Consensus prostate antigens, nucleic acid molecules encoding the same, and vaccines and uses comprising the same

Abstract

To provide a consensus prostate antigen, a nucleic acid molecule encoding the same, a vaccine containing the same and use thereof.SOLUTION: Provided herein are consensus amino acid sequences of prostate antigens capable of breaking tolerance in a target species, including PSA, PSMA, STEAP, and PSCA antigens. Also provided are nucleic acid sequences encoding a consensus amino acid sequence of one or more of the prostate antigens PSA, PSMA, STEAP, and PSCA, as well as genetic constructs / vectors and vaccines expressing such sequences. Also provided herein are methods of generating an autoimmune response against prostate cancer cells by administering one or more of the provided vaccines, proteins, and / or nucleic acid sequences.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to nucleic acid sequences encoding consensus prostate proteins and fragments thereof; improved prostate cancer vaccines, improved methods of eliciting an immune response against prostate cancer cells, and improved methods of prophylactically and / or therapeutically immunizing individuals against prostate cancer. [Background technology]

[0002] Prostate cancer is an important therapeutic immune target. The development of immunotherapeutic strategies is complicated by the need to develop immunogens capable of eliciting a strong immune response, preferably including a CTL response.

[0003] The direct administration of nucleic acid molecules as a vaccination against animal and human diseases has been investigated, with much of the effort focused on effective and efficient delivery methods of nucleic acids in order to obtain the required expression of the desired antigen, resulting in an immunogenic response and ultimately the success of this approach.

[0004] DNA vaccines have many conceptual advantages over conventional vaccine delivery methods, such as live attenuated virus or recombinant protein-based vaccines. DNA vaccines are safe, stable, easily manufactured, well tolerated in humans, and show little evidence of plasmid integration in preclinical studies [Non-Patent Document 1; Non-Patent Document 2]. In addition, DNA vaccines are suitable for repeated administration due to the fact that vaccine efficacy is not affected by pre-existing antibody titers against the vector [3]. However, a major obstacle to the clinical adoption of DNA vaccines is the diminished immunogenicity of the platform when transferred to larger animals [4]. Recent advances in DNA vaccine immunogen engineering, such as codon optimization, RNA optimization, and the addition of immunoglobulin leader sequences, have improved the expression and immunogenicity of DNA vaccines [Andre, S., et al., Increased immune response elicited by DNA vaccination with asynthetic gp120 sequence with optimized codon usage. J Virol, 1998. 72(2):pp.1497-503; Deml, L., et al., Multiple effects of codon usage optimization on expression and immunogenicity of DNA candidate vaccines encoding the human immunodeficiency virus type 1 Gag protein. J Virol, 2001. 75(22):pp.10991-1001; Laddy, DJ, et al., Immunogenicity of novel consensus-based DNA vaccines against influenza. Vaccine, 2007]. 25(16):p.2984-9; Frelin, L., et al., Codon optimization and mRNA amplification effectively enhances the immunogenicity of the hepatitis C virus nonstructural3 / 4Agene. GeneTher, 2004. 11(6): p.522-33].

[0005] Recent technological advances in plasmid delivery systems (including techniques such as electroporation) have improved the expression and immunogenicity of DNA vaccines [Hirao, LA, et al., Intradermal / subcutaneous immunization by electroporation improves plasmid vaccine delivery and potency in pigs and rhesus macaques. Vaccine, 2008.26(3): pp.440-8; Luckay, A., et al., Effect of plasmid DNA vaccine design and in vivo electroporation on the resulting vaccine-specific immune responses in rhesus macaques. J Virol, 2007.81(10): pp.5257-69; Ahlen, G., et al., In vivo electroporation enhances the immunogenicity of hepatitis C virus nonstructural 3 / 4A DNA by increased local DNA uptake and protein expression,inflammation, andinfiltration of CD3+ T cells. JImmunol,2007.179(7): p.4741-53].

[0006] In addition, previous studies have suggested that the use of consensus immunogens can broaden the cellular immune response compared with single native antigens [Yan, J., et al., Enhanced cellular immune responses elicited by an engineered HIV-1 subtype B consensus-based envelope DNA vaccine. Mol Ther, 2007. 15(2): pp. 411-21; Rolland, M., et al., Reconstruction and function of ancestral center-of-tree human immunodeficiency virus type 1 proteins. J Virol, 2007. 81(16): pp. 8507-14]. However, immune tolerance to cancer antigens remains a challenge. The destruction of the immune system and the resulting autoimmunity are barriers to cancer vaccines.

[0007] There remains a need for nucleic acid constructs encoding prostate cancer antigens and for compositions useful in eliciting an immune response against prostate cancer, thereby breaking immune tolerance. There remains a need for economical and effective prophylactic and therapeutic vaccines against prostate cancer. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Martin, T., et al., Plasmid DNA malariavaccine: the potentialforgenomic integration after intramuscular injection. HumGeneTher, (1999)10(5):p.759~68 [Non-patent document 2] Nichols,WW, et al., Potential DNA vaccine integration into host cellgenome.Ann N YAcad Sci,(1995)772:p.30~9 [Non-patent document 3] Chattergoon, M., J. Boyer, and DB Weiner, Geneticimmunization: anewera in vaccines and immune therapeutics. FASEB J(1997)11(10):p.753~63 [Non-patent document 4] Liu, MA and JB Ulmer, Human clinical trials of plasmidDNA vaccines.AdvGenet(2005)55:p.25~40 Summary of the Invention [Means for solving the problem]

[0009] Some embodiments of the present invention include nucleic acid molecules comprising a coding sequence that encodes one or more proteins selected from the group consisting of: a) SEQ ID NO:2; a protein that is 98% homologous 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 a protein that is 98% homologous 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. a) 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: a) SEQ ID NO: 4; a protein 98% homologous 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 c) 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 1, 2, and 275 of SEQ ID NO: 6 are conserved; c) SEQ ID NO: 6; a protein 98% homologous 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, with the proviso that 0, 350, 475, 499, 569, 613, 624, 653, 660, 663, 733, and 734 are conserved; d) SEQ ID NO: 8; a protein that is 98% homologous to SEQ ID NO: 8, with the proviso 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) SEQ ID NO:10; a protein 98% homologous 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) SEQ ID NO:12; a protein 98% homologous 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 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 98% identical to amino acids 19-131 of SEQ ID NO: 14; or a protein having a signal peptide linked to an immunogenic fragment of amino acids 19-131 of SEQ ID NO: 14, wherein the fragment comprises at least 110 amino acid residues of SEQ ID NO: 14 and is linked to a signal peptide. In some embodiments, the nucleic acid is selected from those encoding proteins a), b), c), or d);

[0010] In another aspect, the invention includes a method of treating an individual diagnosed with prostate cancer, comprising administering to the individual a nucleic acid molecule described herein.

[0011] In another embodiment, there is provided a protein selected from the group consisting of: a) SEQ ID NO:2; a protein that is 98% homologous 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 a protein that is at least 261% homologous 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. b) an immunogenic fragment of SEQ ID NO: 2 comprising amino acids corresponding to amino acid residues 21, 86, 127, 129, 154, 156, 182, 195, 206, 218, 220, 237, 249, 255, 265, 271, and 275 of SEQ ID NO: 4; a protein 98% homologous 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 c) 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 SEQ ID NO: 6; a protein 98% homologous 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, with the proviso that 75, 499, 569, 613, 624, 653, 660, 663, 733, and 734 are conserved; d) SEQ ID NO: 8; a protein that is 98% homologous to SEQ ID NO: 8, with the proviso 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) SEQ ID NO: 10; a protein 98% homologous 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) SEQ ID NO: 12; a protein 98% homologous 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 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 98% identical to amino acids 19-131 of SEQ ID NO: 14; or a protein having a signal peptide linked to an immunogenic fragment of amino acids 19-131 of SEQ ID NO: 14, wherein the fragment comprises at least 110 amino acid residues of SEQ ID NO: 14 and is linked to a signal peptide. In some embodiments, the protein is selected from the group comprising proteins a), b), c), or d);

[0012] Some aspects of the present invention include methods of treating an individual diagnosed with prostate cancer, the methods comprising delivering to the individual a protein described herein.

[0013] Another aspect of the invention is a pharmaceutical composition comprising a nucleic acid molecule provided herein and a pharmaceutically acceptable excipient. For example, the present invention provides the following: (Item 1) A nucleic acid molecule comprising a coding sequence encoding one or more proteins selected from the group comprising: a) SEQ ID NO: 2, a protein that is 98% homologous 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 that comprises 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 98% homologous 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 that is 98% homologous 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 that is 98% homologous to SEQ ID NO: 8, provided that amino acids 21, 31, 32, 49, 64, 75, 96, 128, 174, 240, 337, 367, 492, 516, 565, 586, 630, 641, 670, 677, 680, 750, and 751 of SEQ ID NO: 8 are conserved, or an immunogenic fragment of SEQ ID NO: 8 comprising amino acids corresponding to at least 752 amino acid residues of SEQ ID NO: 8, provided that amino acids 21, 31, 32, 49, 64, 75, 96, 128, 174, 240, 337, 367, 492, 516, 565, 586, 630, 641, 670, 677, 680, 750, and 751 of SEQ ID NO: 8 are conserved; e) SEQ ID NO: 10, a protein 98% homologous 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) SEQ ID NO: 12, a protein 98% homologous 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) SEQ ID NO: 14, a protein that is 98% homologous to SEQ ID NO: 14, or an immunogenic fragment of SEQ ID NO: 14 that contains amino acids corresponding to at least 129 amino acid residues of SEQ ID NO: 14. (Item 2) 2. The nucleic acid molecule according to item 1, encoding one or more proteins selected from the group comprising elements a), b), c), or d). (Item 3) 2. The nucleic acid molecule according to item 1, encoding one or more proteins selected from the group comprising at least one selected from element a) or b) and at least one selected from element c) or d). (Item 4) 4. The nucleic acid molecule according to any one of items 1 to 3, encoding one or more proteins selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14. (Item 5) 4. The nucleic acid molecule according to any one of items 1 to 3, encoding one or more proteins selected from the group comprising SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8. (Item 6) The nucleic acid molecule according to the item, comprising one or more sequences selected from the group comprising: a) SEQ ID NO: 1 or a coding sequence 98% homologous to SEQ ID NO: 1; b) SEQ ID NO: 3 or a coding sequence 98% homologous to SEQ ID NO: 3; c) nucleotides 1 to 2250 of SEQ ID NO:5, or a coding sequence that is 98% homologous to nucleotides 1 to 2250 of SEQ ID NO:5; d) nucleotides 1 to 2301 of SEQ ID NO:7, or a coding sequence that is 98% homologous to nucleotides 1 to 2301 of SEQ ID NO:7; e) SEQ ID NO: 9 or a coding sequence 98% identical to SEQ ID NO: 9; f) SEQ ID NO: 11, or a coding sequence 98% identical to SEQ ID NO: 11; or g) SEQ ID NO: 13 or a coding sequence that is 98% homologous to SEQ ID NO: 13. (Item 7) 7. The nucleic acid molecule according to item 6, comprising one or more nucleotide sequences selected from the group comprising elements a), b), c), or d). (Item 8) 7. The nucleic acid molecule according to item 6, comprising one or more nucleotide sequences selected from the group comprising at least one selected from element a) or b), and at least one selected from element c) or d). (Item 9) 9. The nucleic acid molecule according to any one of Items 6 to 8, comprising one or more nucleotide sequences selected from the group comprising SEQ ID NO: 1, SEQ ID NO: 3, nucleotides 1 to 2250 of SEQ ID NO: 5, nucleotides 1 to 2301 of SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13. (Item 10) 10. The nucleic acid molecule according to any one of items 1 to 9, wherein the nucleic acid molecule is a plasmid. (Item 11) 11. The nucleic acid molecule according to any one of items 1 to 10, wherein the nucleic acid molecule is an expression vector and the sequence encoding the one or more proteins is operably linked to a regulatory element. (Item 12) 12. A method for treating an individual diagnosed with prostate cancer, the method comprising administering to the individual a nucleic acid molecule according to any one of items 1 to 11. (Item 13) A protein selected from the group consisting of: a) SEQ ID NO: 2, a protein that is 98% homologous 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 that comprises 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 98% homologous 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 that is 98% homologous 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 that is 98% homologous to SEQ ID NO: 8, provided that amino acids 21, 31, 32, 49, 64, 75, 96, 128, 174, 240, 337, 367, 492, 516, 565, 586, 630, 641, 670, 677, 680, 750, and 751 of SEQ ID NO: 8 are conserved, or an immunogenic fragment of SEQ ID NO: 8 comprising amino acids corresponding to at least 752 amino acid residues of SEQ ID NO: 8, provided that amino acids 21, 31, 32, 49, 64, 75, 96, 128, 174, 240, 337, 367, 492, 516, 565, 586, 630, 641, 670, 677, 680, 750, and 751 of SEQ ID NO: 8 are conserved; e) SEQ ID NO: 10, a protein 98% homologous 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) SEQ ID NO: 12, a protein 98% homologous 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) SEQ ID NO: 14, a protein 98% homologous 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 protein having a signal peptide linked to amino acids 19 to 131 of SEQ ID NO: 14, or a protein having a signal peptide linked to an amino acid sequence that is 98% homologous to amino acids 19 to 131 of SEQ ID NO: 14, or a protein having a signal peptide linked to an immunogenic fragment of amino acids 19 to 131 of SEQ ID NO: 14, wherein the fragment comprises at least 110 amino acid residues of SEQ ID NO: 14 and is linked to a signal peptide. (Item 14) 14. The protein according to item 13, encoding one or more proteins selected from the group comprising elements a), b), c), or d). (Item 15) 14. The protein according to item 13, encoding one or more proteins selected from the group comprising at least one selected from element a) or b) and at least one selected from element c) or d). (Item 16) 16. The protein according to any one of items 13 to 15, encoding a protein selected from the group comprising SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14. (Item 17) 16. The protein according to any one of items 13 to 15, encoding a protein selected from the group comprising SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8. (Item 18) 18. A method for treating an individual diagnosed with prostate cancer, comprising delivering to said individual a protein according to any one of items 13 to 17. (Item 19) 12. A pharmaceutical composition comprising the nucleic acid molecule according to any one of items 1 to 11 and a pharmaceutically acceptable excipient. [Brief explanation of the drawings]

