEQ ID No. 2 are conserved. In some embodiments, an isolated nucleic acid molecule can encode a protein comprising a signal peptide linked to a protein having 98% homology to SEQ ID No.6, so long as amino acids 14, 15, 32, 47, 58, 79, 111, 157, 223, 320, 350, 475, 499, 569, 613, 624, 653, 660, 663, 733, and 734 of SEQ ID No.6 are conserved. In some embodiments, the isolated nucleic acid sequence can encode a protein comprising a signal peptide linked to a protein having 98% homology to SEQ ID No. 10. In the case where a coding sequence for a signal peptide is provided, the signal peptide is linked to the peptide sequence to replace the N-terminal methionine shown in the sequence shown (the coding sequence for the signal peptide typically includes an initiation codon encoding the N-terminal methionine). In some embodiments, the isolated nucleic acid molecule can encode a protein comprising a signal peptide linked to a protein having 98% homology to amino acids 19-131 of SEQ ID NO. 14. In some embodiments, the isolated nucleic acid molecule can encode a protein comprising a signal peptide linked to an immunogenic fragment of SEQ ID NO:2 (comprising amino acids corresponding to at least 256 amino acid residues of SEQ ID NO:2) so long as amino acids 69, 78, 80, 82, 102, 110, 137, 139, 165, 189, 203, 220, 232, and 248 of SEQ ID NO:2 are conserved. In some embodiments, the isolated nucleic acid molecule can encode a protein comprising a signal peptide linked to an immunogenic fragment of SEQ ID No.6 (comprising amino acids corresponding to at least 735 amino acid residues of SEQ ID No. 6) so long as amino acids 14, 15, 32, 47, 58, 79, 111, 157, 223, 320, 350, 475, 499, 569, 613, 624, 653, 660, 663, 733, and 734 of SEQ ID No.6 are conserved. In some embodiments, an isolated nucleic acid molecule can encode a protein comprising a signal peptide linked to an immunogenic fragment of SEQ ID NO:10 (comprising amino acids corresponding to at least 333 amino acid residues of SEQ ID NO: 10). In some embodiments, the isolated nucleic acid molecule can encode a protein comprising a signal peptide linked to a protein having an immunogenic fragment linked to amino acids 19-131 of SEQ ID No. 14, a signal peptide comprising a signal peptide of at least 110 amino acid residues of SEQ ID No. 14.

Provided herein are genetic constructs that can comprise a nucleic acid sequence encoding a consensus prostate antigen disclosed herein, including consensus protein sequences, sequences having homology to common protein sequences, fragments of consensus protein sequences, and sequences homologous to fragments of common protein sequences. The gene construct may be present in the cell as a functional extrachromosomal molecule. The genetic construct may be a linear minichromosome (minichromosome), including a centromere, telomere, or plasmid or cosmid.

The genetic construct may also be part of the genome of a recombinant viral vector (recombinant adenovirus, recombinant adeno-associated virus and recombinant vaccinia virus). The genetic construct may be part of genetic material in an attenuated live microorganism or a recombinant microbial vector that is live in a cell.

The genetic construct may comprise regulatory elements for gene expression of the coding sequence of the nucleic acid. The regulatory element may be a promoter, an enhancer, an initiation codon, a stop codon or a polyadenylation signal.

The nucleic acid sequence may constitute a genetic construct which may be a vector. The vector may be capable of expressing the antigen in a cell of the mammal in an amount effective to elicit an immune response in the mammal. The vector may be recombinant. The vector may comprise a heterologous nucleic acid encoding an antigen. The vector may be a plasmid. The vectors can be used to transfect cells with nucleic acids encoding antigens, and the transformed host cells can be cultured, and the cells maintained under conditions in which the antigens can be expressed.

In some embodiments, the coding sequences for a single consensus prostate antigen are provided on a single vector. In some embodiments, the coding sequences for multiple consensus prostate antigens are provided on a single vector. In some embodiments, compositions are provided that comprise coding sequences for multiple consensus prostate antigens (one antigen per vector or multiple antigens per vector) on multiple vectors.

In some embodiments, the coding sequences for two or more different consensus prostate antigens can be provided on a single vector. In some embodiments, the coding sequence may have a separate promoter to control expression. In some embodiments, a coding sequence may have a single promoter to control expression and an IRES sequence separating the coding sequences. The presence of the IRES sequence results in separate translation of the transcription product. In some embodiments, the coding sequence may have a single promoter to control expression and a coding sequence that encodes a proteolytic cleavage peptide sequence that separates the coding sequences of the antigens. A single translation product is produced which is subsequently processed by a protease that recognizes the protease cleavage site to produce separate protein molecules. The protease cleavage sites used are generally recognized by proteases that are endogenously present in the cell in which expression takes place. In some embodiments, a separate coding sequence for a protease may be included to provide for the production of the protease required to process the polyprotein translation product. In some embodiments, the vector comprises coding sequences for 1,2, 3,4, 5,6, or all 7 consensus prostate antigens.

In each and every case shown herein, the stability and high expression level of the coding sequence can be optimized. In some cases, the codons are selected to reduce secondary structure formation of the RNA (e.g., due to intramolecular bonding).

The vector may comprise a heterologous nucleic acid encoding an antigen and may further comprise a start codon, which may be located upstream of the antigen encoding sequence, and a stop codon, which may be located downstream of the antigen encoding sequence. The initiation and termination codons can be in frame with the antigen coding sequence. The vector may further comprise a promoter operably linked to the antigen coding sequence. The promoter operably linked to the antigen-encoding sequence may be a promoter from simian virus 40(SV40), Mouse Mammary Tumor Virus (MMTV) promoter, Human Immunodeficiency Virus (HIV) promoter such as Bovine Immunodeficiency Virus (BIV) Long Terminal Repeat (LTR) promoter, moloney virus promoter, Avian Leukemia Virus (ALV) promoter, Cytomegalovirus (CMV) promoter such as CMV early promoter, EB virus (EBV) promoter or Rous Sarcoma Virus (RSV) promoter. The promoter may also be a promoter from a human gene such as human actin, human myosin, human hemoglobin, human muscle creatine or human metallothionein. The promoter may also be a tissue-specific promoter, such as a muscle-or skin-specific promoter, natural or synthetic. Examples of such promoters are described in U.S. patent application publication No. us20040175727, the contents of which are incorporated herein by reference in their entirety.

The vector may also comprise a polyadenylation signal, which may be downstream of the consensus prostate antigen coding sequence. The polyadenylation signal may be the SV40 polyadenylation signal, the LTR polyadenylation signal, the bovine growth hormone (bGH) polyadenylation signal, the human growth hormone (hGH) polyadenylation signal, or the human β -globin polyadenylation signal. The SV40 polyadenylation signal may be a polyadenylation signal from the pCEP4 vector (nvoltiproo, san diego, ca).

The vector may also comprise an enhancer upstream of the consensus prostate antigen coding sequence. Enhancers may be necessary for DNA expression. The enhancer may be human actin, human myosin, human hemoglobin, human muscle creatine or a viral enhancer such as from CMV, HA, RSV or EBV. Enhancement of polynucleotide function is described in U.S. Pat. Nos. 5,593,972, 5,962,428 and WO94/016737 (the contents of each of which are incorporated herein by reference in their entirety).

The vector may also comprise a mammalian origin of replication for maintaining the vector extrachromosomally in the cell and for generating multiple copies of the vector. The plasmid may be pVAX1, pCEP4, or pREP4 from elviteluo (san diego, ca), which may comprise an EB virus origin of replication and a nuclear antigen EBNA-1 coding region that can produce high copy episomal replication without integration. The backbone of the vector may be pAV 0242. The vector may be a replication-defective adenovirus type 5 (Ad5) vector.

The vector may also comprise regulatory sequences well adapted to the expression of the gene in the mammalian or human cell to which the vector is administered. The consensus prostate antigen coding sequence may comprise codons that allow for more efficient transcription of the coding sequence in a host cell.

