Cancer vaccines and methods of treatment using the same

Abstract

To provide compositions and methods for treating cancer and in particular vaccines against tumor growth.SOLUTION: Disclosed herein are compositions and methods for treating cancer and in particular vaccines that treat and provide protection against tumor growth.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Technical Field Disclosed herein are cancer therapeutic compositions and methods for treating cancer, particularly vaccines that treat and provide protection against tumor growth. [Background technology]

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 61 / 799,952, filed March 15, 2013, the entire teachings of which are incorporated herein by reference.

[0003] background Cancer is one of the leading causes of death worldwide and in the United States, and is the second most common cause of death, accounting for approximately one-quarter of all deaths. Cancer arises from a single cell that transforms from a normal cell into a tumor cell. Such transformation is often a multistep process that progresses from a precancerous lesion to a malignant tumor. Many factors contribute to this progression, including aging, hereditary contributions, and exposure to external agents such as physical carcinogens (e.g., ultraviolet light and ionizing radiation), chemical carcinogens (e.g., asbestos, components of tobacco smoke), and biological carcinogens (e.g., certain viruses, bacteria, and parasites).

[0004] Cancer prevention, diagnosis, and treatment can take many different forms. Prevention can include testing for predisposing factors (e.g., specific gene mutations), modifying habits (e.g., smoking, diet, and amount of exercise), and vaccination against viruses (e.g., human papillomavirus, hepatitis B virus, etc.). Treatment can include chemotherapy, radiation therapy, and surgical removal of tumors or cancerous tissue. Despite the availability of numerous prevention and treatment methods, such methods often have limited success in effectively preventing and / or treating common cancers.

[0005] Thus, there is a need for the identification and development of cancer prevention and / or treatment compositions and methods to facilitate clinical management of protection against disease progression. Additionally, there is a need for more effective treatments to slow disease progression and / or reduce mortality in subjects afflicted with cancer. Summary of the Invention

[0006] Summary of the Invention The present invention relates to vaccines comprising nucleic acid or amino acid sequences of one or more cancer antigens that stimulate an immune response against specific cancers or tumors associated with specific cancers that are no longer self-antigens. The vaccines can further comprise immune checkpoint inhibitors, such as anti-PD-1 and anti-PDL-1 antibodies, that prevent the suppression of any component of the immune system, such as presentation by MHC classes, presentation by and / or differentiation of T cells, presentation by and / or differentiation of B cells, or any cytokine, chemokine, or signaling for immune cell proliferation and / or differentiation. The one or more cancer antigens of the vaccines can be one or more amino acid sequences, or an amino acid sequence that is 95% or more identical to the amino acid sequence of tyrosinase (Tyr), tyrosinase-related protein 1 (TYRP1), tyrosinase-related protein 2 (TYRP2), melanoma-associated antigen 4 protein (MAGEA4), growth hormone-releasing hormone (GH-R), or growth hormone-releasing hormone (GH-R). The nucleic acid may encode an amino acid sequence selected from the group consisting of an amino acid sequence 95% or more identical to the amino acid sequence of Human Hormone (GHRH), an amino acid sequence 95% or more identical to the amino acid sequence of MART-1 / Melan-A antigen (MART-1 / Melan-A), an amino acid sequence 95% or more identical to the amino acid sequence of a cancer-testis antigen (NY-ESO-1), an amino acid sequence 95% or more identical to the amino acid sequence of a cancer-testis antigen II (NY-ESO-2), an amino acid sequence 95% or more identical to the amino acid sequence of PRAME, an amino acid sequence 95% or more identical to the amino acid sequence of WT1, an amino acid sequence 95% or more identical to the amino acid sequence of hTERT, or a combination thereof. The vaccine may further comprise a nucleic acid encoding one or more antigens selected from the group consisting of PSA, PSMA, STEAP, PSCA, MAGE A1, gp100, a viral antigen, and a combination thereof.