[0014] [Figure 1] 1 shows the results of in vitro translation performed to confirm the expression of PSA antigen and PSMA antigen. [Figure 2A]Cellular immunogenicity data are shown. Cellular immunogenicity of PSA antigen was measured by interferon-γ ELISpot. [Figure 2B] Cellular immunogenicity data are shown. Cellular immunogenicity of PSA antigen was measured by interferon-γ ELISpot. [Figure 3] CD4+ T cell responses characterized by flow cytometry are displayed by graphs showing PSA-specific (left panel), PSMA-specific (middle panel), and total vaccine-specific (right panel) cytokine production: % IFNγ-producing CD4+ T cells (Figure 3A), % IL-2-producing CD4+ T cells (Figure 3B), and % TNFα-producing CD4+ T cells (Figure 3C). [Figure 4] CD8+ T cell responses characterized by flow cytometry are displayed by graphs showing PSA-specific (left panel), PSMA-specific (middle panel), and total vaccine-specific (right panel) cytokine production: % IFNγ-producing CD8+ T cells (Figure 4A), % IL-2-producing CD8+ T cells (Figure 4B), and % TNFα-producing CD8+ T cells (Figure 4C). [Figure 5] ELISA data for PSA-specific antibodies one week after the final immunization are shown. (FIG. 5A) PSA IgG endpoint titers. (FIG. 5B) Representative IgG titer curves. DETAILED DESCRIPTION OF THE INVENTION

[0015] Detailed Description of the Invention Provided herein are consensus sequence prostate proteins and isolated nucleic acid molecules encoding same, in particular the prostate antigens prostate-specific antigen (PSA), prostate-specific membrane antigen (PSMA), prostate antigen six-transmembrane epithelial antigen (STEAP), and prostate-specific stem cell antigen (PSCA).

[0016] The prostate cancer antigens described herein are consensus sequences derived from a pool of homologous antigens from multiple species, including the target species of the vaccine. The species selected for the antigen sequence comparison to form the consensus are selected based on the proximity of the species on a phylogenetic tree, such as H. sapiens (human), M. mulatta (rhesus macaque), and M. fascicularis (cynomolgus macaque). Consensus antigens are not identical to native prostate antigens, but they share close sequence identity, with sequences sharing at least 85%, preferably 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. These consensus cancer antigens described herein can break tolerance (or induce autoimmunity) in the target species, generating an effective immune response against the prostate cancer antigen. Methods for generating DNA vaccines based on consensus cancer antigens are provided herein.

[0017] Some embodiments of the present invention include nucleic acid molecules comprising a coding sequence that encodes one or more proteins selected from the group consisting of: a) SEQ ID NO:2; a protein that is 98% homologous 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 a protein that is 98% homologous 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. a) 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: a) SEQ ID NO: 4; a protein 98% homologous 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 c) 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 1, 2, and 275 of SEQ ID NO: 6 are conserved; c) SEQ ID NO: 6; a protein 98% homologous 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, with the proviso that 0, 350, 475, 499, 569, 613, 624, 653, 660, 663, 733, and 734 are conserved; d) SEQ ID NO: 8; a protein that is 98% homologous to SEQ ID NO: 8, with the proviso 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) SEQ ID NO:10; a protein 98% identical 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) SEQ ID NO:12; a protein 98% identical 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) SEQ ID NO: 14; a protein 98% homologous to SEQ ID NO: 14; 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 to 131 of SEQ ID NO: 14; a protein having a signal peptide linked to an amino acid sequence 98% homologous to amino acids 19 to 131 of SEQ ID NO: 14;Alternatively, a protein having a signal peptide linked to an immunogenic fragment of amino acids 19-131 of SEQ ID NO: 14, the fragment comprising at least 110 amino acid residues of SEQ ID NO: 14 and linked to 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): STEAP consensus antigen sequence 1 (SEQ ID NO: 10) and STEAP consensus antigen sequence 2 (SEQ ID NO: 12). One consensus protein sequence for PSCA is disclosed: PSCA consensus antigen sequence (SEQ ID NO: 14). SEQ ID NO: 14 includes an IgE signal peptide. In some embodiments, the PSCA consensus antigen can 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 those encoding proteins a), b), c), or d) above. In other embodiments, the nucleic acid molecule is a nucleic acid molecule encoding one or more proteins selected from the group including at least one selected from those encoding proteins a) or b) and at least one selected from those encoding proteins c) or d).

[0018] The nucleic acid molecule may further be a molecule encoding one or more proteins selected from the group comprising SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, or SEQ ID NO:14, preferably the group comprising SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:8. In some embodiments, the nucleic acid molecule may be a nucleic acid molecule encoding one or more proteins selected from the group comprising 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.

[0019] In another embodiment, there is provided a protein selected from the group consisting of: a) SEQ ID NO:2; a protein that is 98% homologous 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 a protein that is at least 256 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) an immunogenic fragment of SEQ ID NO: 2 comprising amino acids corresponding to amino acid residues 21, 86, 127, 129, 154, 156, 182, 195, 206, 218, 220, 237, 249, 255, 265, 271, and 275 of SEQ ID NO: 4; a protein 98% homologous 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 c) 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 SEQ ID NO: 6; a protein 98% homologous 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, with the proviso that 75, 499, 569, 613, 624, 653, 660, 663, 733, and 734 are conserved; d) SEQ ID NO: 8; a protein that is 98% homologous to SEQ ID NO: 8, with the proviso 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) SEQ ID NO:10; a protein 98% homologous 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) SEQ ID NO:12; a protein 98% homologous 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) an immunogenic fragment of SEQ ID NO: 12 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 98% identical to amino acids 19-131 of SEQ ID NO: 14; or a protein having a signal peptide linked to an immunogenic fragment of amino acids 19-131 of SEQ ID NO: 14, wherein the fragment comprises at least 110 amino acid residues of SEQ ID NO: 14 and is linked to a signal peptide. In some embodiments, the protein is selected from the group comprising proteins a), b), c), or d). In other embodiments, the protein is a protein encoding one or more proteins selected from the group comprising at least one selected from proteins a) or b) and at least one selected from proteins c) or d).

[0020] The protein may further be a protein selected from the group comprising SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14, preferably the group comprising SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8. In some embodiments, the protein may be a protein selected from the group comprising 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.

[0021] Nucleic acid coding sequences were generated to improve and optimize expression. The codons used in these nucleic acid molecules were selected to generate RNA with reduced secondary structure formation due to intramolecular hybridization. The nucleic acid sequence encoding PSA consensus antigen sequence 1 (SEQ ID NO: 1) and the nucleic acid sequence encoding PSA consensus antigen sequence 2 (SEQ ID NO: 3) are disclosed. Similarly, the nucleic acid coding sequence for PSMA consensus antigen sequence 1 (SEQ ID NO: 5, or nucleotides 1-2250 of SEQ ID NO: 5) and the nucleic acid coding sequence for PSMA consensus antigen sequence 2 (SEQ ID NO: 7, or nucleotides 1-2301 of SEQ ID NO: 7) are provided, as well as the nucleic acid coding sequence for STEAP consensus antigen sequence 1 (SEQ ID NO: 9), STEAP consensus antigen sequence 2 (SEQ ID NO: 11), and the nucleic acid coding sequence for PSCA consensus antigen sequence (SEQ ID NO: 13). Also provided are nucleic acid sequences that are 98% homologous to SEQ ID NO:1, which encode PSA consensus antigen sequence 1 (SEQ ID NO:2) or a protein that is up to 98% homologous to SEQ ID NO:2, preferably a protein that includes 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 that are 98% homologous to SEQ ID NO:3, which encode PSA consensus antigen sequence 2 (SEQ ID NO:4) or a protein that is up to 98% homologous to SEQ ID NO:4, preferably a protein that includes 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.Similarly, a nucleic acid sequence that is 98% homologous to nucleotide 2250 of SEQ ID NO:5 and encodes PSMA consensus antigen sequence 1 (SEQ ID NO:6) or a protein that is at most 98% homologous to SEQ ID NO:6, preferably a protein 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 of SEQ ID NO:6; or a nucleic acid sequence that is 98% homologous to nucleotide 2301 of SEQ ID NO:7 and encodes PSMA consensus antigen sequence 1 (SEQ ID NO:8) or a protein that is at most 98% homologous to SEQ ID NO:8, preferably amino acids 20, 30, 31, 48, 63, 74, 95, 127, 173, 239, 336, 366 of SEQ ID NO:8. , 491, 515, 564, 585, 629, 640, 669, 676, 679, 749, and 750, as well as nucleotides 98% homologous to SEQ ID NO:9 that encode STEAP consensus antigen sequence 1 (SEQ ID NO:10) or a protein up to 98% homologous to SEQ ID NO:10, nucleotides 98% homologous to SEQ ID NO:11 that encode STEAP consensus antigen sequence 2 (SEQ ID NO:12) or a protein up to 98% homologous to SEQ ID NO:12, and nucleotides 98% homologous to SEQ ID NO:13 that encode a protein up to 98% homologous to the PSCA consensus antigen sequence (SEQ ID NO:14) or 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 that encodes SEQ ID NO:4; nucleotides 1-2301 of SEQ ID NO:7 that encode SEQ ID NO:8; SEQ ID NO:11 that encodes SEQ ID NO:12; and SEQ ID NO:13 that encodes SEQ ID NO:14).

[0022] Compositions containing nucleic acid molecules comprising the coding sequences of the isolated nucleic acid molecules provided herein can be useful in inducing an immune response against prostate proteins when administered to animals. Compositions containing one or more of these nucleic acid sequences can be used as vaccines or vaccine components to prevent or treat prostate cancer. Similarly, compositions containing consensus proteins can be useful in inducing an immune response against prostate proteins when administered to animals. Combinations of compositions containing nucleic acid molecules comprising the coding sequences of the isolated nucleic acid molecules provided herein can be useful in inducing an immune response against prostate proteins, and can be used collectively as vaccines or vaccine components to prevent or treat prostate cancer. Similarly, compositions containing consensus proteins can be useful in inducing an immune response against prostate proteins when administered to animals. Compositions containing one or more of these consensus proteins can be used as vaccines or vaccine components to prevent or treat prostate cancer.

[0023] Vaccines are provided that contain the nucleic acid sequences provided herein. In some embodiments, vaccines are provided that contain a nucleic acid sequence encoding one or more consensus protein 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 eliciting an immune response using a nucleic acid sequence 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.

[0024] Vaccines are provided that include 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. Also provided are methods of eliciting 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.

[0025] Methods for protecting an individual from prostate cancer or treating an individual with confirmed prostate cancer are provided. The methods include administering to the individual an effective amount of one or more nucleic acid molecules comprising one or more nucleic acid sequences provided herein. In some methods, delivery of the nucleic acid molecules is facilitated by electroporation of the target tissue or tissue that will receive the nucleic acid molecules. The nucleic acid sequences are expressed in the individual's cells, eliciting an immune response against the prostate protein encoded by the nucleic acid sequences.