The vector may be pSE420 (novacula, san diego, ca) which can be used to produce proteins in e. The vector may also be pYES2 (Yinwettro, san Diego, Calif.) which can be used to produce proteins in a Saccharomyces cerevisiae strain of yeast. The vector may also have a MAXBAC^TMA complete baculovirus expression system (nvoltiproto, san diego, ca) can be used to produce proteins in insect cells. The vector may also be pcDNA I or pcDNA3 (Yinwettro, san Diego, Calif.), which may beFor the production of proteins in mammalian cells, such as Chinese Hamster Ovary (CHO) cells. The vector may be an expression vector or system for producing the protein by conventional techniques and readily available starting materials, including Sambrook et al, molecular cloning and A laboratory Manual, 2 nd edition, Cold spring harbor (1989), which is incorporated herein by reference in its entirety.

The vaccine may comprise one or more of the prostate antigens shown herein and/or the vaccine may comprise one or more nucleic acid sequences encoding one or more of the consensus prostate antigens selected from this group. The vaccine may comprise a combination of one or more of the consensus prostate antigens shown herein with other immunogenic prostate proteins having sequences other than the consensus sequences described herein (including the native sequences), and/or the vaccine may comprise a combination of one or more nucleic acid sequences encoding one or more of the consensus prostate antigens selected from this group with nucleic acid molecules encoding other prostate antigens having sequences other than the consensus sequences described herein.

Without being bound by scientific theory, however, a vaccine that can be used to elicit an immune response (humoral, cellular, or both) broadly against prostate cancer cells can comprise one or more of the following nucleic acid sequences encoding one or more proteins selected from the group consisting of: consensus PSA antigen 1, consensus PSA antigen 2, consensus PSMA antigen 1, consensus PSMA antigen 2, consensus STEAP antigen 1, consensus STEAP antigen 2, and consensus PSCA antigen 1. Coding sequences can also include those provided herein that comprise homologous sequences, fragments, and homologous sequences to fragments.

Some embodiments provide methods of generating an immune response against prostate cancer cells comprising administering to an individual one or more compositions collectively comprising one or more coding sequences or combinations described herein. Some embodiments provide methods of prophylactically vaccinating an individual with prostate cancer comprising administering one or more compositions collectively comprising one or more coding sequences or combinations described herein. Some embodiments provide a method of therapeutically vaccinating an individual having prostate cancer, the method comprising administering one or more compositions collectively comprising one or more coding sequences or combinations described herein.

4. Pharmaceutical composition

Provided herein are pharmaceutical compositions according to the invention comprising about 1 nanogram to about 10mg of DNA. In some embodiments, the pharmaceutical composition according to the invention comprises: 1) at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 95, 100 nanograms, or at least 1,5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 495, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 440, 460, 455, 480, 470, 500, 475, 220, 230, 240, 250, 255, 260, 265, 270, 275, 280, 150, 240, 150, 440, 450, 625. 630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695, 700, 705, 710, 715, 720, 725, 730, 735, 740, 745, 750, 755, 760, 765, 770, 775, 780, 785, 790, 795, 800, 805, 810, 815, 820, 825, 830, 835, 840, 845, 850, 855, 860, 865, 870, 875, 880, 885, 890, 895.900, 905, 910, 915, 920, 925, 930, 935, 940, 945, 950, 955, 960, 965, 970, 975, 980, 985, 990, 995, or 1000 micrograms, or at least 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10mg or more; and 2) up to and including 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 95, or 100 nanograms up to and including 1,5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 440, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 495, 370, 375, 380, 385, 405, 410, 395, 400, 405, 410, 415, 450, 470, 455, 470, 500, 475, 445, 365, 475, 440, 470, 475, 445, 200, 205, 220, 230, 235, 240, 245, 250, 255, 265, 270, 610. 615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695, 700, 705, 710, 715, 720, 725, 730, 735, 740, 745, 750, 755, 760, 765, 770, 775, 780, 785, 790, 795, 800, 805, 810, 815, 820, 825, 830, 835, 840, 845, 850, 855, 860, 865, 870, 875, 880, 885, 890, 895.900, 905, 910, 915, 920, 925, 930, 935, 940, 945, 950, 960, 965, 970, 975, 980, 985, 990, 995, or 1000 micrograms, or up to and including 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9.5, or 10 mg. In some embodiments, a pharmaceutical composition according to the invention comprises about 5 nanograms to about 10mg of DNA. In some embodiments, a pharmaceutical composition according to the invention comprises about 25 nanograms to about 5mg of DNA. In some embodiments, the pharmaceutical composition comprises about 50 nanograms to about 1mg of DNA. In some embodiments, the pharmaceutical composition comprises about 0.1 to about 500 micrograms of DNA. In some embodiments, the pharmaceutical composition comprises about 1 to about 350 micrograms of DNA. In some embodiments, the pharmaceutical composition comprises about 5 to about 250 micrograms of DNA. In some embodiments, the pharmaceutical composition comprises about 10 to about 200 micrograms of DNA. In some embodiments, the pharmaceutical composition comprises about 15 to about 150 micrograms of DNA. In some embodiments, the pharmaceutical composition comprises about 20 to about 100 micrograms of DNA. In some embodiments, the pharmaceutical composition comprises about 25 to about 75 micrograms of DNA. In some embodiments, the pharmaceutical composition comprises about 30 to about 50 micrograms of DNA. In some embodiments, the pharmaceutical composition comprises about 35 to about 40 micrograms of DNA. In some embodiments, the pharmaceutical composition comprises about 100 to about 200 micrograms of DNA. In some embodiments, the pharmaceutical composition comprises about 10 micrograms to about 100 micrograms of DNA. In some embodiments, the pharmaceutical composition comprises about 20 micrograms to about 80 micrograms of DNA. In some embodiments, the pharmaceutical composition comprises about 25 micrograms to about 60 micrograms of DNA. In some embodiments, the pharmaceutical composition comprises about 30 nanograms to about 50 micrograms of DNA. In some embodiments, the pharmaceutical composition comprises about 35 nanograms to about 45 micrograms of DNA. In some preferred embodiments, the pharmaceutical composition comprises about 0.1 to about 500 micrograms of DNA. In some preferred embodiments, the pharmaceutical composition comprises about 1 to about 350 micrograms of DNA. In some preferred embodiments, the pharmaceutical composition comprises about 25 to about 250 micrograms of DNA. In some preferred embodiments, the pharmaceutical composition comprises about 100 to about 200 micrograms of DNA.

The pharmaceutical composition according to the invention is formulated according to the mode of administration to be used. In the case where the pharmaceutical compositions are injectable pharmaceutical compositions, they are sterile, pyrogen-free and particle-free. Preferably, isotonic formulations are used. In general, additives for isotonicity may include sodium chloride, glucose, mannitol, sorbitol, and lactose. In some cases, isotonic solutions, such as phosphate buffered saline, are preferred. Stabilizers include gelatin and albumin. In some embodiments, a vasoconstrictor is added to the formulation.

Preferably the pharmaceutical composition is a vaccine, more preferably a DNA vaccine.

The vaccine may be a DNA vaccine. The DNA vaccine may comprise a plurality of identical or different plasmids comprising nucleic acid coding sequences for one or more consensus prostate antigens. The DNA vaccine may comprise one or more nucleic acid sequences encoding one or more consensus prostate antigens. When the DNA vaccine comprises more than one coding sequence for a consensus prostate antigen, all such sequences may be present on a single plasmid, or each such sequence may be present on a different plasmid.

In some embodiments, the vaccine can comprise a nucleic acid sequence encoding one or more of the consensus prostate antigens in combination with one or more of the consensus prostate antigens.

DNA vaccines are disclosed in U.S. patent nos. 5,593,972, 5,739,118, 5,817,637, 5,830,876, 5,962,428, 5,981,505, 5,580,859, 5,703,055, and 5,676,594 (which are incorporated herein by reference in their entirety). The DNA vaccine may also comprise elements or agents that inhibit its integration into the chromosome. The vaccine may be RNA of prostate antigen. An RNA vaccine can be introduced into the cells.