[0007] The present invention further relates to a method for preventing or treating cancer in a subject in need thereof, comprising administering to the subject in need thereof a vaccine comprising a specific number of cancer antigens for treating or preventing a specific cancer, the method comprising administering to the subject in need thereof a vaccine comprising a CMV cancer antigen for treating or preventing glioblastoma, or administering to the subject in need thereof a vaccine comprising a CMV cancer antigen in combination with one or more of the cancer antigens hTERT, NY-ESO-1, MAGE-A1, or WT1 for treating or preventing glioblastoma, or administering to the subject in need thereof a vaccine comprising one or more of the cancer antigens PSA, PSMA, or STEAP for treating or preventing prostate cancer, or administering to the subject in need thereof a vaccine comprising one or more of the cancer antigens PSA, PSMA, or STEAP in combination with the cancer antigens PSA, PSMA, or STEAP for treating or preventing prostate cancer. administering a vaccine comprising one or more of the cancer antigens tyrosinase, PRAME, or GP-100 in combination with one or more of the cancer antigens hTERT, NY-ESO-1, MAGE-A1, or WT1 to treat or prevent melanoma to a subject in need of the above-mentioned preventive or therapeutic method; administering a vaccine comprising one or more of the cancer antigens tyrosinase, PRAME, or GP-100 in combination with one or more of the cancer antigens hTERT, NY-ESO-1, MAGE-A1, or WT1 to treat or prevent melanoma to a subject in need of the above-mentioned preventive or therapeutic method; administering a vaccine comprising one or more of the cancer antigens HPV 16 E6 or HPV 16 E7 to treat or prevent head and neck cancer to a subject in need of the above-mentioned preventive or therapeutic method;administering a vaccine comprising E7 in combination with one or more of the cancer antigens hTERT, NY-ESO-1, MAGE-A1, or WT1; administering a vaccine comprising one or more of the cancer antigens tyrosinase, PRAME, or GP-100 to treat or prevent melanoma to a subject in need of the above preventive or therapeutic method; administering a vaccine comprising tyrosinase, PRAME, or GP-100 in combination with one or more of the cancer antigens hTERT, NY-ESO-1, MAGE-A1, or WT1 to treat or prevent melanoma to a subject in need of the above preventive or therapeutic method; administering a vaccine comprising one or more of the cancer antigens HPV 6, HPV 11, or HPV 16 to treat or prevent anal cancer to a subject in need of the above preventive or therapeutic method; administering a vaccine comprising HBV core antigen, HBV surface antigen, HCV NS34A, HCV NS5A, HCV NS5B, or HCV NS4B in combination with one or more of the cancer antigens hTERT, NY-ESO-1, MAGE-A1, or WT1 to a subject in need of the above preventive or therapeutic method for treating or preventing liver cancer; or administering a vaccine comprising HBV core antigen, HBV surface antigen, HCV NS34A, HCV NS5A, HCV NS5B, or HCV NS4B in combination with one or more of the cancer antigens hTERT, NY-ESO-1, MAGE-A1, or WT1 to a subject in need of the above preventive or therapeutic method for treating or preventing liver cancer; administering a vaccine comprising HPV 16 E6 / E7 or HPV 18 E6 / E7 or HPV 18 E7 / E7 or HPV 18 E8 / E8 or HPV 18 E9 / E9 or HPV 18 E10 / E11 or HPV 18 E11 / E12 or HPV 18 E12 / E13 or HPV 18 E13 / E14 or HPV 18 E14 / E15 or HPV 18 E15 / E16 or HPV 18 E16 / E17 or HPV 18 E18 / E19 or HPV 18 E20 / E21 or HPV 18 E21 / E22 or HPV 18 E22 / E23 or HPV 18 E30 / E31 or HPV 18 E31 / E32 or HPV 18 E40 / E41 or HPV 18 E50 / E51 or HPV 18 E6 / E7 or HPV 18 E16 / E18 or HPV 18 E18 / E19 or HPV 18 E22 / E16 or HPV 18 E3 or administering a vaccine containing one or more of HPV 16 E6 / E7 or HPV 18 E6 / E7 to a subject in need of the above prevention or treatment method to treat or prevent cervical cancer.or administering to a subject in need thereof a vaccine comprising E6 / E7 in combination with one or more of the cancer antigens hTERT, NY-ESO-1, MAGE-A1, or WT1, or administering to a subject in need thereof a vaccine comprising one or more of the cancer antigens PRAME, WT-1, or hTERT in combination with one or more of the cancer antigens NY-ESO-1 or MAGE-A1 in order to treat or prevent blood cancer, or administering to a subject in need thereof a vaccine comprising PRAME, WT-1, or hTERT in combination with one or more of the cancer antigens NY-ESO-1 or MAGE-A1 in order to treat or prevent blood cancer, and the administering steps (a) to (i) may further comprise combining with an immune checkpoint inhibitor selected from the group consisting of an anti-PD-1 antibody, an anti-PD-L1 antibody, and a combination thereof. [Brief explanation of the drawings]