[0026] 1.Definition The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0027] When numerical ranges are recited herein, each intervening number is expressly contemplated to the same degree of precision. For example, the range 6 to 9 contemplates the numbers 7 and 8 in addition to 6 and 9, and the range 6.0 to 7.0 expressly contemplates 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.

[0028] a. adjuvant As used herein, the term "adjuvant" refers to any molecule added to the DNA plasmid vaccines described herein for the purpose of enhancing the immunogenicity of the antibodies encoded by the DNA plasmid and encoding nucleic acid sequences described below.

[0029] b.Antibodies As used herein, the term "antibody" refers to antibodies of the classes IgG, IgM, IgA, IgD, or IgE, or fragments or derivatives thereof (including Fab, F(ab')2, and Fd), as well as single-chain, bispecific, diabody, bifunctional, and derivatives thereof. The antibody may be an antibody isolated from mammalian serum, a polyclonal antibody, an affinity-purified antibody, or a mixture thereof, which exhibits sufficient binding specificity for the desired epitope or a sequence derived therefrom.

[0030] c. coding sequence As used herein, the term "coding sequence" or "encoding nucleic acid" refers to a nucleic acid (RNA or DNA molecule) comprising a nucleotide sequence that encodes a protein. The coding sequence can further comprise initiation and termination signals operably linked to regulatory elements comprising a promoter and polyadenylation signal capable of directing expression in cells of an individual or mammal to which the nucleic acid is administered.

[0031] D. complement As used herein, the terms "complement" or "complementary" mean that a nucleic acid may exhibit Watson-Crick (e.g., AT / U and CG) or Hoogsteen base pairing between nucleotides or nucleotide analogs of a nucleic acid molecule.

[0032] e. consensus or consensus sequence As used herein, the term "consensus" or "consensus sequence" refers to a polypeptide sequence based on a comparative sequence analysis of multiple subtypes of a particular prostate antigen. Nucleic acid sequences encoding the consensus polypeptide sequence can be prepared. Vaccines containing proteins containing consensus sequences and / or nucleic acid molecules encoding such proteins can be used to induce broad immunity against specific prostate antigens.

[0033] f. Electroporation The terms "electroporation," "electropermeabilization," and "electrokinetic enhancement" ("EP"), used interchangeably herein, refer to the use of transmembrane electric field pulses to generate microscopic pathways (pores) in biological membranes. The presence of these pathways allows the passage of plasmids, oligonucleotides, siRNA, drugs, ions, and water from one side of the cell membrane to the other.

[0034] g. Fragment The term "fragment," as used herein with respect to a nucleic acid sequence, refers to a nucleic acid sequence encoding a polypeptide capable of eliciting an immune response in a mammal that cross-reacts with a full-length prostate antigen, or a fragment thereof. The fragment may be a DNA fragment selected from at least one of various nucleotide sequences encoding a consensus amino acid sequence and constructs containing the sequence. The DNA fragment may include a coding sequence for an immunoglobulin leader, such as an IgE sequence or an IgG sequence. The DNA fragment may encode a protein fragment as described below.

[0035] The term "fragment" when used in reference to a polypeptide sequence means 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).

[0036] The human PSA sequence is approximately 261 amino acids. A fragment of PSA consensus antigen 1 may contain at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably 98% or 99%, of SEQ ID NO:2, provided that it contains one or more of amino acids 69, 78, 80, 82, 102, 110, 137, 139, 165, 189, 203, 220, 232, and 248. A fragment of PSA consensus antigen 1 may contain 255, 256, 257, 258, 259, or 260 amino acids of SEQ ID NO:2, but preferably contains 256 or more amino acids. Fragments 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, provided that they contain 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 optionally exclude amino acids 1-17. In some embodiments, a fragment of PSA consensus antigen 2 may optionally include one or more of amino acids 1 to 17, and amino acids 18 to 278, and may include 255, 256, 257, 258, 259, or 260 amino acids of SEQ ID NO:4, but preferably includes 274 or more amino acids.

[0037] The human PSMA sequence is approximately 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, provided that it contains 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. A fragment of PSMA consensus antigen 1 may comprise 745, 746, 747, 748, or 749 amino acids of SEQ ID NO: 6, but preferably contains 735 or more amino acids. 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 it contains 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 optionally exclude amino acids 1-16. In some embodiments, a fragment of PSA consensus antigen 2 may optionally include one or more of amino acids 1-16, and amino acids 17-766, and a fragment of PSMA consensus antigen 2 may include 760, 761, 762, 763, 764, or 765 amino acids of SEQ ID NO:8, but preferably includes 751 or more amino acids.

[0038] The human STEAP sequence is approximately 339 amino acids. The consensus STEAP sequence can include the amino acid sequence of an immunoglobulin leader, such as IgE or IgG. Consensus STEAP antigen 2 contains an 18-amino acid leader sequence in place of the methionine at position 1. A fragment of PSMA consensus antigen 2 can include the leader sequence and can comprise 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 can include amino acids 1-350, 1-351, 1-352, 1-353, 1-354, or 1-355 of SEQ ID NO: 12.

[0039] The human PSCA sequence is approximately 114 amino acids. The consensus STEAP sequence can include the amino acid sequence of an immunoglobulin leader, such as IgE or IgG. The consensus PSCA antigen contains an 18-amino acid leader sequence in place of the methionine at position 1. Fragments of the PSCA consensus antigen can include the leader sequence and can contain 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. Fragments of the PSMA consensus antigen 1 can include amino acids 1-125, 1-126, 1-127, 1-128, 1-129, or 1-130 of SEQ ID NO: 14.

[0040] h. Gene constructs As used herein, the term "genetic construct" refers to a DNA or RNA molecule comprising a nucleotide sequence encoding a protein. The coding sequence comprises a start and stop signal operably linked to regulatory elements, including a promoter and polyadenylation signal, capable of directing expression in the cells of an individual or mammal to which the nucleic acid is administered. As used herein, the term "expressible form" refers to a genetic construct comprising the necessary regulatory elements operably linked to a coding sequence encoding a protein such that the coding sequence is expressed when present in the cells of an individual.

[0041] i.Homology Multiple sequence alignments and homology phylogenetic trees were generated using ClustalW, a general-purpose multiple sequence alignment program for DNA or proteins.

[0042] j. Same As used herein, the terms "identical" or "identity" in the context of two or more nucleic acid or polypeptide sequences means that the sequences share a specified percentage of identical residues over a specified region. To calculate this percentage, optimally align the two sequences, compare the two sequences over a specified region, determine the number of positions where identical residues occur in both sequences to obtain the number of matching positions, divide the number of matching positions by the total number of positions in the specified region, and multiply the result by 100 to obtain the percent sequence identity. If the two sequences are different in length or the alignment results in one or more cohesive ends, and only a single sequence is included in the specified comparison region, the residues of the single sequence are included in the denominator but not the numerator. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. Identity calculations can be performed manually or using computer sequence algorithms such as BLAST and BLAST 2.0.

[0043] K. immune response As used herein, the term "immune response" refers to activation of a host's immune system, e.g., a mammal's immune system, in response to the introduction of an antibody, such as a prostate consensus antibody. This immune response can be cellular or humoral in form, or both.

[0044] l.Nucleic acid As used herein, the term "nucleic acid" or "oligonucleotide" or "polynucleotide" refers to at least two nucleotides covalently linked to each other. By describing a single strand, the sequence of the complementary strand is also defined. Thus, a nucleic acid also encompasses the complementary strand of the described single strand. Many variants of a nucleic acid can be used for the same purpose as a given nucleic acid. Thus, a nucleic acid also encompasses substantially identical nucleic acids and their complements. A single strand provides a probe that can hybridize to a target sequence under stringent hybridization conditions. Thus, a nucleic acid also encompasses a probe that hybridizes under stringent hybridization conditions.

[0045] Nucleic acids can be single-stranded or double-stranded, or can contain portions of both double-stranded and single-stranded sequences. Nucleic acids can be genomic DNA, cDNA, RNA, or hybrids, and can contain combinations of deoxyribonucleotides and ribonucleotides, including combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine. Nucleic acids can be obtained by chemical synthesis or recombinant methods.

[0046] m. Functionally linked As used herein, the term "operably linked" means that expression of a gene is under the control of a promoter that is spatially connected to the gene. The promoter can be located 5' (upstream) or 3' (downstream) of the gene under its control. The distance between the promoter and the gene can be approximately 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, changes in this distance can be accommodated without loss of promoter function.

[0047] n.Promoter As used herein, the term "promoter" refers to a synthetic or naturally occurring molecule capable of conferring, activating, or enhancing intracellular expression of a nucleic acid. A promoter can contain one or more specific transcriptional regulatory sequences to further enhance expression and / or modify its spatial and / or temporal expression. A promoter can also contain distal enhancer or repression elements, which can be located as far as thousands of base pairs from the transcription start site. Promoters can be derived from sources including viruses, bacteria, fungi, plants, insects, and animals. A promoter can regulate the expression of genetic components constitutively or differentially with respect to the cell, tissue, or organ in which expression occurs, or with respect to the developmental stage in which expression occurs, or can regulate the expression of genetic components in response to external stimuli, such as physiological stress, pathogens, metal ions, inducers, etc. Representative examples of promoters include the bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operator promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMVIE promoter, SV40 early promoter, SV40 late promoter, and CMVIE promoter.

[0048] o. Stringent hybridization conditions As used herein, the term "stringent hybridization conditions" refers to the 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 therefore will vary from situation to situation. Stringent conditions are those that meet the melting point (T) for a particular sequence at a defined ionic strength pH. m ) can be selected to be about 5 to 10°C lower than T m is the temperature (under defined ionic strength, pH, and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (because the target sequence is present in excess, T m(In this case, 50% of the probes are occupied at equilibrium.) Stringent conditions include a salt concentration of less than about 1.0 M sodium ion, e.g., about 0.01 to 1.0 M sodium ion (or other salt), at pH 7.0 to 8.3, and a temperature of at least about 30°C for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C for long probes (e.g., greater than about 50 nucleotides). Stringent conditions can also be achieved by adding 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, 5x SSC, and 1% SDS, incubation at 42°C; or 5x SSC, 1% SDS, incubation at 65°C, followed by a wash in 0.2x SSC, 0.1% SDS at 65°C.

[0049] p. Substantially complementary As used herein, the term "substantially complementary" refers to any of the following: 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, 85, 90, 95, 100, 180, 270, 360, 450, 540, 630, 720, 810, 900, 990, 1080, 1170, 1260, 1350, 1440, 1530, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5100, 5200, 5300, 5400, 5500, 5600, 5700, 5800, 5900, 6000, 6100, 620 It means that a first sequence is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the complement of a second sequence over a region of 0, 1620, 1710, 1800, 1890, 1980, 2070 or more nucleotides or amino acids, or that the two sequences hybridize under stringent hybridization conditions.

[0050] q. Substantially identical As used herein, the term "substantially identical" means any of the following: 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, 85, 90, 95, 100, 180, 270, 360, 450, 540, 630, 720, 810, 900, 990, 1080, 1170, 1260, 1350, 144 It means that a first sequence and a second sequence are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical over a region of 0, 1530, 1620, 1710, 1800, 1890, 1980, 2070 or more nucleotides or amino acids, or, with respect to nucleic acids, where a first sequence is substantially complementary to the complement of a second sequence.

[0051] r. subtype or serotype The terms "subtype" or "serotype," as used interchangeably herein with respect to prostate cancer antigens, refer to genetic variants of prostate cancer antigens, with one subtype (or variant) being recognized by the immune system as distinct from another subtype.

[0052] s. mutant The term "variant," as used herein with respect to nucleic acids, means (i) a portion or fragment of a reference nucleotide sequence; (ii) the complement of a reference nucleotide sequence or a portion thereof; (iii) a nucleic acid that is substantially identical to a reference nucleic acid or its complement; or (iv) a nucleic acid that hybridizes under stringent conditions to a reference nucleic acid, its complement, or a substantially identical sequence thereof.