The vaccine may be a recombinant vaccine comprising the above-described genetic construct or antigen. The vaccine may further comprise one or more consensus prostate antigens in the form of one or more protein subunits, or one or more attenuated viral particles comprising one or more consensus prostate antigens. Attenuated vaccines can be live attenuated vaccines, killed vaccines and vaccines that use recombinant vectors to deliver foreign genes encoding one or more consensus prostate antigens, as well as subunit and glycoprotein vaccines. Examples of live attenuated vaccines, vaccines that use recombinant vectors to deliver prostate antigens, subunit vaccines, and glycoprotein vaccines are described in U.S. Pat. nos. 4,510,245; 4,797,368; 4,722,848; 4,790,987, respectively; 4,920,209, respectively; 5,017,487, respectively; 5,077,044, respectively; 5,110,587; 5,112,749, respectively; 5,174,993; 5,223,424, respectively; 5,225,336, respectively; 5,240,703, respectively; 5,242,829, respectively; 5,294,441, respectively; 5,294,548, respectively; 5,310,668, respectively; 5,387,744, respectively; 5,389,368, respectively; 5,424,065, respectively; 5,451,499, respectively; 5,453,364, respectively; 5,462,734, respectively; 5,470,734, respectively; 5,474,935, respectively; 5,482,713, respectively; 5,591,439, respectively; 5,643,579, respectively; 5,650,309, respectively; 5,698,202, respectively; 5,955,088, respectively; 6,034,298; 6,042,836, respectively; 6,156,319 and 6,589,529, each of which is incorporated herein by reference.

The vaccines provided are useful for inducing immune responses, including therapeutic or prophylactic immune responses. Antibodies to the consensus prostate antigen and/or killer T cells can be generated. Such antibodies and cells can be isolated.

The vaccine may further comprise a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient may be a functional molecule such as a vehicle, adjuvant, carrier or diluent. The pharmaceutically acceptable excipient may be a transfection facilitating agent which may comprise surfactants such as Immune Stimulating Complexes (ISCOMS), freund's incomplete adjuvant, LPS analogues including monophosphoryl lipid a, muramyl peptides, quinone analogues, vesicles (vesicles) such as squalene and squalene, hyaluronic acid, lipids, liposomes, calcium ions, viral proteins, polyanions, polycations or nanoparticles or other known transfection facilitating agents.

The transfection facilitating agent is polyanion, polycation, including poly-L-glutamic acid (LGS) or lipid. The transfection facilitating agent is poly-L-glutamate, more preferably poly-L-glutamate is present in the vaccine at a concentration of less than 6 mg/ml. The transfection facilitating agent may also include surfactants such as Immune Stimulating Complexes (ISCOMS), freunds incomplete adjuvant, LPS analogs including monophosphoryl lipid a, muramyl peptides, quinone analogs, and vesicles such as squalene and squalene, and hyaluronic acid may also be administered with the gene construct. In some embodiments, the DNA vector vaccine may further comprise a transfection facilitating agent such as lipids, liposomes, including lecithin liposomes as a DNA-liposome mixture or other liposomes known in the art (see, e.g., W09324640), calcium ions, viral proteins, polyanions, polycations or nanoparticles, or other known transfection facilitating agents. Preferably, the transfection facilitating agent is a polyanion, polycation, including poly-L-glutamate (LGS) or lipid. The concentration of transfection reagent in the vaccine is less than 4mg/ml, less than 2mg/ml, less than 1mg/ml, less than 0.750mg/ml, less than 0.500mg/ml, less than 0.250mg/ml, less than 0.100mg/ml, less than 0.050mg/ml or less than 0.010 mg/ml.

The pharmaceutically acceptable excipient may be an adjuvant. Adjuvants may be other genes expressed in surrogate plasmids, or delivered as proteins with the above plasmids in vaccines. The adjuvant may be selected from: alpha-interferon (IFN-. alpha.), beta. -interferon (IFN-. beta.), gamma-interferon, platelet-derived growth factor (PDGF), TNF. alpha., TNF. beta., GM-CSF, Epidermal Growth Factor (EGF), cutaneous T-cell attracting chemokine (CTACK), epithelial thymus-expressing chemokine (TECK), mucosa-associated epithelial chemokine (MEC), IL-12, IL-15, MHC, CD80, CD86 (including IL-15 with deleted signal sequence and optionally including signal peptide from IgE). The adjuvant can be IL-12, IL-15, IL-28, CTACK, TECK, Platelet Derived Growth Factor (PDGF), TNF α, TNF β, GM-CSF, Epidermal Growth Factor (EGF), IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-18, or a combination thereof.

Other genes that may be useful adjuvants include genes encoding: MCP-1, MIP-la, MIP-1P, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, pl50.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, mutant forms of IL-18, CD40, CD40L, angiogenic factors, fibroblast growth factors, IL-7, nerve growth factors, vascular endothelial growth factors, Fas, TNF receptors, Flt, ApoApo-1, P55, WSL-1, DR3, AIMP, Apo-3, LARD, NGRF, DR4, DR5, KILLER, TRAIL-2, TRICK2, TRIJCK 6, caspase, FOUN-3, FOICE-3, FORC, Sp-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, inactivated NIK, SAP K, SAP-1, JNK, interferon-responsive genes, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK ligands, Ox40, Ox40 ligands, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1, TAP2, and functional fragments thereof.

The vaccine may also comprise a gene vaccine facilitator as described in U.S. sequence No.021,579 filed on 1/4 of 1994 (which is incorporated herein by reference in its entirety).

5. Delivery method

Provided herein are methods for delivering pharmaceutical formulations, preferably vaccines, to provide gene constructs and consensus prostate antigens comprising consensus antigens of epitopes that make the consensus prostate antigens particularly effective immunogens against which immune responses against prostate cancer cells can be induced. Methods of delivering vaccines or vaccinations to induce therapeutic and/or prophylactic immune responses may be provided. Vaccines can be delivered to individuals to modulate the activity of the immune system and enhance the immune response of mammals.

After delivery of the vaccine into a mammal, thereby delivering the vector to the cells of the mammal, the transfected cells will express and secrete the corresponding prostate consensus protein. Such secreted proteins or synthetic antigens will be recognized by the immune system, which will elicit an immune response that may include: antibodies raised against the antigen and T cell immune responses specific for the antigen. In some examples, a mammal vaccinated with a vaccine discussed herein will have an immune system that has been exposed to an antigen. Vaccines can be delivered to individuals to modulate the activity of the individual's immune system, thereby enhancing the immune response.

Vaccines can be delivered in the form of DNA vaccines, methods of delivering DNA vaccines are described in U.S. patent nos. 4,945,050 and 5,036,006, both of which are incorporated herein by reference.