[0008] [Figure 1A] FIG. 1 shows the construction of pTyr. [Figure 1B] FIG. 1 shows the construction of pTyr. [Figure 1C] FIG. 1 shows the construction of pTyr. [Figure 1D] FIG. 1 shows the construction of pTyr. [Figure 1E] FIG. 1 shows the construction of pTyr. [Figure 2] (A) and (B) show the immunization strategy and induction of cell-mediated immune responses by vaccination with Tyr DNA, respectively. [Figure 3] FIG. 1 shows flow fluorescence-activated cell sorting (FACS) of control and immunized mice. [Figure 4] FIG. 1 shows the induction of tyrosinase-specific antibodies in immunized mice. [Figure 5] Figure 1 shows Kaplan-Meier survival curves and tumor volume curves after tumor challenge in control and immunized mice, respectively. [Figure 6] FIG. 1 shows MDSC cell numbers in immunized and non-immunized mice. [Figure 7] FIG. 1 shows staining for MDSCs in mice immunized with pVax1 and pTyr. [Figure 8A] FIG. 1 shows secretion of MCP-1 by MDSCs. [Figure 8B] FIG. 1 shows secretion of MCP-1 by MDSCs. [Figure 9] FIG. 1 shows the phylogenetic relationships of Tyr nucleotide sequences among the animals listed. [Figure 10A] (A) Schematic representation of the plasmid map of pPRAME (also known herein as pGX1411). [Figure 10B] (B) Staining of RD and 293T cells with DAPI for nuclei and for the consensus PRAME antigen. [Figure 10C] (C) Western blotting for consensus PRAME antigen in lysates from untransfected cells ("control"), cells transfected with pVAX ("pVAX"), and cells transfected with pPRAME ("PRAME-pVAX"). [Figure 11A] FIG. 1 shows a graph plotting interferon gamma (IFN-γ) spot forming units (SFU) / 10 6 splenocytes against mouse group. [Figure 11B] FIG. 1 shows a graph plotting interferon gamma (IFN-γ) spot forming units (SFU) / 10 6 splenocytes against mouse group. [Figure 12A] (A) Schematic representation of the plasmid map of pNY-ESO-1 (also known herein as pGX1409). [Figure 12B](B) Staining of cells for nuclei with DAPI and for the consensus NY-ESO-1 antigen. [Figure 12C] (C) Western blotting for consensus NY-ESO-1 antigen in RD and 293T lysates from untransfected cells ("control"), cells transfected with pVAX ("pVAX"), and cells transfected with pNY-ESO-1 ("pNY-ESO-1"). [Figure 13] FIG. 1 shows a graph plotting interferon-gamma (IFN-γ) spot-forming units (SFU) / 10 6 splenocytes against mouse group. [Figure 14] FIG. 1 shows a graph plotting interferon-gamma (IFN-γ) spot-forming units (SFU) / 10 6 splenocytes against mouse group. [Figure 15] FIG. 1 is a schematic diagram showing some of the different cancers and their associated cancer antigen(s). DETAILED DESCRIPTION OF THE INVENTION

[0009] Detailed Description The present invention relates to vaccines that can be customized for specific cancers and tumors. For use in vaccines, consensus antigen sequences have been designed for specific cancer-associated antigens, such as tyrosinase (Tyr), preferentially expressed antigen in melanoma (PRAME), tyrosinase-related protein 1 (Tyrp1), cancer-testis antigen (NY-ESO-1), hepatitis B virus antigen, and Wilms' tumor 1 antigen (WT-1), enabling customized vaccines for the prevention and treatment of specific cancers. For example, tyrosinase antigens can be used in vaccines for the prevention or treatment of melanoma. The vaccines of the present invention can provide any combination of specific cancer antigens for the specific prevention or treatment of cancer in a subject in need of treatment.