[0053] The term "variant" as used with respect to a peptide or polypeptide refers to a protein that differs in amino acid sequence by amino acid insertion, deletion, or conservative substitution, but retains at least one biological activity. A variant can also refer to a protein having an amino acid sequence substantially identical to that of a reference protein, and that retains at least one biological activity. Conservative amino acid substitutions, i.e., the replacement of one amino acid with another amino acid of similar properties (e.g., hydrophilicity, degree, and distribution of charged regions), are recognized in the art as typically resulting in minor changes. Such minor changes can be identified, in part, by examining the hydropathic index, as understood in the art. Kyte et al., J. Mol. Biol. 157:105-132 (1982). Hydropathicity of Amino Acids The hydrophilicity index is based on a consideration of its hydrophobicity and charge. It is known in the art that amino acids with similar hydrophilicity indices can be substituted and still maintain protein function. In one embodiment, amino acids with hydrophilicity indices within ±2 are substituted. The hydrophilicity of amino acids can also be used to identify substitutions that result in proteins that retain biological function. By considering the hydrophilicity of amino acids in the context of a peptide, the maximum local average hydrophilicity of the peptide can be calculated, a useful measure that has been reported to correlate well with antigenicity and immunogenicity. See U.S. Pat. No. 4,554,101, which is incorporated herein by reference in its entirety. As is understood in the art, substituting amino acids with similar hydrophilicity values ​​results in peptides that retain biological activity, such as immunogenicity. Substitutions can be made using amino acids with hydrophilicity values ​​within ±2 of each other. Both the hydrophilicity index and hydrophilicity value of an amino acid are affected by the specific side chain of that amino acid. Consistent with these findings, amino acid substitutions that are compatible with biological function are understood to depend on the relative similarity of the amino acids, particularly the relative similarity of the side chains of the amino acids, as manifested by hydrophobicity, hydrophilicity, charge, size, and other properties.

[0054] t.vector The term "vector" as used herein refers to a nucleic acid sequence that includes a replication origin.Vector can be vector, bacteriophage, bacterial artificial chromosome or yeast artificial chromosome.Vector can be DNA vector or RNA vector.Vector can be a self-replicating extrachromosomal vector, preferably DNA plasmid.

[0055] 2. Consensus Prostate Antigen Provided herein is a consensus antigen capable of eliciting an immune response against a prostate antigen in a mammal. The consensus antigen can contain epitopes that make the antigen particularly effective as an immunogen capable of eliciting against prostate cancer cells. The consensus prostate antigen can include a full-length translation product, a variant thereof, a fragment thereof, or a combination thereof.

[0056] Seven different consensus prostate antigens were designed. Two of these consensus prostate antigens are consensus PSA antigen 1 (SEQ ID NO: 2) and consensus PSA antigen 2 (SEQ ID NO: 4). Two of these consensus prostate antigens are consensus PSMA antigen 1 (SEQ ID NO: 6) and consensus PSMA antigen 2 (SEQ ID NO: 8). Two of these consensus prostate antigens are consensus STEAP antigen 1 (SEQ ID NO: 10) and consensus STEAP antigen 2 (SEQ ID NO: 12). One of these consensus prostate antigens is consensus PSCA antigen (SEQ ID NO: 14). Proteins may include sequences homologous to these prostate antigens, fragments of these prostate antigens, and proteins having sequences homologous to fragments of these prostate antigens.

[0057] Consensus PSA antigen 1 (SEQ ID NO:2) is about 91% homologous to the human PSA sequence, about 95% homologous to M. fascicuaris PSA, and about 96% homologous to M. mulatta PSA. Consensus PSA antigen 1 differs from the human PSA sequence at amino acids 69, 78, 80, 82, 102, 110, 137, 139, 165, 189, 203, 220, 232, and 248 of SEQ ID NO:2.

[0058] Consensus PSA antigen 2 (SEQ ID NO: 4) is approximately 90-91% homologous to the human PSA sequence, approximately 95% homologous to M. fascicuaris PSA, and approximately 95% homologous to M. mulatta PSA. Consensus PSA antigen 2 contains a leader sequence at its N-terminus. Consensus PSA antigen 2 also differs from the human PSA sequence at 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.

[0059] Consensus PSMA antigen 1 (SEQ ID NO: 6) is approximately 96% homologous to the human PSMA sequence and approximately 94% homologous to M. mulatta PSMA. Consensus PSMA antigen 1 differs from the human PSMA sequence at 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.

[0060] Consensus PSMA antigen 2 (SEQ ID NO: 8) is approximately 96% homologous to the human PSA sequence and approximately 94% homologous to M. mulatta PSA. Consensus PSMA antigen 2 contains a leader sequence at its N-terminus. Consensus PSMA antigen 2 also differs from the human PSA sequence at 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.

[0061] Consensus STEAP antigen 1 (SEQ ID NO: 10) is approximately 94% homologous to some human STEAP sequences and approximately 99% homologous to other human STEAP sequences, and is also approximately 94% homologous to M. mulatta PSMA.

[0062] Consensus STEAP antigen 2 (SEQ ID NO: 12) is approximately 88% homologous to some human STEAP sequences and approximately 94% homologous to other human STEAP sequences. Consensus STEAP antigen 2 (SEQ ID NO: 12) is also approximately 94% homologous to M. mulatta PSMA. Consensus STEAP antigen 2 contains a leader sequence at its N-terminus.

[0063] The consensus PSCA antigen (SEQ ID NO: 14) is approximately 87% homologous to human PSCA. The consensus PSCA antigen (SEQ ID NO: 14) differs from human PSCA by containing a leader sequence at its N-terminus.

[0064] The protein may have a sequence that is 98% homologous 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).

[0065] The protein may have a sequence that is 99% homologous 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).

[0066] As noted above, some embodiments include a leader sequence at the N-terminus. In some embodiments, this 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 omitted from the sequence. Similarly, in some embodiments of the nucleic acid sequences provided herein, SEQ ID NO: 15 (the sequence encoding SEQ ID NO: 16) is omitted from the sequence.

[0067] Thus, some embodiments relate to proteins comprising a signal peptide linked to SEQ ID NO:2, SEQ ID NO:6, or SEQ ID NO:10 in place of the claimed N-terminal methionine (the coding sequence for a signal peptide typically includes an initiation codon that encodes an 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 98% homologous 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 98% identical 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 proteins comprising a signal peptide linked to a protein 98% identical to SEQ ID NO:10. In all cases where the signal peptide is linked at the N-terminus, it is linked in place of the N-terminal methionine as claimed (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 98% identical 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, with the proviso 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 linked to a protein having a signal peptide linked to an immunogenic fragment of amino acids 19-131 of SEQ ID NO: 14, wherein the immunogenic fragment comprises at least 110 amino acid residues of SEQ ID NO: 14.

[0068] 3. Gene Sequences, Constructs, and Plasmids Nucleic acid molecules encoding consensus amino acid sequences were generated to optimize stability and expression in humans. Codon selection was based, inter alia, on an attempt to minimize intramolecular interactions and secondary structure formation, and was determined using codons that result in improved expression. Vaccines can include one or more nucleic acid sequences encoding one or more consensus versions of immunogenic proteins selected from this group of sequences generated to optimize stability and expression in humans. Nucleic acid sequences were generated that incorporated an IgE leader coding sequence at the 5' end of the optimized consensus-encoding nucleic acid sequence, and the nucleic acid sequences encoded proteins with an IgE leader sequence at the N-terminus of the consensus amino acid sequence. In some embodiments, the nucleic acid sequence encoding the IgE leader is SEQ ID NO: 15.

[0069] The provided nucleic acid sequences encode PSA consensus antigen sequence 1 (protein sequence of SEQ ID NO:2; nucleic acid sequence of SEQ ID NO:1), PSA consensus antigen sequence 2 (protein sequence of SEQ ID NO:4; nucleic acid sequence of SEQ ID NO:3), PSMA consensus antigen sequence 1 (protein sequence of SEQ ID NO:6; nucleic acid sequence having nucleotides 1-2250 of SEQ ID NO:5), PSMA consensus antigen sequence 2 (protein sequence of SEQ ID NO:8; nucleic acid sequence having nucleotides 1-2301 of SEQ ID NO:7), STEAP consensus antigen sequence 1 (protein sequence of SEQ ID NO:10; nucleic acid sequence of SEQ ID NO:9), STEAP consensus antigen sequence 2 (protein sequence of SEQ ID NO:12; nucleic acid sequence of SEQ ID NO:11), or PSCA consensus antigen sequence (protein sequence of SEQ ID NO:14; nucleic acid sequence of SEQ ID NO:13). The nucleic acid sequence of SEQ ID NO:5, which encodes PSMA consensus antigen sequence 1, contains, in addition to the nucleotides encoding PSMA, an additional 9 codons (27 nucleotides) immediately prior to the stop codon that encode an 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 and is particularly useful for detecting protein expression using commercially available anti-HA tag antibodies. In addition to encoding SEQ ID NO:6, SEQ ID NO:5 encodes an additional nine amino acid sequence (SEQ ID NO:32), which is linked at its N-terminal end to the C-terminus of SEQ ID NO:6. In some embodiments, the PSMA-1 consensus antigen comprises a protein encoded by SEQ ID NO:5 and having the amino acid sequence of SEQ ID NO:6, which is linked at its C-terminal end to the N-terminus of SEQ ID NO:32. In some embodiments, the PSMA-1 consensus antigen comprises a protein encoded by nucleotides 1-2250 of SEQ ID NO:5 and 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'-terminal end. The nucleic acid sequence of SEQ ID NO:7, which encodes PSMA consensus antigen sequence 2, contains, in addition to the nucleotides encoding the IgE signal linked to the PSMA protein, an additional 9 codons (27 nucleotides) immediately prior to the stop codon that encode an HA tag (SEQ ID NO:32) not shown in SEQ ID NO:8.In addition to encoding SEQ ID NO:8, SEQ ID NO:7 encodes an additional 9 amino acid sequence (SEQ ID NO:32), which is linked at its N-terminal position 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, which is linked at its C-terminal position 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'-terminal position.

[0070] The isolated nucleic acid molecule encodes a protein having a sequence 98% identical to PSA consensus antigen sequence 1 (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; amino acids 21, 86, 127, 129, 154, 156, 182, 195, 206, 21 of SEQ ID NO: 4; a protein having a sequence 98% homologous to PSA consensus antigen sequence 2 (SEQ ID NO: 4), provided that amino acids 8, 220, 237, 249, 255, 265, 271, and 275 are conserved; amino acids 14, 15, 32, 47, 58, 79, 111, 157, 223, 320, 350, 475, 499, 569, 613, 624, 653, 660, 663, 733, and 73 of SEQ ID NO: 6; a protein having a sequence 98% identical to PSMA consensus antigen sequence 1 (SEQ ID NO: 6), provided that amino acids 4, 5, 6, 7, 9, 12, 7, 17, 23, 33, 36, 49, 51, 56, 58, 62, 64, 66, 67, 67, 67, 74, and 750 of SEQ ID NO: 8 are conserved; a protein having a sequence 98% identical to STEAP consensus antigen sequence 1 (SEQ ID NO: 10); a protein having a sequence 98% identical to STEAP consensus antigen sequence 2 (SEQ ID NO: 12); or a protein having a sequence 98% identical to PSCA consensus antigen sequence (SEQ ID NO: 14), 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.

[0071] The isolated nucleic acid molecule encodes a protein having a sequence that is 99% homologous to PSA consensus antigen sequence 1 (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, and amino acids 21, 86, 127, 129, 154, 156, 182, 195, 206, 21 of SEQ ID NO: 4 are conserved. a protein having a sequence 99% homologous to PSA consensus antigen sequence 2 (SEQ ID NO: 4), provided that amino acids 8, 220, 237, 249, 255, 265, 271, and 275 are conserved; amino acids 14, 15, 32, 47, 58, 79, 111, 157, 223, 320, 350, 475, 499, 569, 613, 624, 653, 660, 663, 733, and 73 of SEQ ID NO: 6; a protein having a sequence 99% identical to PSMA consensus antigen sequence 1 (SEQ ID NO: 6), provided that amino acids 4, 5, 6, 7, 9, 12, 7, 17, 23, 33, 36, 49, 51, 56, 58, 62, 64, 66, 67, 67, 74, and 750 of SEQ ID NO: 8 are conserved; a protein having a sequence 99% identical to STEAP consensus antigen sequence 1 (SEQ ID NO: 10); a protein having a sequence 99% identical to STEAP consensus antigen sequence 2 (SEQ ID NO: 12); or a protein having a sequence 99% identical to PSCA consensus antigen sequence (SEQ ID NO: 14), 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.