The vaccine can be administered to a mammal to elicit an immune response in the mammal. The mammal may be a human, non-human primate, cow, pig, sheep, goat, antelope, bison, buffalo, bovidae, deer, hedgehog, elephant, llama, alpaca, mouse, rat or chicken, preferably a human, cow, pig or chicken.

a. Combination therapy

The pharmaceutical composition, preferably a vaccine, may be administered in combination with one or more other prostate proteins or genes. The vaccine may be administered in combination with a protein or a gene encoding an adjuvant including: alpha-interferon (IFN-. alpha.), beta. -interferon (IFN-. beta.), gamma-interferon, IL-12, IL-15, IL-28, CTACK, TECK, platelet-derived growth factor (PDGF), TNF. alpha., TNF. beta., GM-CSF, Epidermal Growth Factor (EGF), IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-18, MCP-1, MIP-la, MIP-1P, IL-8, TES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, pl50.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, PDGF-1, VLA-1, Mac-1, PLCAM 50.95, PECAM-1, ICAM-2, ICAM-3, and so on, M-CSF, G-CSF, IL-4, mutant forms of IL-18, CD40, CD40L, vascular growth factor, fibroblast growth factor, IL-7, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Flt, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, caspase, Fos, c-jun, Sp-1, ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, inactivated NIK, SAP K, SAP-1, JNK, interferon response gene, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK ligand, Ox40, Ox40 ligand, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1 or TAP2 or a functional fragment thereof.

b. Route of administration

The vaccine can be administered by different routes including oral, parenteral, sublingual, transdermal, rectal, transmucosal, topical, by inhalation, by buccal administration, intrapleural, intravenous, intraarterial, intraperitoneal, subcutaneous, intramuscular, intranasal, epidural, and intraarticular, or a combination thereof. For veterinary use, the compositions may be administered as a suitably acceptable formulation in accordance with normal veterinary practice. Veterinarians can readily determine the dosage regimen and route of administration that is most appropriate for a particular animal. The vaccine may be administered using a conventional syringe, a needleless injection device, a "microprojectile bombardent gene gun" or other physical methods such as electroporation ("EP"), "hydrodynamic methods" or ultrasound.

Vectors for vaccines can be delivered to mammals using several well-known techniques, including DNA injection (also known as DNA vaccination) with and without in vivo electroporation, liposome-mediated, nanoparticle-facilitated recombinant vectors such as recombinant adenoviruses, recombinant adeno-associated viruses, and recombinant vaccines. The prostate antigen can be delivered by DNA injection in conjunction with in vivo electroporation.

c. Electroporation

Administration of the vaccine by electroporation of the plasmid of the vaccine can be achieved using an electroporation device that can be configured to deliver an energy pulse to the desired tissue of the mammal that is effective to cause reversible pore formation on the cell membrane, and in some embodiments the energy pulse is a constant current similar to the current input preset by the user.

In some embodiments in which electroporation is used, the electroporation device may include an electroporation component and an electrode component or handling component (handle assembly). The electroporation component may include and integrate one or more of the various elements of the electroporation device, including: a controller, a current waveform generator, an impedance detector, a waveform recorder, an input element, a status reporting element, a communication port, a memory component, a power supply, and a power switch. Electroporation may be carried out using an in vivo electroporation device such asEP systems (Nenovi pharmaceutical, Brucel, Pa.) or Elgen electroporators (Nenovi pharmaceutical, Brucel, Pa.) were implemented to facilitate transfection of cells with plasmids.

The electroporation component may be used as one element of the electroporation apparatus, and the other elements are separate elements (or components) in communication with the electroporation component. The electroporation component may be used as more than one element of the electroporation device that may be in communication with other elements of the electroporation device that are separate from the electroporation component. The elements of the electroporation apparatus that exist as a component of one motor or machine apparatus may not be limited as the elements may be used as one apparatus or as separate elements communicating with each other. The electroporation component may be capable of delivering an energy pulse that produces a constant current in a desired tissue and includes a feedback mechanism. The electrode assembly may include an electrode array having a plurality of electrodes in a spatial arrangement, wherein the electrode assembly receives an energy pulse from the electroporation component and delivers the energy pulse to a desired tissue through the electrodes. At least one of the plurality of electrodes is neutral during delivery of the energy pulse and measures an impedance of the desired tissue, communicating the impedance to the electroporation component. A feedback mechanism may receive the measured impedance and may adjust the energy pulse delivered through the electroporation component to maintain a constant current.

The plurality of electrodes may deliver the energy pulses in a distributed pattern. The plurality of electrodes may deliver the energy pulses in a decentralized pattern by controlling the electrodes under a programmed sequence, and the programmed sequence is input to the electroporation component by a user. The programming sequence may include a plurality of sequentially delivered pulses, wherein each pulse of the plurality of pulses is delivered with at least two active electrodes (one neutral electrode measuring impedance), and wherein subsequent pulses of the plurality of pulses are delivered with different electrodes of the at least two active electrodes (one neutral electrode measuring impedance).

The feedback mechanism may be performed using hardware or software. The feedback mechanism may be performed by an analog closed loop circuit. Feedback occurs once every 50, 20, 10 or 1 mus, but is preferably real-time or instantaneous (i.e., substantially simultaneously, as determined by available techniques for determining reaction time). The neutral electrode may measure impedance in the desired tissue and communicate the impedance to a feedback mechanism, and the feedback mechanism reacts to the impedance and adjusts the energy pulse to maintain the constant current at a value similar to the preset current. The feedback mechanism can continuously and instantaneously maintain a constant current during the delivery of the energy pulse.

Examples of electroporation devices and methods that can facilitate the delivery of the DNA vaccines of the present invention include those described in U.S. Pat. No.7,245,963 to Draghia-Akli et al, U.S. patent publication 2005/0052630 to Smith et al, the contents of which are incorporated herein by reference in their entirety. Other electroporation devices and electroporation methods that may be used to facilitate DNA vaccine delivery include those provided in co-pending and commonly owned U.S. patent application serial No.11/874072 filed on 10/17/2007, which is hereby incorporated by reference in its entirety for the benefit of 35USC119(e) claims U.S. provisional application serial No.60/852,149 filed on 10/17/2006 and 60/978,982 filed on 10/2007.

U.S. Pat. No.7,245,963 to Draghia-Akli et al describes a standard electrode system and its use for facilitating the introduction of biomolecules into cells of selected tissues in vivo or in plants. A standard electrode system may include a plurality of needle electrodes; subcutaneous needles; an electrical plug providing an electrically conductive connection from the programmable constant current pulse controller to the plurality of pin electrodes; and a power source. An operator may grasp the plurality of needle electrodes secured to the carrier structure and securely insert them into selected tissue of the body or plant. The biomolecules are then delivered to the selected tissue via a subcutaneous needle. The programmable constant current pulse controller is activated and a constant current electrical pulse is applied to the plurality of needle electrodes. The applied constant current electrical pulse facilitates the introduction of the biomolecule into the cell between the plurality of electrodes. U.S. Pat. No.7,245,963 is incorporated herein by reference in its entirety.

U.S. patent publication 2005/0052630, filed by Smith et al, describes an electroporation device that can be used to efficiently facilitate the introduction of biomolecules into cells of selected tissues in vivo or in plants. The electroporation devices include electrically powered devices ("EKD devices") whose operation is specified by software or firmware. The EKD device generates a series of encodable constant current pulse patterns between the electrodes of the array based on user control and input of pulse parameters and allows current waveform data to be stored and retrieved. The electroporation device further comprises a replaceable electrode disk having an array of needle electrodes, a central injection channel for the injection needle, and a movable guide disk. U.S. patent publication 2005/0052630 is incorporated herein by reference in its entirety.

The electrode arrays and methods described in U.S. Pat. No.7,245,963 and U.S. patent publication 2005/0052630 are applicable not only to deep penetration into tissues such as muscles, but also to deep penetration into other tissues or organs. Due to the configuration of the electrode array, the injection needle (for delivering the selected biomolecules) can also be inserted completely into the target organ and injected perpendicular to the target tissue at the area pre-delineated by the electrodes. The electrodes described in U.S. Pat. No.7,245,963 and U.S. Pat. publication 2005/005263 are preferably 20mm long and 21 gauge (gauge).

Further, included in some embodiments are incorporated electroporation devices and uses thereof, among those described in the following patents: U.S. patent 5,273,525, published 28.1993, U.S. patent 6,110,161, published 8.29.2001, 6,261,281, published 17.7.2001, 6,958, 060, published 25.10.2005, and U.S. patent 6,939,862, published 6.9.2005. Further, patents covering the subject matter are provided in U.S. patent 6,697,669 published on 24.2.2004, which relates to the delivery of DNA using any of a variety of devices, and U.S. patent 7,328,064 published on 5.2.2008, which depicts the methods of injecting DNA included herein. The above patents are incorporated herein by reference in their entirety. Another embodiment of an electroporation device for use with the cancer antigens described herein is the Elgen EP instrument (nnowei pharmaceutical, brubell, pa).

d. Method for preparing vaccine

Provided herein are methods for preparing DNA plasmids comprising the DNA vaccines discussed herein. After the final subcloning step into a mammalian expression plasmid, the DNA plasmid can be used to inoculate a cell culture in a large-scale fermentor using methods known in the art.