[0010] One method for designing the amino acid sequence of the nucleic acid and the recombinant cancer antigen encoded thereby is by introducing mutations that change specific amino acids in the entire amino acid sequence of the unmutated cancer antigen. The introduction of mutations does not significantly alter the cancer antigen so that it cannot be universally applied to mammalian subjects, preferably human or canine subjects, but the introduction of mutations alters the cancer antigen sufficiently that the resulting amino acid sequence exceeds the acceptable range, i.e., it is considered a foreign antigen that can elicit an immune response. Another method can be to create a recombinant cancer antigen that has at least 85% and up to 99% amino acid sequence identity, preferably at least 90% and up to 98%, more preferably at least 93% and up to 98%, or even more preferably at least 95% and up to 98% sequence identity, to its corresponding unmutated cancer antigen. In some examples, the recombinant cancer antigen has 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to its corresponding unmutated cancer antigen. The unmutated cancer antigen is an antigen normally associated with a particular cancer or cancer tumor. Depending on the cancer antigen, the consensus sequence for the cancer antigen can be across mammals, within a subtype of a species, or across viral strains or serotypes. Some cancer antigens do not vary significantly from their wild-type amino acid sequence. Some cancer antigens have nucleic acid / amino acid sequences that vary significantly between species, making it impossible to generate a consensus sequence. In these cases, a recombinant cancer antigen that will elicit an immune response beyond acceptable limits is generated when the recombinant cancer antigen has at least 85% and up to 99% amino acid sequence identity, preferably at least 90% and up to 98%, more preferably at least 93% and up to 98%, and even more preferably at least 95% and up to 98% sequence identity, to its corresponding unmutated cancer antigen. In some examples, the recombinant cancer antigen has 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to its corresponding unmutated cancer antigen.The above approaches can be combined so that the final recombinant cancer antigen has a percent similarity to the amino acid sequence of the unmutated cancer antigen as discussed above.

[0011] The recombinant cancer antigens can induce antigen-specific T cell and / or high-titer antibody responses, thereby inducing or eliciting an immune response directed against or reactive to cancers or tumors expressing the antigen. In some embodiments, the induced or elicited immune response can be a cellular immune response, a humoral immune response, or both a cellular and humoral immune response. In some embodiments, the induced or elicited cellular immune response can include the induction or secretion of interferon-gamma (IFN-γ) and / or tumor necrosis factor alpha (TNF-α). In other embodiments, the induced or elicited immune response can reduce or inhibit one or more immunosuppressive factors that promote the growth of tumors or cancers that express the antigen, including, but not limited to, factors that downregulate MHC presentation, antigen-specific regulatory T cells (Tregs), cytokines such as PD-L1, FasL, IL-10, and TFG-β, soluble factors produced by tumor-associated macrophages, tumor-associated fibroblasts, immunosuppressive cells, CTLA-4, PD-1, MDSC, MCP-1, and factors that upregulate immune checkpoint molecules.

[0012] The above vaccines may be further combined with antibodies against checkpoint inhibitors, such as PD-1 and PDL-1, to increase stimulation of both cellular and humoral immune responses. Using anti-PD-1 or anti-PDL-1 antibodies prevents PD-1 or PDL-1 from suppressing T cell and / or B cell responses. Overall, designing cancer antigens that are recognized by the immune system helps overcome other forms of immunosuppression by cancer cells, and these vaccines can be used in combination with suppressive or blocking therapies (such as anti-PD-1 and anti-PDL-1 antibody therapy) to further increase T cell and / or B cell responses.