[0072] The isolated nucleic acid molecule can encode a protein having a sequence 98% identical to a sequence encoding PSA consensus antigen sequence 1 (SEQ ID NO: 1), a sequence encoding PSA consensus antigen sequence 2 (SEQ ID NO: 3), a sequence encoding PSMA consensus antigen sequence 1 (SEQ ID NO: 5, preferably nucleotides 1-2250 of SEQ ID NO: 5), a sequence encoding PSMA consensus antigen sequence 2 (SEQ ID NO: 7, preferably nucleotides 1-2301 of SEQ ID NO: 7), a sequence encoding STEAP consensus antigen sequence 1 (SEQ ID NO: 9), a sequence encoding STEAP consensus antigen sequence 2 (SEQ ID NO: 11), or a sequence encoding a PSCA consensus antigen sequence (SEQ ID NO: 13).

[0073] The isolated nucleic acid molecule can encode a protein having a sequence 99% identical to a sequence encoding PSA consensus antigen sequence 1 (SEQ ID NO: 1), a sequence encoding PSA consensus antigen sequence 2 (SEQ ID NO: 3), a sequence encoding PSMA consensus antigen sequence 1 (SEQ ID NO: 5, preferably nucleotides 1-2250 of SEQ ID NO: 5), a sequence encoding PSMA consensus antigen sequence 2 (SEQ ID NO: 7, preferably nucleotides 1-2301 of SEQ ID NO: 7), a sequence encoding STEAP consensus antigen sequence 1 (SEQ ID NO: 9), a sequence encoding STEAP consensus antigen sequence 2 (SEQ ID NO: 11), or a sequence encoding a PSCA consensus antigen sequence (SEQ ID NO: 13).

[0074] The isolated nucleic acid molecule can encode a protein comprising a leader sequence at its N-terminus. In some embodiments, the nucleic acid molecule can encode the IgE leader sequence of SEQ ID NO: 16. In some embodiments, the 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, instead of the claimed N-terminal methionine (the coding sequence for a signal peptide typically includes an initiation codon that encodes an 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 98% homologous 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. In some embodiments, the isolated nucleic acid molecule can encode a protein comprising a signal peptide linked to a protein 98% homologous 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. In some embodiments, the isolated nucleic acid molecule can encode a protein comprising a signal peptide linked to a protein 98% homologous to SEQ ID NO:10. If a coding sequence for a signal peptide is provided, the signal peptide is linked to the peptide sequence in place of the N-terminal methionine set forth in the displayed sequence (signal peptide coding sequences typically include an initiation codon that encodes an N-terminal methionine). In some embodiments, the isolated nucleic acid molecule can encode a protein comprising a signal peptide linked to a protein 98% homologous 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, with the proviso 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. 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, with the proviso 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. 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: 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 a signal peptide linked to an immunogenic fragment of amino acids 19-131 of SEQ ID NO: 14, wherein the immunogenic fragment comprises at least 110 amino acid residues of SEQ ID NO: 14.

[0075] Provided herein is a genetic construct that can contain a nucleic acid sequence encoding the consensus prostate antigen disclosed herein (including a consensus protein sequence, a sequence homologous to the consensus protein sequence, a fragment of the consensus protein sequence, or a sequence homologous to a fragment of the consensus protein sequence). The genetic construct can exist within a cell as a functional extrachromosomal molecule. The genetic construct can be a linear minichromosome containing a centromere, telomere, plasmid, or cosmid.

[0076] The genetic construct may be part of the genome of a recombinant viral vector, including recombinant adenovirus, recombinant adenovirus-associated virus, and recombinant vaccinia. The genetic construct may be part of the genetic material of an attenuated live microorganism or a recombinant microbial vector that lives intracellularly.

[0077] A gene construct can include regulatory elements for gene expression of a nucleic acid coding sequence. Regulatory elements can be promoters, enhancers, initiation codons, stop codons, or polyadenylation signals.

[0078] The nucleic acid sequence may constitute a genetic construct, which may be a vector. The vector may be capable of expressing an effective amount of an antigen in the cells of a mammal to induce an immune response in the mammal. The vector may be recombinant. The vector may contain a heterologous nucleic acid encoding the antigen. The vector may be a plasmid. The vector may be useful for transfecting cells with a nucleic acid encoding the antigen, and the transformed host cells are cultured and maintained under conditions in which the antigen is expressed.

[0079] In some embodiments, the coding sequence for a single consensus prostate antigen is provided in a single vector. In some embodiments, the coding sequences for multiple consensus prostate antigens are provided in a single vector. In some embodiments, compositions comprising coding sequences for multiple consensus prostate antigens are provided in multiple vectors, one antigen per vector or multiple antigens per vector.

[0080] In some embodiments, coding sequences for two or more consensus prostate antigens may be provided in a single vector. In some embodiments, the coding sequences may have separate promoters controlling expression. In some embodiments, the coding sequences may have a single promoter controlling expression using an IRES sequence separating the coding sequences. The presence of an IRES sequence results in separate translation of the transcripts. In some embodiments, the coding sequences may have a single promoter controlling expression using a coding sequence encoding a proteolytic peptide sequence separating the antigen coding sequences. A single translation product is generated, which is then processed by a protease that recognizes the protease cleavage site to generate separate protein molecules. The protease cleavage site used is typically recognized by a protease endogenously present in the cell in which expression occurs. In some embodiments, separate coding sequences for proteases may be included to produce the proteases required for processing the polyprotein translation product. In some embodiments, the vector contains coding sequences for one, two, three, four, five, six, or all seven of the seven consensus prostate antigens.

[0081] In each of the examples described herein, the coding sequence may be optimized for stability and high expression levels, and in some examples, codons are selected to reduce the formation of secondary RNA structures, such as those formed by intramolecular bonds.

[0082] The vector can contain a heterologous nucleic acid encoding an antigen and can further contain an initiation codon, which can be located upstream of the antigen-encoding sequence, and a termination codon, which can be located downstream of the antigen-encoding sequence. The initiation and termination codons can be in-frame with the antigen-encoding sequence. The vector can also contain a promoter operably linked to the antigen-encoding sequence. The promoter operably linked to the antigen-encoding sequence can be a promoter derived from simian virus 40 (SV40), a mouse mammary tumor virus (MMTV) promoter, a human immunodeficiency virus (HIV) promoter such as bovine immunodeficiency virus (BIV), a long terminal repeat (LTR) promoter, a Moloney virus promoter, an avian leukosis virus (ALV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate-early promoter, an Epstein-Barr virus (EBV) promoter, or a Rous sarcoma virus (RSV) promoter. The promoter can also be a promoter derived from a human gene such as human actin, human myosin, human hemoglobin, human muscle creatine, or human metallothionein. The promoter may be a natural or synthetic tissue-specific promoter, such as a muscle- or skin-specific promoter. Examples of such promoters are described in U.S. Patent Application Publication No. 20040175727, the entire contents of which are incorporated herein by reference.

[0083] The vector may contain a polyadenylation signal that may be located downstream of the consensus prostate antigen coding sequence. The polyadenylation signal may be an SV40 polyadenylation signal, an LTR polyadenylation signal, a bovine growth hormone (bGH) polyadenylation signal, a human growth hormone (hGH) polyadenylation signal, or a human β-globin polyadenylation signal. The SV40 polyadenylation signal may be a polyadenylation signal derived from the pCEP4 vector (Invitrogen, San Diego, CA).

[0084] The vector may contain an enhancer upstream of the consensus prostate antigen coding sequence.The enhancer may be required for DNA expression.The enhancer may be human actin, human myosin, human hemoglobin, human muscle creatine, or a viral enhancer such as an enhancer derived from CMV, HA, RSV, or EBV.Polynucleotide function enhancers are described in U.S. Patent Nos. 5,593,972, 5,962,428, and WO 94 / 016737, the entire contents of each of which are incorporated by reference.

[0085] The vector can also contain a mammalian origin of replication to maintain the vector extrachromosomally and produce multiple copies of the vector within the cell. The vector can be pVAX1, pCEP4, or pREP4 from Invitrogen (San Diego, CA), which contain the Epstein-Barr virus origin of replication and the nuclear antigen EBNA-1 coding region and can produce high copy numbers of episomal replication without integration. The vector backbone can be pAV0242. The vector can be a replication-deficient adenovirus type 5 (Ad5).

[0086] The vector may contain regulatory sequences that may be sufficient for gene expression in mammalian or human cells into which the vector is administered. The consensus prostate antigen coding sequence may contain codons that may increase the transcription efficiency of the coding sequence in the host cell.

[0087] The vector may be pSE420 (Invitrogen, San Diego, CA), in which case it can be used to produce the protein in Escherichia coli (E. coli). The vector may be pYES2 (Invitrogen, San Diego, CA), in which case it can be used to produce the protein in Saccharomyces cerevisiae strains of yeast. The vector may be the MAXBAC™ complete baculovirus expression system (Invitrogen, San Diego, CA), in which case it can be used to produce the protein in insect cells. The vector may be pcDNAI or pcDNA3 (Invitrogen, San Diego, CA), in which case it can be used to produce the protein in mammalian cells, such as Chinese hamster ovary (CHO) cells. The vector may be an expression vector or expression system for producing the protein using conventional methods and readily available starting materials (including Sambrook et al., Molecular Cloning and Laboratory Manual, Second Ed., Cold Spring Harbor (1989)), which is incorporated by reference in its entirety. body is incorporated).

[0088] The vaccine may comprise one or more of the prostate antigens described herein and / or the vaccine may comprise one or more nucleic acid sequences encoding one or more consensus prostate antigens selected from this group. The vaccine may comprise one or more of the consensus prostate antigens described herein in combination with other immunogenic prostate proteins using sequences (including naturally occurring sequences) other than the consensus sequences disclosed herein and / or the vaccine may comprise one or more nucleic acid sequences encoding one or more consensus prostate antigens selected from this group in combination with nucleic acid molecules encoding other prostate antigens using sequences other than the consensus sequences disclosed herein.

[0089] Without being bound by scientific theory, a vaccine that can be used to elicit a broad immune response (humoral, cellular, or both) against prostate cancer cells can include 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 also include coding sequences provided herein, including homologous sequences, fragments, and homologous sequences of fragments.

[0090] 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 against prostate cancer, comprising administering one or more compositions collectively comprising one or more coding sequences or combinations described herein. Some embodiments provide methods of therapeutically vaccinating an individual with prostate cancer, comprising administering one or more compositions collectively comprising one or more coding sequences or combinations described herein.

[0091] 4. Pharmaceutical Compositions Provided herein are pharmaceutical compositions according to the present invention comprising about 1 nanogram to about 10 mg of DNA. In some embodiments, the pharmaceutical compositions according to the present invention comprise: 1) at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 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, 160, 170, 180, 190, 210, 220, 230, 240, 250, 260, 270, 280, 290, 310, 320, 330, 340, 350, 360, 370, 380, 390, 410, 420, 430, 440, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 210 55, 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, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 605, 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, 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, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 mg or more, 2) up to 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nanograms (inclusive), or up to 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, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 410, 420, 430, 440, 450, 460, 470, 480, 490, 510, 520, 530, 540, 550, 560, 570, 0, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445 , 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 605, 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, 955, 960, 965, 970, 975, 980, 985, 990, 995, or 1,000 micrograms (inclusive), or a maximum of 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 mg, inclusive. In some embodiments, a pharmaceutical composition according to the present invention comprises about 5 nanograms to about 10 mg of DNA. In some embodiments, a pharmaceutical composition according to the present invention comprises about 25 nanograms to about 5 mg of DNA. In some embodiments, a pharmaceutical composition contains about 50 nanograms to about 1 mg of DNA. In some embodiments, a pharmaceutical composition contains about 0.1 to about 500 micrograms of DNA. In some embodiments, a pharmaceutical composition contains about 1 to about 350 micrograms of DNA. In some embodiments, a pharmaceutical composition contains about 5 to about 250 micrograms of DNA. In some embodiments, a pharmaceutical composition contains about 10 to about 200 micrograms of DNA. In some embodiments, a pharmaceutical composition contains about 15 to about 150 micrograms of DNA. In some embodiments, a pharmaceutical composition contains about 20 to about 100 micrograms of DNA. In some embodiments, a pharmaceutical composition contains about 25 to about 75 micrograms of DNA. In some embodiments, a pharmaceutical composition contains about 30 to about 50 micrograms of DNA. In some embodiments, the pharmaceutical composition contains about 35 to about 40 micrograms of DNA. In some embodiments, the pharmaceutical composition contains about 100 to about 200 micrograms of DNA. In some embodiments, the pharmaceutical composition contains about 10 micrograms to about 100 micrograms of DNA. In some embodiments, the pharmaceutical composition contains about 20 micrograms to about 80 micrograms of DNA. In some embodiments, the pharmaceutical composition contains about 25 micrograms to about 60 micrograms of DNA. In some embodiments, the pharmaceutical composition contains about 30 nanograms to about 50 micrograms of DNA. In some embodiments, the pharmaceutical composition contains about 35 nanograms to about 45 micrograms of DNA. In some preferred embodiments, the pharmaceutical composition contains about 0.The pharmaceutical composition contains 1 to about 500 micrograms of DNA. In some preferred embodiments, the pharmaceutical composition contains about 1 to about 350 micrograms of DNA. In some preferred embodiments, the pharmaceutical composition contains about 25 to about 250 micrograms of DNA. In some preferred embodiments, the pharmaceutical composition contains about 100 to about 200 micrograms of DNA.