DNA plasmids for use with the EP apparatus of the invention can be formulated or prepared using a combination of known equipment and techniques, but preferably they are prepared using the optimized plasmid preparation techniques described in the approved co-pending U.S. provisional application serial No.60/939,792 filed on 23/5/2007. In some embodiments, the DNA plasmids used in these studies can be formulated at a concentration of greater than or equal to 10 mg/mL. In addition to the equipment and protocols described in U.S. serial No.60/939,792 (including the equipment and protocols described in the approved patent, U.S. patent No.7,238,522 issued on 7/3/2007), the manufacturing techniques also include or incorporate various equipment and protocols that are generally known to those skilled in the art. The above applications and patents (U.S. serial No.60/939,792 and U.S. patent No.7,238,522, respectively) are incorporated by reference herein in their entirety.

Examples

The invention is further illustrated in the following examples. It should be understood that these examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Accordingly, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such variations are also intended to fall within the scope of the appended claims. .

Example 1

Common immunogens for PSA and PSMA were designed based on the available full-length human and cynomolgus sequences in the GenBank database previously described in Laddy, d.j., Yan, j., Corbitt, n., Kobasa, d., Kobinger, g.p., Weiner, D.B, (2007) immunogenicity of novel consensus-based DNA vaccines against avian influenza 25,2984, and Laddy, d.j., Yan, j., Kutzler, m., Kobasa, d., kogerbin, g.p., Khan, a.s., Greenhouse, j., saesai, n.y., Draghia-dri, r, Weiner, D.B. (2008).

The consensus antigen sequence was synthesized by GeneScript (pistatavir, new jersey). The HA tag was included at the C-terminus of the antigen sequence. The mRNA stability and codon usage of the antigen sequence in humans were optimized. The final sequence was cloned into pVAX1 vector (Yinvelo Roots, Calsbad, Calif.) between BamHI and XhoI sites.

Consensus PSA antigen 1(SEQ ID NO:2) was generated. This sequence, comprising 261 amino acids, was compared to each PSA sequence shown in table 1. PSA sequences used included two human sequences, sequences from cynomolgus monkeys, and sequences from cynomolgus monkeys. Table 1 includes SEQ ID NO and accession numbers for each sequence compared to consensus PSA antigen 1(SEQ ID NO: 2).

TABLE 1

Multiple sequence alignments of homo sapiens (SEQ ID NO:17 and SEQ ID NO:18), cynomolgus monkey (SEQ ID NO:20) and cynomolgus monkey (SEQ ID NO:19) PSA sequences with consensus PSA antigen 1(SEQ ID NO:2) were generated. KLK3 (kallikrein 3) is a gene encoding PSA and is pseudonymized by PSA. PSA antigen 1 has 91% homology to homo sapiens full-length PSA protein sequence, 96% homology to cynomolgus monkey full-length PSA protein sequence, and 95% homology to cynomolgus monkey full-length PSA protein sequence.

Example 2

Consensus PSA antigen 2(SEQ ID NO:4) was produced. This sequence, which contained 279 amino acids including the IgE leader sequence, was compared to each of the PSA sequences shown in table 2. PSA sequences used included two human sequences, sequences from cynomolgus monkeys, and sequences from cynomolgus monkeys. Table 2 includes SEQ ID NOs and accession numbers for each sequence compared to consensus PSA antigen 2(SEQ ID NO: 4).

TABLE 2

Multiple sequence alignments of homo sapiens (SEQ ID NO:17 and SEQ ID NO:18), cynomolgus monkey (SEQ ID NO:21) and cynomolgus monkey (SEQ ID NO:19) PSA sequences with consensus PSA antigen 1(SEQ ID NO:4) were generated. KLK3 (kallikrein 3) is a gene encoding PSA and is pseudonymized by PSA. PSA antigen 1 has 90-91% homology with homo sapiens full-length PSA protein sequence, 95% homology with cynomolgus monkey full-length PSA protein sequence, and 95% homology with macaque partial PSA protein sequence.

Example 3

Consensus PSMA antigen 1(SEQ ID NO:6) was generated. This sequence, comprising 750 amino acids, was compared to each PSMA sequence shown in table 3. The PSMA sequence used included two human sequences and a sequence from cynomolgus monkey. Table 3 includes SEQ ID NO and accession numbers for each sequence compared to consensus PSMA antigen 1(SEQ ID NO: 6).

TABLE 3

Multiple sequence alignments of the wisdom human and cynomolgus PSMA sequences with PSMA antigen 1 were generated. The PSMA antigen 1 consensus sequence (SEQ ID NO:6) has 96% homology with homo sapiens PSMA protein sequence (SEQ ID NO:22 and SEQ ID NO:23) and 94% homology with cynomolgus monkey full length PSMA protein sequence (SEQ ID NO: 24).

Example 4

Consensus PSMA antigen 2(SEQ ID NO:8) was generated. This sequence, comprising 766 amino acids including the IgE leader sequence, was compared to each of the PSMA sequences shown in table 4. The PSMA sequence used included two human sequences and a sequence from cynomolgus monkey. Table 4 includes SEQ ID NOs and accession numbers for each sequence compared to consensus PSMA antigen 2(SEQ ID NO: 8).

TABLE 4

Multiple sequence alignments of homo sapiens (SEQ ID NO:22 and SEQ ID NO:23) and cynomolgus PSMA sequences (SEQ ID NO:24 and SEQ ID NO:25) with PSMA antigen 2 were generated. The PSMA antigen 2 consensus sequence (SEQ ID NO:8) has 96% homology to homo sapiens PSMA protein sequence and 94% homology to cynomolgus PSMA protein sequence.

Example 5

Consensus STEAP antigen 1(SEQ ID NO:10) was generated. This sequence, containing 339 amino acids, was compared to each of the STEAP sequences shown in table 5. The STEAP sequences used included two full-length human sequences, a full-length sequence from cynomolgus monkey, and two shorter human sequences. Table 5 includes the common STEAP antigen 1(SEQ ID NO:10) for each sequence of SEQ ID NO and accession number.

TABLE 5

Multiple sequence alignments of homo sapiens and cynomolgus macaque STEAP sequences with a common STEAP antigen 1 were generated. The STEAP antigen 1 consensus sequence (SEQ ID NO:10) has 99% homology with the human full-length isoform STEAP1 protein sequences (SEQ ID NO:26 and SEQ ID NO:27), 94% homology with the shorter homo sapiens isoform STEAP1 protein sequence (SEQ ID NO:29 and SEQ ID NO:30), and 94% homology with the cynomolgus monkey full-length STEAP1 protein sequence (SEQ ID NO: 28).

Example 6

Consensus STEAP antigen 2 was generated (SEQ ID NO: 12). This sequence, comprising 356 amino acids, was compared to each of the STEAP sequences shown in table 6. The STEAP sequences used included two full-length human sequences, a full-length sequence from cynomolgus monkey, and two shorter human sequences. Table 6 includes the common STEAP antigen 2(SEQ ID NO:12) for each sequence of SEQ ID NO and accession number.

TABLE 6

Multiple sequence alignments of homo sapiens and cynomolgus macaque STEAP1 sequences with consensus STEAP1 antigen 2 were generated. The STEAP1 antigen 2 consensus sequence (SEQ ID NO:12) has 94% homology with the full length human isoform STEAP1 protein sequence (SEQ ID NO:26 and SEQ ID NO:27), 88% homology with the shorter homo sapiens isoform STEAP1 protein sequence (SEQ ID NO:29 and SEQ ID NO:30), and 94% homology with the cynomolgus monkey full length STEAP1 protein sequence (SEQ ID NO: 28).