[0013] 1.Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In the case of conflict, the present specification, including definitions, will control. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. It should be noted that the materials, methods, and examples disclosed herein are illustrative only and are not intended to be limiting. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0014] As used herein, the terms "comprise(s), "include(s), "having," "has," "can," "contain(s)," and variations thereof are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms "a," "and," and "the" may also have plural meanings unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments that "comprising," "consisting of," and "consisting essentially of" the embodiments or elements set forth herein, whether or not explicitly stated.

[0015] For the recitation of numerical ranges herein, each intervening number is expressly contemplated to the same degree of precision. For example, in the range 6 to 9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and in the range 6.0 to 7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are expressly contemplated.

[0016] As used herein, "adjuvant" refers to any molecule added to the DNA plasmid vaccines described herein to enhance the immunogenicity of the antigen encoded by the DNA plasmid and encoding nucleic acid sequence described herein below.

[0017] As used herein, "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 antibodies, diabodies, bispecific antibodies, bifunctional antibodies, and derivatives thereof. The antibody may be an antibody isolated from a mammalian serum sample, a polyclonal antibody, an affinity-purified antibody, or a mixture thereof that exhibits sufficient binding specificity for a desired epitope or a sequence derived therefrom.

[0018] As used herein, "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 the cells of an individual or mammal to which the nucleic acid is administered.

[0019] As used herein, "complement" or "complementary" means that a nucleic acid can exhibit Watson-Crick (e.g., AT / U and CG) or Hoogsteen base pairing between nucleotides or nucleotide analogs of a nucleic acid molecule.

[0020] As used herein, "consensus" or "consensus sequence" refers to a polypeptide sequence based on an alignment analysis of multiple sequences for the same gene from different organisms. Nucleic acid sequences encoding consensus polypeptide sequences can be prepared. Vaccines containing proteins comprising consensus sequences and / or nucleic acid molecules encoding such proteins can be used to induce broad immunity against antigens.

[0021] As used interchangeably herein, "electroporation," "electropermeabilization," and "electrokinetic enhancement" ("EP") refer to the use of transmembrane electric field pulses to generate microchannels (pores) in biological membranes. The presence of these microchannels allows biomolecules, such as plasmids, oligonucleotides, siRNA, drugs, ions, and water, to pass from one side of the cell membrane to the other.

[0022] As used herein, the term "fragment" in reference to a nucleic acid sequence refers to a nucleic acid sequence, or a portion thereof, that encodes a polypeptide capable of eliciting an immune response in a mammal that cross-reacts with an antigen described herein. The fragment can be a DNA fragment selected from at least one of the various nucleotide sequences encoding protein fragments described below. The fragment can comprise at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of one or more of the nucleic acid sequences described below. In some embodiments, the fragments are at least 20 nucleotides or more, at least 30 nucleotides or more, at least 40 nucleotides or more, at least 50 nucleotides or more, at least 60 nucleotides or more, at least 70 nucleotides or more, at least 80 nucleotides or more, at least 90 nucleotides or more, at least 100 nucleotides or more, at least 150 nucleotides or more, at least 200 nucleotides or more, at least 250 nucleotides or more, at least 30 ... It can comprise at least 350 nucleotides or more, at least 400 nucleotides or more, at least 450 nucleotides or more, at least 500 nucleotides or more, at least 550 nucleotides or more, at least 600 nucleotides or more, at least 650 nucleotides or more, at least 700 nucleotides or more, at least 750 nucleotides or more, at least 800 nucleotides or more, at least 850 nucleotides or more, at least 900 nucleotides or more, at least 950 nucleotides or more, at least 1000 nucleotides or more.

[0023] "Fragment" or "immunogenic fragment" as used with respect to a polypeptide sequence refers to a polypeptide capable of eliciting an immune response in a mammal that cross-reacts with an antigen described herein. The fragment can be a polypeptide fragment selected from at least one of the following various amino acid sequences: A fragment of a consensus protein can comprise at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the consensus protein. In some embodiments, a fragment of a consensus protein can comprise at least 20 amino acids or more, at least 30 amino acids or more, at least 40 amino acids or more, at least 50 amino acids or more, at least 60 amino acids or more, at least 70 amino acids or more, at least 80 amino acids or more, at least 90 amino acids or more, at least 100 amino acids or more, at least 110 amino acids or more, at least 120 amino acids or more, at least 130 amino acids or more, at least 140 amino acids or more, at least 150 amino acids or more, at least 160 amino acids or more, at least 170 amino acids or more, or at least 180 amino acids or more of a protein sequence disclosed herein.