[0092] The pharmaceutical composition according to the present invention is formulated according to the mode of administration to be used. In the case of an injectable pharmaceutical composition, the pharmaceutical composition is sterile, pyrogen-free, and particulate-free. Preferably, an isotonic formulation is used. Generally, isotonic additives can include sodium chloride, glucose, mannitol, sorbitol, and lactose. In some cases, an isotonic solution such as phosphate-buffered saline is suitable. Stabilizers include gelatin and albumin. In some embodiments, a vasoconstrictor is added to the formulation.

[0093] The pharmaceutical composition is preferably a vaccine, more preferably a DNA vaccine.

[0094] The vaccine may be a DNA vaccine. The DNA vaccine may comprise multiple identical or different plasmids containing 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 comprises coding sequences for multiple consensus prostate antigens, all of the sequences may be present on a single plasmid, or each sequence may be present on a separate plasmid.

[0095] In some embodiments, the vaccine may comprise a nucleic acid sequence encoding one or more consensus prostate antigens in combination with one or more consensus prostate antigens.

[0096] 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 further comprise an element or reagent that inhibits integration into chromosomes. The vaccine may be an RNA of a prostate antigen. This RNA vaccine can be introduced into cells.

[0097] The vaccine may be a recombinant vaccine containing the above-mentioned gene construct or antigen. The vaccine may contain one or more consensus prostate antigens in the form of one or more protein subunits, or may contain one or more attenuated virus particles containing one or more consensus prostate antigens. The attenuated vaccine may be a live attenuated vaccine, a killed vaccine, or a vaccine that uses a recombinant vector to deliver a foreign gene encoding one or more consensus prostate antigens, as well as a subunit vaccine and a glycoprotein vaccine. Examples of live attenuated vaccines, vaccines using recombinant vectors to deliver prostate antigens, subunit vaccines, and glycoprotein vaccines are described in U.S. Patent Nos. 4,510,245, 4,797,368, 4,722,848, 4,790,987, 4,920,209, 5,017,487, 5,077,044, 5,110,587, 5,112,749, 5,174,993, 5,223,424, 5,225,336, 5,240,703, 5,242,829, 5,294,441, 5,294,548, Nos. 5,310,668, 5,387,744, 5,389,368, 5,424,065, 5,451,499, 5,453,364, 5,462,734, 5,470,734, 5,474,935, 5,482,713, 5,591,439, 5,643,579, 5,650,309, 5,698,202, 5,955,088, 6,034,298, 6,042,836, 6,156,319, and 6,589,529, each of which is incorporated herein by reference.

[0098] The provided vaccines can be used to induce immune responses, including therapeutic and preventative immune responses. Antibodies and / or killer T cells directed against consensus prostate antigens can be generated. Such antibodies and cells can be isolated.

[0099] 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-enhancing agent, which may include surfactants such as immune stimulating complexes (ISCOMS), Freund's incomplete adjuvant, LPS analogs including monophosphoryl lipid A, muramyl peptides, quinone analogs, vesicles such as squalene and squalene, hyaluronic acid, lipids, liposomes, calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other known transfection-enhancing agents.

[0100] The transfection-facilitating agent may be a polyanion, a polycation, including poly-L-glutamate (LGS), or a lipid. The transfection-facilitating agent is poly-L-glutamate, and more preferably, poly-L-glutamate is present in the vaccine at a concentration of less than 6 mg / ml. Transfection-facilitating agents may include surfactants such as immune-stimulating complexes (ISCOMS), Freund's incomplete adjuvant, LPS analogs including monophosphoryl lipid A, muramyl peptides, quinone analogs, and vesicles such as squalene and squalene; hyaluronic acid may also be used for co-administration with the genetic construct. In some embodiments, the DNA vector vaccine may include a transfection-facilitating agent, such as a lipid, liposome (including lecithin liposomes or other liposomes known in the art as DNA liposome mixtures) (see, e.g., WO 9324640), calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other known transfection-facilitating agents. Preferably, the transfection-facilitating agent is a polyanion, a polycation, including poly-L-glutamate (LGS), or a lipid. The concentration of the transfection agent in the vaccine is less than 4 mg / ml, less than 2 mg / ml, less than 1 mg / ml, less than 0.750 mg / ml, less than 0.500 mg / ml, less than 0.250 mg / ml, less than 0.100 mg / ml, less than 0.050 mg / ml, or less than 0.010 mg / ml.

[0101] The pharmaceutically acceptable excipient may be an adjuvant. The adjuvant may be another gene expressed on an alternative plasmid, or may be combined with the above plasmid and delivered as a protein within the vaccine. The adjuvant may be selected from the group consisting of: α-interferon (IFN-α), β-interferon (IFN-β), γ-interferon, platelet-derived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermal growth factor (EGF), cutaneous T-cell-attracting chemokine (CTACK), epithelial thymus-expressed chemokine (TECK), mucosal-associated epithelial chemokine (MEC), IL-12, IL-15, MHC, CD80, CD86 (including IL-15 lacking its signal sequence and optionally containing a signal peptide derived from IgE). The adjuvant may be IL-12, IL-15, IL-28, CTACK, TECK, platelet-derived growth factor (PDGF), TNFα, TNFβ, 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.

[0102] Other genes that may be useful adjuvants include those encoding MCP-1, MIP-1a, 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, mutated forms of IL-18, and 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, DR 4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, inactive NIK, SAP K, SAP-1, JNK, interferon response genes, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK ligand, Ox40, Ox40 ligand, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1, TAP2, and functional fragments thereof.

[0103] The vaccine may further comprise a genetic vaccine facilitator as described in US patent application Ser. No. 021,579, filed Apr. 1, 1994, which is incorporated by reference in its entirety.

[0104] 5.Delivery method To provide gene constructs and consensus prostate antigens containing epitopes that make the pharmaceutical preparations particularly effective immunogens and capable of inducing an immune response against prostate cancer cells, methods for delivering pharmaceutical preparations (preferably vaccines) are provided herein. Methods for delivering vaccines to induce therapeutic and / or prophylactic immune responses, i.e., vaccination methods, can be provided. By delivering vaccines to individuals, the activity of the mammalian immune system can be modulated and the immune response can be enhanced.

[0105] When the vaccine is delivered to a mammal, and then the vector is introduced into the cells of the mammal, the transfected cells express and secrete the corresponding prostate consensus protein.The secreted protein or synthetic antigen is recognized by the immune system, and an immune response is initiated.The immune response can include the production of antibodies against the antigen and antigen-specific T cell responses.In some examples, the mammal that is vaccinated with the vaccine discussed herein has a primed immune system.By delivering a vaccine to an individual, the activity of the individual's immune system can be adjusted, and thus the immune response can be enhanced.

[0106] The vaccine can be delivered in the form of a DNA vaccine, and methods for delivering DNA vaccines are described in US Pat. Nos. 4,945,050 and 5,036,006, both of which are incorporated by reference in their entireties.

[0107] The vaccine can be administered to a mammal to elicit an immune response in the mammal, which may be a human, non-human primate, cow, pig, sheep, goat, antelope, bison, buffalo, bovine, deer, hedgehog, elephant, llama, alpaca, mouse, rat, or chicken, preferably a human, cow, pig, or chicken.

[0108] Combination therapy The pharmaceutical composition, preferably the vaccine, can be administered in combination with one or more other prostate proteins or genes. The vaccine can be administered in combination with proteins or genes encoding adjuvants, including α-interferon (IFN-α), β-interferon (IFN-β), γ-interferon, IL-12, IL-15, IL-28, CTACK, TECK, platelet-derived growth factor (PDGF), TNFα, TNFβ, 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-1α, MIP-1p, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MAbCAM- 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 form 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 ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, inactive NIK, SAP K, SAP-1, JNK, interferon response genes, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK ligand, Ox40, Ox40 ligand, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1, or TAP2, or a functional fragment thereof.

[0109] b. Route of administration Vaccines can be administered by a variety of routes, including orally, parenterally, sublingually, transdermally, rectally, transmucosally, topically, by inhalation, buccal administration, intrapleurally, intravenously, intraarterially, intraperitoneally, subcutaneously, intramuscularly, intranasally, intrathecally, intraarticularly, or a combination thereof. For use in animals, the compositions can be administered in any suitably acceptable formulation in accordance with normal veterinary practice. A veterinarian can readily determine the optimal dosing regimen and route of administration for an individual animal. Vaccines can be administered by conventional syringes, needleless injection devices, "microparticle gene guns," or other physical methods such as electroporation ("EP"), "hydrodynamic methods," or ultrasound.

[0110] Several well-known techniques can be used to deliver vaccine vectors to mammals, including DNA injection (also referred to as DNA vaccination), with or without in vivo electroporation, liposome-mediated, nanoparticle-facilitated, recombinant vectors (e.g., recombinant adenovirus, recombinant adenovirus-associated virus, recombinant vaccinia, etc.) Prostate antigens can be delivered via DNA injection and with in vivo electroporation.

[0111] c. electroporation Vaccination via electroporation of vaccine plasmids can be performed using an electroporation device that can be configured to deliver an energy pulse to a desired mammalian tissue effective to form reversible pores in the cell membrane, which in some embodiments is a constant current similar to a user-preset current input.

[0112] In some embodiments utilizing electroporation, the electroporation device may include an electroporation component and an electrode assembly or handle assembly. The electroporation component may include or incorporate one or more of the various components of an electroporation device, including a controller, a current waveform generator, an impedance tester, a waveform recorder, an input element, a status reporting element, a communication port, a memory element, a power supply, and a power switch. Electroporation can be performed using an in vivo electroporation device, such as the CELLECTRA® EP system (Inobio Pharmaceuticals, Inc., Blue Bell, PA), the Elgen electroporator (Inobio Pharmaceuticals, Inc., Blue Bell, PA), or the like, to facilitate transfection of cells with plasmids.

[0113] The electroporation component may function as one element of an electroporation apparatus, with the remaining elements being separate elements (or components) that communicate with the electroporation component. The electroporation component may function as more than one element of an electroporation apparatus and may communicate with additional elements of the electroporation apparatus separate from the electroporation component. The elements of the electroporation apparatus can function as a single device or as separate elements that communicate with each other, so there is no restriction on the elements of the electroporation apparatus being part of a single electromechanical or mechanical device. The electroporation component may be capable of delivering an energy pulse that generates a constant current within the desired tissue and includes a feedback mechanism. The electrode assembly may include an electrode array having multiple spatially arranged electrodes that receive the energy pulse from the electroporation component and deliver the energy pulse to the desired tissue via the electrodes. At least one of the multiple electrodes is neutral during delivery of the energy pulse and measures the impedance within the desired tissue and transmits the impedance to the electroporation component. A feedback mechanism can receive impedance measurements and adjust the energy pulses delivered from the electroporation component to maintain a constant current.

[0114] The plurality of electrodes can deliver energy pulses in a distributed pattern via control of the electrodes under a programmed sequence, the programmed sequence being input into the electroporation component by a user. The programmed sequence can include multiple energy pulses delivered in sequence, each pulse of the plurality of pulses being delivered by at least two active electrodes with one indifferent electrode measuring impedance, and each subsequent pulse of the plurality of pulses being delivered by another one of the at least two active electrodes with one indifferent electrode measuring impedance.

[0115] The feedback mechanism can be implemented in either hardware or software. The feedback mechanism can be implemented by an analog closed-loop circuit. This feedback occurs every 50 μs, 20 μs, 10 μs, or 1 μs, but is preferably real-time feedback or instantaneous (i.e., substantially instantaneous as determined by available techniques for determining response time). The indifferent electrode can measure the impedance in the desired tissue and communicate that impedance to the feedback mechanism, which responds to the impedance and adjusts the energy pulse to maintain a constant current similar to the preset current. The feedback mechanism can maintain a constant current continuously and instantaneously during the delivery of the energy pulse.