Example 7

Consensus PSCA antigen was generated (SEQ ID NO: 14). This sequence, comprising 131 amino acids including the IgE leader sequence, was compared to each of the PSCA sequences shown in table 7. The PSCA sequence used was a full-length human sequence. Table 7 includes SEQ ID NOs and accession numbers of the sequences used for comparison to the consensus PSCA antigen (SEQ ID NO: 14).

TABLE 7

Multiple sequence alignments of homo sapiens PSCA sequence (SEQ ID NO:31) with the consensus PSCA antigen (SEQ ID NO:14) were generated. The PSCA antigen consensus sequence has 87% homology to full-length homo sapiens PSCA.

Example 8

In vitro translation was performed to confirm the expression of PSA and PSMA antigens. Use ofThe rapid coupling of the transcription/translation system and 35S-methionine (Promega). The pVAX vector alone (negative control) or the pVAX backbone together with the PSA or PSMA antigen insert and 35S-methionine was added to the reaction mixture according to the manufacturer' S instructions. The reaction was carried out at 30 ℃ for 2 hours. The labeled proteins were immunoprecipitated using an anti-HA affinity gel (Sigma, St. Louis, Mo.) by rotation overnight in radioimmunoprecipitation assay (RIPA) buffer at 4 ℃. The immunoprecipitated proteins were electrophoresed on SDS-PAGE gels, followed by fixing and drying the gels. Expression of the 35S-labeled protein was detected by autoradiography. The results are shown in FIG. 1.

Example 9

Cellular immunogenicity of PSA and PSMA antigens was determined by interferon gamma ELISpot.

Female 4 to 6 week old BALB/c mice were purchased from Jackson laboratories (Berbour, Maine). All animals were housed in a temperature controlled, periodic light facility at pennsylvania university. Animal Care was performed according to the guidelines of the institute of health and Care and Use Committee (Institutional Care and Use Committee) of the american national institute of health and pennsylvania university.

For cellular immunogenicity studies, intramuscular injections were followed by useAn adaptive constant current electroporation device (nover pharmaceutical, brubel, pa) delivers 10 or 20 μ g of each antigen to the anterior tibialis of Balb/c mice by electroporation. Mice (n = 5/group) received 2 immunizations at weeks 0 and 2. Two 0.1 amp constant current square wave pulses were delivered through a triangular 3-electrode array consisting of 26-gauge solid stainless steel electrodes. Each pulse is 52 milliseconds in length with a 1 second delay between pulses. Mice received a total of 2 immunizations (the immunizations were administered at 2 week intervals). Mice were sacrificed humanely 1 week after the second immunization for analysis of cellular and humoral immune responses.

Cells and responses were assessed 1 week after the last immunization (week 5). Antigen-specific secretion of IFN γ was determined using ELISpot assay. Mouse IFN gamma capture antibody (R)&System D, minneapolis, mn) was used to coat flat bottom Immobilon-P plates (millibo, birrica, ma) at 4 ℃ overnight. Splenocytes were aseptically isolated and resuspended in R10 medium (royal park memorial Institute medium1640 (Rosewell park Institute medium1640) supplemented with 10% fetal bovine serum, 1% antibiotic-antimycotic and 0.1% 2-mercaptoethanol). 2X10 from immunized mice⁵Individual splenocytes were added to each well of a 96 well plate and stimulated in the presence of R10 (negative control), concanavalin a (positive control) (sigma, st louis, missouri) or antigen-specific peptide mixture at 37 ℃ in 5% CO2 for overnight. The next day, the mouse IFN γ detection antibody (R)&System D, minneapolis, mn) was added to the plates, which were then incubated overnight at 4 ℃. The next day, streptavidin-ALP (monoclonal antibody technology Inc. (MabTech), Sweden) was added to the plates for 2 hrsAntigen-specific spots were visualized using BCIP/NPT substrates (monoclonal antibody technologies, Sweden). PSA and PSMA peptides are full-length 15-mer peptides covering a consensus immunogen, do not include the HA tag or leader sequence, overlap by 11 amino acids, and are synthesized by GenScript (pistatavir, new jersey). For each peptide, PSA and PSMA peptides were used at a final concentration of 1.0. mu.g/mL. IFN γ ELISpot was used to assess antigen-specific cellular responses 1 week after the last 1 immunization. For PSA, the IFN γ response was similar for the 10 μ g (772.2+/-138.2SFU) and 20 μ g (771.1+/-155.2SFU) vaccine doses (FIG. 2A). In contrast, for 20 μ g of vaccine (1585.0+/-194.0SFU), there was a dose-dependent increase in PSMA-specific FN γ response compared to 10 μ g of vaccine (1047.2+/-160.7SFU) (FIG. 2B). Minimal background was observed for PSA or PSMA responses in naive mice.

Example 10

Vaccine-induced CD4+ and CD8+ T cell production of IFN gamma, IL-2 and TNF alpha

Flow cytometry was used to further characterize the cellular immunogenicity of co-delivery of PSA and PSMA vaccines. Antigen-specific CD4+ and CD8+ T cell production of IFN γ, IL-2 and TNF α of PSA and PSMA components of the total vaccine-specific response and the total vaccine-specific response were determined (n = 5).

Cellular immune responses were measured by intracellular cytokine staining and flow cytometry using the CytoFix/CytoPerm kit according to the manufacturer's instructions (BD bioscience, san diego, ca). Splenocytes harvested from immunized mice were washed with PBS and subsequently resuspended in R10 medium to a final concentration of 107 cells/ml. Cells were seeded in 96-well round bottom plates in a volume of 100. mu.l, and 100. mu.l additional R10 medium (negative control), medium containing antigen-specific peptide mixtures or medium containing myristol-phorbol-acetate (PMA,10ng/ml) and ionomycin (250 ng/ml; positive control) (Sigma, St. Louis, Mo.) was added and the plates incubated at 37 ℃ for 6 hours with 5% CO 2. All stimulation media contained 1 μ g/μ L of each GolgiPlug and GolgiStop (BD biosciences, san diego, ca). At the end of the incubation period, plates were pelleted by centrifugation and washed 2 times with PBS. Cells were then stained with vital violet dye (LIVE/DEAD violet vital dye, nvolterogen, carlsbad, ca) for 30 minutes at 4 ℃. After washing with PBS as above, cells were externally stained with anti-CD4PerCPCy5.5 and anti-CD 8APC at 4 ℃ for 30 min, followed by fixation and permeabilization. anti-CD 3PE-Cy5, anti-IL-2 PE, anti-IFN γ AlexaFluor-700 and anti-TNF α FITC (BD bioscience, san Diego, Calif.) were added and incubated at 4 ℃ for an additional 30 minutes. The cells were washed with PBS for the last time and fixed in 1% PFA.

Co-delivery of PSA and PSMA vaccines induced potent CD4+ secretion of IFN γ, IL-2, and TNF α. The percentage of CD4+ T cells that produced PSA-specific (0.21%) and PSMA-specific (0.24%) IFN γ contributed equally to the overall vaccine-specific CD4+ T cell IFN γ response (0.44%) (fig. 3A). IL-2(1.08%) producing PSMA-specific CD4+ T cells constituted the majority of the total percentage of CD4+ T cells producing vaccine-specific IL-2(1.40%) (FIG. 3B). The percentage of PSA (0.31%) and PSMA (0.29%) induced CD4+ T cell production by TNF α contributed equally to the total vaccine-specific response (0.60%) (fig. 3C). Overall, CD4+ T cell responses were well balanced between PSA and PSMA, except PSMA induced most of the vaccine-specific CD4+ T cell IL-2 production.