[0024] As used herein, the term "genetic construct" refers to a DNA or RNA molecule comprising a nucleotide sequence that encodes a protein. The coding sequence includes start and stop signals operably linked to regulatory elements, including a promoter and polyadenylation signal, that are capable of directing expression in the cells of an individual to whom the nucleic acid molecule is administered. As used herein, the term "expressible form" refers to a genetic construct that includes the necessary regulatory elements operably linked to a coding sequence that encodes a protein such that the coding sequence is expressed when present in the cells of an individual.

[0025] As used herein, the term "homology" refers to the degree of complementarity. There can be partial or complete homology (i.e., identity). A partially complementary sequence that at least partially inhibits a completely complementary sequence from hybridizing to a target nucleic acid is referred to using the functional term "substantially homologous." When used in reference to a double-stranded nucleic acid sequence, such as a cDNA or genomic clone, the term "substantially homologous" refers to a probe that can hybridize to a strand of the double-stranded nucleic acid sequence under low stringency conditions. When used in reference to a single-stranded nucleic acid sequence, the term "substantially homologous" refers to a probe that can hybridize to (i.e., is the complement of) a single-stranded nucleic acid template sequence under low stringency conditions.

[0026] As used herein, "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, the two sequences are optimally aligned, compared over a specified region, and the number of positions where identical residues occur in both sequences is determined to obtain the number of matching positions. The number of matching positions is divided by the total number of positions in the specified region, and the result is multiplied 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.

[0027] As used herein, "immune response" means activation of a host's immune system, e.g., a mammal's immune system, in response to the introduction of an antigen. This immune response can be cellular or humoral in form, or both.

[0028] As used herein, "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 depicted 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 probes that hybridize under stringent hybridization conditions.

[0029] Nucleic acids can be single-stranded or double-stranded, and can contain portions of both double-stranded and single-stranded sequences. Nucleic acids can be DNA, 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.

[0030] As used herein, "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.

[0031] As used herein, "peptide," "protein," or "polypeptide" can refer to a linked sequence of amino acids, which can be natural, synthetic, or modified natural and synthetic, or a combination thereof.

[0032] As used herein, a "promoter" refers to a synthetic or naturally occurring molecule capable of conferring, activating, or enhancing the expression of a nucleic acid in a cell. 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 a bacteriophage T7 promoter, a bacteriophage T3 promoter, an SP6 promoter, a lac operator promoter, a tac promoter, an SV40 late promoter, an SV40 early promoter, an RSV-LTR promoter, a CMV IE promoter, an SV40 early promoter, an SV40 late promoter, and a CMV IE promoter.

[0033] "Signal peptide" and "leader sequence" are used interchangeably herein and refer to an amino acid sequence that can be attached to the amino terminus of a protein described herein. Generally, a signal peptide / leader sequence directs the localization of a protein. As used herein, a signal peptide / leader sequence preferably facilitates secretion of the protein from the cell in which it is produced. The signal peptide / leader sequence is often cleaved from the rest of the protein (often referred to as the mature protein) upon secretion from the cell. The signal peptide / leader sequence is attached to the amino terminus (i.e., N-terminus) of the protein.

[0034] As used herein, "stringent hybridization conditions" refers to the conditions under which a first nucleic acid sequence (e.g., a probe) hybridizes 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 vary from situation to situation. Stringent conditions can be selected to be approximately 5-10°C lower than the melting temperature (Tm) for a particular sequence at a defined ionic strength and pH. Tm can be 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, at Tm 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., more 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. Examples of stringent hybridization conditions include: 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.

[0035] As used herein, "subject" means a mammal desiring or needing to be immunized with a vaccine described herein. The mammal may be a human, chimpanzee, dog, cat, horse, cow, mouse, or rat.