[0116] Examples of electroporation devices and methods that can facilitate delivery of the DNA vaccines of the present invention are described in U.S. Patent No. 7,245,963 to Draghia-Akli et al. and U.S. Patent Application Publication No. 2005 / 0052630 to Smith et al. Other electroporation devices and methods that can be used to enhance delivery of DNA vaccines include those provided in co-pending and commonly owned U.S. patent application Ser. No. 11 / 874,072, filed October 17, 2007, which claims the benefit under 35 U.S.C. 119(e) of U.S. Provisional Patent Application Ser. No. 60 / 852,149, filed October 17, 2006, and U.S. Provisional Patent Application Ser. No. 60 / 978,982, filed October 10, 2007, all of which are hereby incorporated by reference in their entireties.

[0117] U.S. Patent No. 7,245,963 to Draghia-Akli et al. describes a modular electrode system and its use for promoting the introduction of biomolecules into cells of selected tissues within a body or plant. The modular electrode system may include multiple needle electrodes, hypodermic needles, an electrical connector providing conductive connection from a programmable constant current pulse controller to the multiple needle electrodes, and a power source. An operator can grasp the multiple needle electrodes mounted on a support structure and firmly insert them into selected tissues within a body or plant. The biomolecule is then delivered to the selected tissue via the hypodermic needle. The programmable constant current pulse controller is activated to apply constant current electrical pulses to the multiple needle electrodes. The applied constant current electrical pulses promote the introduction of biomolecules into cells between the multiple electrodes. The entire contents of U.S. Patent No. 7,245,963 are incorporated herein by reference.

[0118] US Patent Application Publication No. 2005 / 0052630, filed by Smith et al., describes a method for treating a vascular endothelial cell in vivo or implanted in vivo. This paper describes an electroporation device that can be used to effectively promote the introduction of biomolecules into selected tissues within a subject. The electroporation device includes an electrokinetic device ("EKD device"), the operation of which is specified by software or firmware. The EKD device generates a series of programmable constant current pulse patterns between electrodes in an array based on user control and input of pulse parameters, and allows for the storage and retrieval of current waveform data. The electroporation device also includes a replaceable electrode disk with a series of needle electrodes, a central injection channel for an injection needle, and a removable guide disk. The entire contents of U.S. Patent Application Publication No. 2005 / 0052630 are incorporated herein by reference.

[0119] The electrode arrays and methods described in U.S. Patent No. 7,245,963 and U.S. Patent Application Publication No. 2005 / 0052630 can be adapted for deep penetration of tissues such as muscle, as well as other tissues or organs. Due to the configuration of the electrode array, the injection needle (for delivery of the biomolecule of choice) is also fully inserted into the target organ, and the injection is administered perpendicularly to the target tissue within the area predefined by the electrodes. The electrodes described in U.S. Patent No. 7,245,963 and U.S. Patent Application Publication No. 2005 / 005263 are preferably 20 mm long and 21 gauge.

[0120] Additionally, in some embodiments incorporating electroporation devices and their use, the electroporation devices described in the following patent documents are contemplated: U.S. Patent No. 5,273,525, issued December 28, 1993; U.S. Patent No. 6,110,161, issued August 29, 2000; U.S. Patent No. 6,261,281, issued July 17, 2001; and U.S. Patent No. 6,958,060, issued October 25, 2005, and U.S. Patent No. 6,939,862, issued September 6, 2005. Also contemplated herein are patents covering subject matter provided in U.S. Patent No. 6,697,669, issued February 24, 2004, which relates to delivery of DNA using any of a variety of devices, and U.S. Patent No. 7,328,064, issued February 5, 2008, which describes a method for injecting DNA. The above patents are incorporated by reference in their entireties. Another embodiment of an electroporation device for use with the cancer antigens described herein is the Elgen EP device (Innovio Pharmaceuticals, Inc., Blue Bell, PA).

[0121] d. Vaccine preparation method The present specification provides a method for preparing the DNA plasmids comprising the DNA vaccines discussed herein.The DNA plasmids after the final step of subcloning into mammalian expression plasmids can be used to inoculate large-scale fermentation tanks with cell cultures by methods known in the art.

[0122] DNA plasmids for use in the EP devices of the present invention can be formulated or manufactured using a combination of known equipment and techniques, but are preferably manufactured using the optimized plasmid manufacturing techniques described in co-pending U.S. Provisional Application No. 60 / 939,792, filed May 23, 2007, and licensed. In some instances, the DNA plasmids used in these studies can be formulated at concentrations of 10 mg / mL or greater. Manufacturing techniques may include or incorporate those described in U.S. Patent Application No. 60 / 939,792, including the manufacturing techniques described in licensed U.S. Patent No. 7,238,522, issued July 3, 2007, as well as various equipment and protocols commonly known to those of skill in the art. The above-referenced patent applications and patents, U.S. Patent Application No. 60 / 939,792 and U.S. Patent No. 7,238,522, are each incorporated herein in their entirety. [Example]

[0123] The present invention is further described in the following examples. While the following examples illustrate preferred embodiments of the present invention, it should be understood that they are provided by way of illustration only. From the above description and the following examples, those skilled in the art will be able to ascertain the essential features of the present invention and make various changes and modifications to the present invention to adapt it to various applications and conditions without departing from the spirit and scope of the present invention. Thus, from the foregoing description, various modifications of the present invention in addition to those shown and described herein will be apparent to those skilled in the art. Such modifications are also intended to fall within the scope of the appended claims.

[0124] Example 1 Laddy, DJ, Yan, J., Corbitt, N., Kobasa,D., Kobinger,GP,Weiner,DB(2007). Immunogenicity of novelconsensus-based DNAvaccinesagainstavianinfluenza. Khan, AS, Greenhouse, J., Sardesai, NY, Draghia-Akli, R., Weiner, DB (2008). Consensus immunogens for PSA and PSMA were designed from full-length human and macaque sequences available in the GenBank database, as previously described.

[0125] Synthesis of the consensus antigen sequence was performed by GeneScript (Piscataway, NJ). An HA tag was included at the C-terminus of the antigen sequence. The antigen sequence was optimized for mRNA stabilization and codon usage in humans. The final sequence was cloned into the BamHI and XhoI sites of the pVAX1 vector (Invitrogen, Carlsbad, CA).

[0126] A consensus PSA antigen 1 (SEQ ID NO: 2) was generated. This sequence contains 261 amino acids and was compared to each of the PSA sequences listed in Table 1. The PSA sequences used included two human sequences, one from M. fascicularis, and one from M. mulatta. Entries in Table 1 include the SEQ ID NO and accession number of each sequence used for comparison with consensus PSA antigen 1 (SEQ ID NO: 2).

[0127] [Table 1]

[0128] Consensus PSA antigen 1 (SEQ ID NO: 2) was used to generate a multiple sequence alignment of the PSA sequences from H. sapiens (SEQ ID NOs: 17 and 18), M. mulatta (SEQ ID NO: 20), and M. facicularis (SEQ ID NO: 19). KLK3 (kallikrein3) is the gene encoding PSA and is another name for PSA. PSA antigen 1 is 91%, 96%, and 95% homologous to the full-length PSA protein sequences of H. sapiens, M. mulatta, and M. facicularis, respectively.

[0129] Example 2 A consensus PSA antigen 2 (SEQ ID NO: 4) was generated. This sequence contains 279 amino acids, including the IgE leader sequence, and was compared to each of the PSA sequences listed in Table 2. The PSA sequences used included two human sequences, one from M. fascicularis, and one from M. mulatta. The entries in Table 2 include the SEQ ID NO and accession number of each sequence used for comparison with consensus PSA antigen 2 (SEQ ID NO: 4).

[0130] [Table 2]

[0131] Consensus PSA antigen 1 (SEQ ID NO: 4) was used to generate a multiple sequence alignment of the PSA sequences from H. sapiens (SEQ ID NOs: 17 and 18), M. mulatta (SEQ ID NO: 21), and M. facicularis (SEQ ID NO: 19). KLK3 (kallikrein3) is the gene encoding PSA and is another name for PSA. PSA antigen 1 is 90-91% and 95% homologous to the full-length PSA protein sequences of H. sapiens and M. facicularis, respectively, and 95% homologous to the partial PSA protein sequence of M. mulatta.

[0132] Example 3 A consensus PSMA antigen 1 (SEQ ID NO: 6) was generated. This sequence contains 750 amino acids and was compared to each of the PSMA sequences listed in Table 3. The PSMA sequences used included two human sequences and a sequence from M. mulatta. Entries in Table 3 include the SEQ ID NO and accession number for each sequence used for comparison to consensus PSMA antigen 1 (SEQ ID NO: 6).

[0133] [Table 3]

[0134] A multiple sequence alignment of the H. sapiens and M. mulatta PSMA sequences was generated using PSMA antigen 1. The PSMA antigen 1 consensus sequence (SEQ ID NO: 6) is 96% homologous to the H. sapiens PSMA sequence (SEQ ID NOs: 22, 23) and 94% homologous to the M. mulatta full-length PSMA protein sequence (SEQ ID NO: 24).

[0135] Example 4 A consensus PSMA antigen 2 (SEQ ID NO: 8) was generated. This sequence contains 766 amino acids, including the IgE leader sequence, and was compared to each of the PSMA sequences listed in Table 4. The PSMA sequences used included two human sequences and a sequence from M. mulatta. Entries in Table 4 include the SEQ ID NO and accession number for each sequence used for comparison to consensus PSMA antigen 2 (SEQ ID NO: 8).

[0136] [Table 4]

[0137] PSMA antigen 2 was used to generate a multiple sequence alignment of H. sapiens (SEQ ID NOs: 22, 23) and M. mulatta PSMA sequences (SEQ ID NOs: 24, 25). The PSMA antigen 2 consensus sequence (SEQ ID NO: 8) is the H. sapiens PSMA antigen. It is 96% homologous to the protein sequence and 94% homologous to the M. mulatta PSMA protein sequence.

[0138] Example 5 A consensus STEAP antigen 1 (SEQ ID NO: 10) was generated. This sequence contains 339 amino acids and was compared to each of the STEAP sequences listed in Table 5. The STEAP sequences used included two full-length human sequences, a sequence derived from M. mulatta, and two less-than-full-length human sequences. Entries in Table 5 include the SEQ ID NO and accession number for each sequence used for comparison to consensus STEAP antigen 1 (SEQ ID NO: 10).

[0139] [Table 5]

[0140] A multiple sequence alignment of the H. sapiens and M. mulatta STEAP sequences was generated using consensus STEAP antigen 1. The STEAP antigen 1 consensus sequence (SEQ ID NO: 10) is 99% homologous to the human full-length isoform (SEQ ID NOs: 26, 27), 94% homologous to the shorter-length H. sapiens isoforms (SEQ ID NOs: 29, 30), and 94% homologous to the M. mulatta full-length STEAP1 protein sequence (SEQ ID NO: 28).

[0141] Example 6 A consensus STEAP antigen 2 (SEQ ID NO: 12) was generated. This sequence contains 356 amino acids and was compared to each of the STEAP sequences listed in Table 6. The STEAP sequences used included two full-length human sequences, a sequence derived from M. mulatta, and two less-than-full-length human sequences. Entries in Table 6 include the SEQ ID NO and accession number for each sequence used for comparison to consensus STEAP antigen 2 (SEQ ID NO: 12).

[0142] [Table 6]

[0143] A multiple sequence alignment of the H. sapiens and M. mulatta STEAP1 sequences was generated using consensus STEAP1 antigen 2. The STEAP1 antigen 2 consensus sequence (SEQ ID NO: 12) is 94% identical to the full-length human isoform (SEQ ID NOs: 26 and 27), 88% identical to the shorter H. sapiens isoforms (SEQ ID NOs: 29 and 30), and 94% identical to the M. mulatta full-length STEAP1 protein sequence (SEQ ID NO: 28).

[0144] Example 7 A consensus PSCA antigen (SEQ ID NO: 14) was generated. This sequence contains 131 amino acids, including the IgE leader sequence, and was compared to the PSCA sequences listed in Table 7. The PSCA sequence used is the full-length human sequence. Entries in Table 7 include the SEQ ID NO and accession number of the sequence used for comparison with the consensus PSCA antigen (SEQ ID NO: 14).

[0145] [Table 7]

[0146] The consensus PSCA antigen (SEQ ID NO: 14) was used to generate a multiple sequence alignment of the H. sapiens PSCA sequence (SEQ ID NO: 31). The PSCA antigen consensus sequence is 87% homologous to the full-length H. sapiens PSCA.