The vaccine induced strong antigen specific CD8+ T cell production of IFN γ and IL-2 and to a lesser extent TNF α production. PSA (0.70%) and PSMA (0.67%) induced robust CD8+ T cell IFN γ production. Indeed, vaccine-specific CD8+ T cell secretion of IFN γ constituted 1.37% of the total CD8+ T cell population (fig. 4A). The vaccine also induced a strong CD8+ T cell IL-2 response (1.54%). Similar to the IL-2 response of CD4+ T cells, the percentage of IL-2 secreting PSMA-specific (1.06%) CD8+ T cells was approximately 2-fold more specific for PSA (0.47%) (FIG. 4B). The total percentage of vaccine-specific CD8+ T cell production of TNF α (0.11%) responded to the PSA component of the vaccine (fig. 4C). In conclusion, there is a high percentage of vaccine specific CD8+ T cell production of IFN γ and IL-2. Similar to the CD4+ T cell response, IFN γ production is equally balanced between PSA and PSMA, and the magnitude of the IL-2 PSMA-specific response is greater than that of the PSA-specific response.

Example 11

PSA-specific IgG seroconversion

Antibody responses may play an important role in tumor immunotherapy. Therefore, we subsequently examined this parameter of the immune response against PSA antigen based on the availability of protein targets.

To determine PSA-specific serum antibody titers, 96-well Nunc-Immuno MaxiSorp plates (Nunc, rochester, n.y.) were coated with 1 μ g/well of recombinant PSA protein diluted in PBS (ferjie garde industry, akton, ma) at 4 ℃. Plates were washed with PBS, 0.05% Tween20(PBST), blocked with 10% BSA/PBST at room temperature for 1 hour, and incubated with serial dilutions of serum from immunized or naive animals for 1 hour at room temperature. The plates were then washed 3 times with PBST and goat anti-mouse IgG (santa cruz, ca) was added at a dilution of 1:5,000 in PBST. Bound enzyme was detected by SigmaFAST o-phenylenediamine dihydrochloride (OPD; Sigma-Aldrich, St.Louis, Mo.) and optical density was measured at 450nm on a Biotek (Wilnoulli, Fomont.) reverse reader shown in FIG. 5B. Endpoint titers were determined as previously described (Frey, a. et al 1998). In short, the upper prediction limit is calculated using the student t-distribution. The mathematical formula for determining the upper prediction limit (upper prediction limit) is expressed as the standard deviation multiplied by a factor based on the number of negative controls (n =5) and the confidence level (95%). The endpoint titer is reported as the reciprocal of the final dilution above the upper predicted limit.

In addition to conferring robust cell-mediated immunity, PSA vaccines induce strong antigen-specific humoral responses. Serum isolated from mice was assayed for antibody titer by ELISA 1 week after the last immunization (n = 5). The average PSA-specific antibody endpoint titers for the vaccine induction were 4,427 (range 1581-15,811) (FIG. 5A). The lifetime of such reactions can also be very important.

Example 12

Prostate specific antigen amino acid sequences available in GenBank include the following: gb _ EAW71923.1_ homo sapiens _ klk3_ CRAb; 001639.1_ homo sapiens _ PSA _ isoform 1_ preproprotein; gb _ AAA59995.1_ homo sapiens _ PSA _ precursor; gb _ AAA60193.1_ wisdom _ PSA; gb _ EAW71933.1_ wisdom _ klk3_ CRA _ l; NP _001025218.1_ homo sapiens _ PSA _ isoform 3_ preproprotein; gb _ CAD54617.1_ wisdom _ PSA; gb _ CAD30844.1_ wisdom _ PSA; gb _ AAA59996.1_ homo sapiens _ PSA _ precursor; gb _ AAD14185.1_ homo sapiens _ PSA; Q6DT45.1_ cynomolgus monkey _ KLK 3; NP _001036241.1_ macaque _ PSA _ precursor; AAZ82258.1_ Kiwi _ PSA; AAZ82255.1_ western gorilla (g.gorilla) _ PSA; gi |163838666| ref | NP _001106216.1| plasma kallikrein [ baboon hunt (Papioanubis) ]; gi |73746696| gb | AAZ82261.1| prostate specific antigen [ baboon hunter ]; i |73746692| gb | AAZ82259.1| prostate specific antigen [ cynomolgus ]; gi |73746694| gb | AAZ82260.1| prostate specific antigen [ mustache longshoscu cephus ]; gi |73746682| gb | AAZ82254.1| prostate specific antigen [ bonobo (Pan paniscus) ]; gi |73746680| gb | AAZ82253.1| prostate specific antigen [ chimpanzees (Pan troglodytes) ]; gi |73746686| gb | AAZ82256.1| prostate specific antigen [ chimpanzee (Pongo pygmaeus) ] and 3746688| gb | AAZ82257.1| prostate specific antigen [ chimpanzee gibbon (nomascous gabriella) ].

The PSMA amino acid sequence available in GenBank includes the following sequences: NP _004467.1_ human _ GCPII _ isoform 1; human _ PSMA _ AAC 83972.1; macaque _ GCPII _ isoform 1XP _001096141.2 and macaque _ GCPII _ isoform 2_ XP _ 002799784.1.

Available in GenBank STEAP amino acid sequence includes the following sequence: NP036581.1_ human _ STEAP 1; EAL24167.1_ human _ STEAP 1; XP001103605.1_ rhesus monkey _ STEAP1_ isoform 3; EAW93751.1_ human _ STEAP1_ CRAb; EAW93749.1_ human _ STEAP1_ CRAa; XP001164838.1_ chimpanzee _ STEAP isoform 2; XP002818311.1_ sumida chimpanzee (p.abelii) _ STEAP 1; NP 001162459.1-baboon east african (p.anubis) _ STEAP 1; NP _999470.1_ boar (s.scrofa) _ STEAP1 and NP _081675.2_ mus (m.musculus) _ STEAP 1.

NP _005663.2_ human _ PSCA is an accession number for the amino acid sequence of PSCA available in GenBank.

Claims (19)

1. A nucleic acid molecule comprising a coding sequence encoding one or more proteins selected from the group consisting of:

a) SEQ ID NO. 2, a protein having 98% homology to SEQ ID NO. 2, provided that amino acids 69, 78, 80, 82, 102, 110, 137, 139, 165, 189, 203, 220, 232 and 248 of SEQ ID NO. 2 are conserved, or an immunogenic fragment of SEQ ID NO. 2 comprising amino acids corresponding to at least 256 amino acid residues of SEQ ID NO. 2, provided that amino acids 69, 78, 80, 82, 102, 110, 137, 139, 165, 189, 203, 220, 232 and 248 of SEQ ID NO. 2 are conserved;

b) SEQ ID NO.4, a protein having 98% homology to SEQ ID NO.4, provided that amino acids 21, 86, 127, 129, 154, 156, 182, 195, 206, 218, 220, 237, 249, 255, 265, 271 or 275 of SEQ ID NO.4 are conserved, or an immunogenic fragment of SEQ ID NO.4 comprising amino acids corresponding to at least 274 amino acid residues of SEQ ID NO.4, provided that amino acids 21, 86, 127, 129, 154, 156, 182, 195, 206, 218, 220, 237, 249, 255, 265, 271 or 275 of SEQ ID NO.4 are conserved;

c) SEQ ID NO 6, a protein having 98% homology to SEQ ID NO 6, provided that amino acids 14, 15, 32, 47, 58, 79, 111, 157, 223, 320, 350, 475, 499, 569, 613, 624, 653, 660, 663, 733 and 734 of SEQ ID NO 6 are conserved, or an immunogenic fragment of SEQ ID NO 6 comprising amino acids corresponding to at least 735 amino acid residues of SEQ ID NO 6, provided that amino acids 14, 15, 32, 47, 58, 79, 111, 157, 223, 320, 350, 475, 499, 569, 613, 624, 653, 660, 663, 733 and 734 of SEQ ID NO 6 are conserved;

d) SEQ ID NO 8, a protein having at least 98% homology to SEQ ID NO 8, provided that amino acids 20, 30, 31, 48, 63, 74, 95, 127, 173, 239, 336, 366, 491, 515, 564, 585, 629, 640, 669, 676, 679, 749 and 750 of SEQ ID NO 8 are conserved, or an immunogenic fragment of SEQ ID NO 8 comprising amino acids corresponding to at least 751 amino acid residues of SEQ ID NO 8, provided that amino acids 20, 30, 31, 48, 63, 74, 95, 127, 173, 239, 336, 366, 491, 515, 564, 585, 629, 640, 669, 676, 679, 749 and 750 of SEQ ID NO 8 are conserved;

e) 10, a protein having 98% homology to SEQ ID No. 10, or an immunogenic fragment of SEQ ID No. 10 comprising amino acids corresponding to at least 333 amino acid residues of SEQ ID No. 10;

f) 12, a protein having 98% homology to SEQ ID No. 12, or an immunogenic fragment of SEQ ID No. 12 comprising amino acids corresponding to at least 349 amino acid residues of SEQ ID No. 12; or

g) 14, a protein having 98% homology to SEQ ID No. 14, or an immunogenic fragment of SEQ ID No. 14 comprising amino acids corresponding to at least 129 amino acid residues of SEQ ID No. 14.

2. The nucleic acid molecule of claim 1 encoding one or more nucleic acids selected from the group consisting of: a protein of component a), b), c) or d).

3. The nucleic acid molecule of claim 1 encoding one or more proteins selected from the group consisting of: at least one selected from components a) or b), and at least one selected from components c) or d).

4. The nucleic acid molecule of any one of claims 1-3 encoding one or more nucleic acids selected from the group consisting of: 2,4, 6, 8, 10, 12 or 14.

5. The nucleic acid molecule of any one of claims 1-3 encoding one or more nucleic acids selected from the group consisting of: 2,4, 6 or 8.

6. The nucleic acid molecule of claim comprising one or more sequences selected from the group consisting of:

a) 1 or a coding sequence having 98% homology to SEQ ID NO 1;

b) 3 or a coding sequence having 98% homology to SEQ ID No. 3;

c) nucleotides 1-2250 of SEQ ID NO.5, or a coding sequence having 98% homology to nucleotides 1-2250 of SEQ ID NO. 5;

d) nucleotides 1-2301 of SEQ ID NO.7, or a coding sequence having 98% homology to nucleotides 1-2301 of SEQ ID NO. 7;

e) 9 or a coding sequence having 98% homology to SEQ ID NO 9;

f) 11 or a coding sequence having 98% homology to SEQ ID NO 11; or

g) 13 or a coding sequence having 98% homology to SEQ ID NO 13.

7. The nucleic acid molecule of claim 6, comprising one or more nucleic acids selected from the group consisting of: the nucleotide sequence of component a), b), c) or d).

8. The nucleic acid molecule of claim 6, comprising one or more nucleotide sequences selected from the group consisting of: at least one selected from components a) or b), and at least one selected from components c) or d).

9. The nucleic acid molecule of any one of claims 6-8, comprising one or more nucleic acids selected from the group consisting of: 1, SEQ ID NO; 3, SEQ ID NO; nucleotides 1-2250 of SEQ ID NO. 5; nucleotides 1-2301 of SEQ ID NO. 7; 9, SEQ ID NO; the nucleotide sequence of SEQ ID NO.11 or SEQ ID NO. 13.

10. The nucleic acid molecule of any one of claims 1-9, wherein the nucleic acid molecule is a plasmid.

11. The nucleic acid molecule of any one of claims 1-10, wherein the nucleic acid molecule is an expression vector and the sequence encoding the further protein is operably linked to a regulatory element.

12. A method of treating a subject diagnosed with prostate cancer, the method comprising administering to the subject a nucleic acid molecule of any one of claims 1-11.

13. A protein selected from the group consisting of:

a) 2, a protein having 98% homology to SEQ ID No. 2, provided that amino acids 69, 78, 80, 82, 102, 110, 137, 139, 165, 189, 203, 220, 232 and 248 of SEQ ID No. 2 are conserved; or an immunogenic fragment of SEQ ID NO. 2 comprising amino acids corresponding to at least 256 amino acid residues of SEQ ID NO. 2, provided that amino acids 69, 78, 80, 82, 102, 110, 137, 139, 165, 189, 203, 220, 232 and 248 of SEQ ID NO. 2 are conserved;

b) 4, a protein having 98% homology to SEQ ID No.4, provided that amino acids 21, 86, 127, 129, 154, 156, 182, 195, 206, 218, 220, 237, 249, 255, 265, 271, or 275 of SEQ ID No.4 are conserved; or an immunogenic fragment of SEQ ID NO.4 comprising amino acids corresponding to at least 274 amino acid residues of SEQ ID NO.4, provided that amino acids 21, 86, 127, 129, 154, 156, 182, 195, 206, 218, 220, 237, 249, 255, 265, 271, or 275 of SEQ ID NO.4 are conserved;

c) SEQ ID NO 6, a protein having 98% homology to SEQ ID NO 6, provided that amino acids 14, 15, 32, 47, 58, 79, 111, 157, 223, 320, 350, 475, 499, 569, 613, 624, 653, 660, 663, 733 and 734 of SEQ ID NO 6 are conserved; or an immunogenic fragment of SEQ ID No.6 comprising amino acids corresponding to at least 735 amino acid residues of SEQ ID No.6, provided that amino acids 14, 15, 32, 47, 58, 79, 111, 157, 223, 320, 350, 475, 499, 569, 613, 624, 653, 660, 663, 733 and 734 of SEQ ID No.6 are conserved;

d) SEQ ID NO 8, a protein having at least 98% homology to SEQ ID NO 8, provided that amino acids 20, 30, 31, 48, 63, 74, 95, 127, 173, 239, 336, 366, 491, 515, 564, 585, 629, 640, 669, 676, 679, 749 and 750 of SEQ ID NO 8 are conserved, or an immunogenic fragment of SEQ ID NO 8 comprising amino acids corresponding to at least 751 amino acid residues of SEQ ID NO 8, provided that amino acids 20, 30, 31, 48, 63, 74, 95, 127, 173, 239, 336, 366, 491, 515, 564, 585, 629, 640, 669, 676, 679, 749 and 750 of SEQ ID NO 8 are conserved;

e) 10, a protein having 98% homology to SEQ ID No. 10; or an immunogenic fragment of SEQ ID NO. 10 comprising amino acids corresponding to at least 333 amino acid residues of SEQ ID NO. 10;

f) 12, a protein having 98% homology to SEQ ID No. 12, or an immunogenic fragment of SEQ ID No. 12 comprising amino acids corresponding to at least 349 amino acid residues of SEQ ID No. 12;

g) 14, a protein having 98% homology to SEQ ID No. 14, or an immunogenic fragment of SEQ ID No. 14 comprising amino acids corresponding to at least 129 amino acid residues of SEQ ID No. 14; or

h) A signal peptide linked to amino acids 19-131 of SEQ ID NO. 14, a protein having a signal peptide linked to an amino acid sequence having 98% homology with amino acids 19-131 of SEQ ID NO. 14, or a signal peptide having an immunogenic fragment linked to amino acids 19-131 of SEQ ID NO. 14, a protein comprising at least 110 amino acid residues of SEQ ID NO. 14 and linked to a fragment of the signal peptide.

14. The protein of claim 13, encoding one or more selected from the group consisting of: a protein of component a), b), c) or d).

15. The protein of claim 13, which encodes one or more proteins selected from the group consisting of: at least one selected from components a) or b), and at least one selected from components c) or d).

16. The protein of any one of claims 13-15, which encodes a polypeptide selected from the group consisting of: 2,4, 6, 8, 10, 12 or 14.

17. The protein of any one of claims 13-15, which encodes a polypeptide selected from the group consisting of: 2,4, 6 or 8.

18. A method of treating a subject diagnosed with prostate cancer, comprising delivering to the subject a protein according to any one of claims 13-17.

19. A pharmaceutical composition comprising the nucleic acid molecule of any one of claims 1-11 and a pharmaceutically acceptable excipient.