[0147] Example 8 To confirm the expression of PSA and PSMA antigens, in vitro translation was performed. The ion System and 35S-methionine (Promega) were used. According to the manufacturer's instructions, the pVAX vector alone (negative control), or the pVAX backbone with the PSA or PSMA antigen insert, and 35S-methionine were added to the reaction mixture. The reaction was carried out at 30°C for 2 hours. The labeled proteins were immunoprecipitated using anti-HA affinity gel (Sigma, St. Louis, MO) by overnight rotation in radioimmunoprecipitation assay (RIPA) buffer at 4°C. The immunoprecipitated proteins were electrophoresed on an SDS-PAGE gel, followed by fixation and drying. Expression of the 35S-labeled proteins was detected by autoradiography. The results are shown in Figure 1.

[0148] Example 9 The cellular immunogenicity of PSA and PSMA antigens was measured by interferon-γ ELISpot.

[0149] Four- to six-week-old female BALB / c mice were purchased from The Jackson Laboratory (Bar Harbor, ME). All animals were housed in a temperature-controlled, photoperiod-controlled facility at the University of Pennsylvania. Animal care was conducted in accordance with the guidelines of the National Institutes of Health and the University of Pennsylvania Institutional Care and Use Committee.

[0150] To test cellular immunogenicity, 10 μg or 20 μg of each antigen was delivered intramuscularly into the tibialis anterior muscle of Balb / c mice, followed by electroporation using a CELLECTRA® adaptive constant current device (Inobio Pharmaceuticals, Blue Bell, PA). Mice (n = 5 per group) were immunized twice, at weeks 0 and 2. Two square-wave pulses of 0.1 ampere constant current were delivered through a triangular three-electrode array consisting of 26-gauge solid stainless steel electrodes. Each pulse was 52 milliseconds long, with a 1-second delay between pulses. Mice were immunized twice, two weeks apart. One week after the second immunization, mice were humanely sacrificed for analysis of cellular and humoral immune responses.

[0151] Cellular responses were assessed one week after the final immunization (week 5). Antigen-specific secretion of IFNγ was measured using ELISpot analysis. Mouse IFNγ capture antibody (R&D Systems, Minneapolis, MN) was used to coat flat-bottom Immobilon-P plates (Millipore, Billerica, MA) overnight at 4°C. Splenocytes were aseptically isolated and resuspended in R10 medium (Roswell Park Memorial Institute Medium 1640 supplemented with 10% fetal bovine serum, 1% antibacterial-antimycotic, and 0.1% 2-mercaptoethanol). 2 × 10 cells obtained from immunized mice were used. 5Splenocytes were added to each well of a 96-well plate and stimulated overnight at 37°C and 5% CO2 in the presence of R10 (negative control), concanavalin A (positive control) (Sigma, St. Louis, MO), or an antigen-specific peptide pool. The following day, a mouse IFNγ detection antibody (R&D Systems, Minneapolis, MN) was added to the plate and then incubated overnight at 4°C. The following day, streptavidin-ALP (Mabtec, Sweden) was added to the plate for 2 hours, and antigen-specific spots were visualized with BCIP / NPT substrate (Mabtec, Sweden). The PSA and PSMA peptides were 15-mer peptides that overlap by 11 amino acids and span the entire length of the consensus immunogen, excluding the HA tag and leader sequence. They were synthesized by GenScript (Piscataway, NJ). Each peptide was used at a final concentration of 1.0 μg / mL. One week after the final immunization, antigen-specific cellular responses were assessed using IFNγ ELISpot. For PSA, IFNγ responses were similar for both 10 μg (772.2 ± 138.2 SFU) and 20 μg (771.1 ± 155.2 SFU) vaccine doses (Figure 2A). In contrast, PSMA-specific IFNγ responses increased dose-dependently, with greater responses for the 20 μg vaccine (1585.0 ± 194.0 SFU) compared with the 10 μg vaccine (1047.2 ± 160.7 SFU) (Figure 2B). Minimal background PSA and PSMA responses were observed in untreated mice.

[0152] Example 10 Vaccine-induced CD4+ and CD8+ T cell production of IFNγ, IL-2, and TNFα We further characterized the cellular immunogenicity of co-delivered PSA and PSMA vaccines using flow cytometry. We measured antigen-specific CD4+ and CD8+ T cell production of IFNγ, IL-2, and TNFα for the total vaccine-specific response and the PSA and PSMA components of the total vaccine-specific response (n=5).

[0153] Cellular immune responses were also measured by intracellular cytokine staining and flow cytometry using the CytoFix / CytoPerm kit according to the manufacturer's instructions (BD Biosciences, San Diego, CA). Splenocytes collected from immunized mice were washed with PBS and then resuspended in R10 medium to a final concentration of 10 cells / ml. Cells were seeded in 100 μl of 96-well round-bottom plates and incubated with 100 μl of R10 medium (negative control), medium containing an antigen-specific peptide pool, or medium containing phorbol myristate acetate (PMA, 10 ng / ml) and ionomycin (250 ng / ml; positive control) (Sigma, St. Louis, MO) at 37°C and 5% CO for 6 hours. All stimulation media contained 1 μg / μL of GolgiPlug and GolgiStop (BD Biosciences, San Diego, CA). At the end of the incubation period, the plates were spun down and washed twice with PBS. Cells were then stained with a purple dye (LIVE / DEAD Violet Viability Dye, Invitrogen; Carlsbad, CA) for 30 minutes at 4°C for visualization. After washing with PBS as above, the exterior of the cells was stained with anti-CD4 PerCPCy5.5 and anti-CD8 APC for 30 minutes at 4°C, followed by fixation and permeabilization. Anti-CD3 PE-Cy5, anti-IL-2 PE, anti-IFNγ AlexaFluor-700, and anti-IFNγ AlexaFluor-700 were used. and anti-TNFα FITC (BD Biosciences, San Diego, CA), and the cells were again incubated for 30 minutes at 4° C. Finally, the cells were washed with PBS and fixed with 1% PFA.

[0154] Co-delivery of PSA and PSMA vaccines elicited robust secretion of IFNγ, IL-2, and TNFα by CD4+ T cells. The proportion of PSA-specific IFNγ-producing CD4+ T cells (0.21%) and the proportion of PSMA-specific IFNγ-producing CD4+ T cells (0.24%) contributed equally to the total vaccine-specific CD4+ T cell IFNγ response (0.44%) (Figure 3A). PSMA-specific IL-2-producing CD4+ T cells (1.08%) accounted for the majority of the total vaccine-specific IL-2-producing CD4+ T cells (1.40%) (Figure 3B). The proportion of PSA-induced TNFα-producing CD4+ T cells (0.31%) and the proportion of PSMA-induced NFα-producing CD4+ T cells (0.29%) contributed equally to the total vaccine-specific response (0.60%) (Figure 3C). Overall, CD4+ T cell responses were balanced between PSA and PSMA, with the exception of vaccine-specific CD4+ T cell IL-2 production, which was largely induced by PSMA.

[0155] The vaccine induced robust antigen-specific IFNγ and IL-2 production by CD8+ T cells and, to a lesser extent, TNFα production. Both PSA (0.70%) and PSMA (0.67%) induced robust IFNγ production by CD8+ T cells. Indeed, IFNγ secretion by vaccine-specific CD8+ T cells accounted for 1.37% of the total CD8+ T cell population (Figure 4A). The vaccine also induced robust IL-2 responses (1.54%) by CD8+ T cells. Similar to the IL-2 responses of CD4+ T cells, the proportion of PSMA-specific CD8+ T cells producing IL-2 (1.06%) was approximately twice as high as that of PSA-specific CD8+ T cells (0.47%) (Figure 4B). The overall proportion of TNFα production by vaccine-specific CD8+ T cells (0.11%) was in response to the PSA component of the vaccine (Figure 4C). In summary, there was a high proportion of vaccine-specific CD8+ T cell IFNγ and IL-2 production. Similar to the CD4+ T cell response, IFNγ production was equally balanced between PSA and PSMA, but the PSMA-specific response was greater in magnitude than the PSA-specific response for IL-2.

[0156] Example 11 PSA-specific IgG seroconversion Antibody responses can play an important role in tumor immunotherapy, therefore, we next investigated this parameter of the immune response to the PSA antigen based on the availability of a protein target.

[0157] To measure PSA-specific serum antibody titers, a 96-well Nunc-Immuno MaxiSorp plates (Nunc, Rochester, NY) were coated overnight at 4°C with 1 μg / well of recombinant PSA protein (Fitzgerald Industries, Acton, MA) diluted in PBS. The plates were washed with PBS, 0.05% Tween 20 (PBST), blocked with 10% BSA / PBST for 1 hour at room temperature, and incubated with serial dilutions of serum from immunized or naive animals for 1 hour at room temperature. The plates were then washed three times with PBST, and goat anti-mouse IgG (Santa Cruz, Santa Cruz, CA) was added at a dilution of 1:5,000 in PBST. Bound enzyme was detected with SigmaFASTO-phenylenediamine dihydrochloride (OPD; Sigma-Aldrich, St. Louis, MO), and absorbance was measured at 450 nm on a Biotek (Winooski, VT) plate reader, as shown in Figure 5B. End-point titers were determined as previously described (Frey, A. et al. 1998). Briefly, the upper prediction limit was calculated using the Student's t distribution. The formula defining the upper predicted limit is expressed as standard deviation × coefficient (coefficient based on the number of negative controls (n=5) and the confidence level (95%)). The final titer was reported as the reciprocal of the last dilution that exceeded the upper predicted limit.

[0158] The PSA vaccine not only conferred robust cell-mediated immunity but also induced strong antigen-specific humoral responses. Antibody titers were measured by ELISA in sera isolated from mice (n = 5) 1 week after the final immunization. The vaccine induced a mean PSA-specific antibody endpoint titer of 4,427 (range 1581–15,811) (Figure 5A). The longevity of these responses may also be important.

[0159] Example 12 GenBank provides a range of genetic backgrounds for the manufacturer:g b_EAW71923.1_H.sapiens_klk3_CRAb;_001639. 1_H.sapiens_PSA_iso1_preproprotein;gb_AAA59995.1_H.sapiens_PSA_precursor;gb_AAA601 93.1_H.sapiens_PSA;gb_EAW71933.1_H.sapiens_klk3_CRA_l;NP_001025218.1_H.sapiens_PSA _iso3_preproprotein;gb_CAD54617.1_H.sapiens_PSA;gb_CAD30844.1_H.sapiens_PSA;gb_AAA 59996.1_H.sapiens_PSA_precursor;gb_AAD14185.1_H.sapiens_PSA;Q6DT45.1_M.fasciculari s_KLK3;NP_001036241.1_M.mulatta_PSA_precursor;AAZ82258.1_M.mulatta_PSA;AAZ82255.1_ G.gorilla_PSA;gi|163838666|ref|NP_001106216.1|plasma kallikrein[Papioanubis];gi|73746696|gb|AAZ82261.1|prostatespecific antigen[Papioanubis];i|73746692|gb|AAZ82259.1| prostatespecific antigen[Erythrocebuspatase];gi|73746694|gb|AAZ82260.1|prostatespecificantigen[Cercopithecuscephus];gi|73746682|gb|AAZ82254.1|prostatespecificantigen[P anpaniscus];gi|73746680|gb|AAZ82253.1|prostatespecificantigen[Panthroglodytes];gi|73746686|gb|AAZ82256.1|prostatespecificantigen[Pongopygmaeus];and also3746688|gb|AAZ82257.1| prostate specific antigen [Nomascus gabriellae]. .

[0160] PSMA amino acid sequences available in GenBank include: NP_004467.1_Human_GCPII_iso1; Human_PSMA_AAC83972.1; M. mulatta_GCPII_iso1XP_001096141.2; and M. mulatta_GCPII_iso2_XP_002799784.1.

[0161] STEAP amino acid sequences available in GenBank include: NP036581.1_Human_STEAP1; EAL24167.1_Human_STEAP1; XP001103605.1_M. mulatta_STEAP1_iso3; EAW93751.1_Human_STEAP1_CRAb; EAW93749.1_Human_STEAP1_CRAa; XP001164838.1_P. troglodytes_STEAPiso2; XP002818311.1_P. abelii_STEAP1; NP001162459.1_P. anubis_STEAP1; NP_999470.1_S. scrofa_STEAP1; and NP_081675.2_M. musculus_STEAP1.

[0162] NP_005663.2_Human_PSCA is the accession number for the PSCA amino acid sequence available in GenBank.

Claims

[Claim 1] The method described in the specification.

Images