[0036] As used herein, "substantially complementary" refers to a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 180, 270, 360, 450, 540, or more nucleotides or amino acids that is substantially complementary to a first sequence. It means that a sequence is at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the complement of a second sequence, or that the two sequences hybridize under stringent hybridization conditions.

[0037] As used herein, "substantially identical" refers to sequences over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 180, 270, 360, 450, 540, or more nucleotides or amino acids. It means that a first sequence and a second sequence are at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, or, with respect to nucleic acids, where a first sequence is substantially complementary to the complement of a second sequence.

[0038] As used herein, "treatment" or "treating" can mean protecting an animal from a disease by preventing, suppressing, repressing, or completely eliminating the disease. Preventing a disease involves administering a vaccine of the invention to an animal before the onset of the disease. Suppressing a disease involves administering a vaccine of the invention to an animal after induction of the disease but before the clinical appearance of the disease. Suppressing a disease involves administering a vaccine of the invention to an animal after the clinical appearance of the disease.

[0039] As used herein, "variant" 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.

[0040] A "variant," as used with respect to a peptide or polypeptide, 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, retaining at least one biological activity. Conservative amino acid substitutions, i.e., replacing one amino acid with another with 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 of an amino acid, as understood in the art. Kyte et al., J. Mol. Biol. 157:105-132 (1982). The hydropathic index of an amino acid is based on consideration of its hydrophobicity and charge. It is known in the art that amino acids with similar hydropathic indices can be substituted and still retain protein function. In one embodiment, amino acids with hydropathic indices of ±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 peptide's maximum local average hydrophilicity can be calculated, a useful measure that has been reported to correlate well with antigenicity and immunogenicity. U.S. Patent No. 4,554,101, which is incorporated herein by reference in its entirety. As understood in the art, substituting amino acids with similar hydrophilicity values ​​results in peptides that retain biological activity, e.g., immunogenicity. Substitutions can be made using amino acids with hydrophilicity values ​​within ±2 of each other. Both the hydropathic index and hydrophilicity value of an amino acid are influenced by the specific side chain of that amino acid. Consistent with this knowledge, amino acid substitutions that are compatible with biological function are understood to depend on the relative similarity of amino acids, particularly the relative similarity of the amino acid's side chains, as manifested by hydrophobicity, hydrophilicity, charge, size, and other properties.

[0041] A variant may be a nucleic acid sequence that is substantially identical over the entire length of the complete gene sequence or a fragment thereof. The nucleic acid sequence may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the entire length of the gene sequence or a fragment thereof. A variant may be an amino acid sequence that is substantially identical over the entire length of the amino acid sequence or a fragment thereof. The amino acid sequence may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the entire length of the amino acid sequence or a fragment thereof.

[0042] As used herein, "vector" refers to a nucleic acid sequence that includes an origin of replication. The vector can be a viral vector, a bacteriophage, a bacterial artificial chromosome, or a yeast artificial chromosome. The vector can be a DNA vector or an RNA vector. The vector is a self-replicating extrachromosomal vector, and preferably a DNA plasmid. The vector can contain or include one or more heterologous nucleic acid sequences.

[0043] 2. Vaccines The present invention relates to an anti-cancer vaccine. The vaccine may contain one or more cancer antigens. The vaccine may prevent tumor growth. The vaccine may reduce tumor growth. The vaccine may prevent tumor cell metastasis. Depending on the cancer antigen, the vaccine may be intended to treat liver cancer, prostate cancer, melanoma, blood cancer, head and neck cancer, glioblastoma, recurrent respiratory papillomatosis, anal cancer, cervical cancer, and brain cancer.

[0044] The first step in developing such a vaccine is to identify cancer antigens that are not recognized by the immune system and are self-antigens. The identified cancer antigens are then changed from self-antigens to foreign antigens so that they can be recognized by the immune system. Redesigning the nucleic acid and amino acid sequences of recombinant cancer antigens from self-antigens to foreign antigens allows the immune system to exceed its tolerance range. To exceed this tolerance range, several redesign methods, as described below, can be applied to cancer antigens.

[0045] The recombinant cancer antigens of the vaccine are not recognized as self-antigens and therefore exceed tolerance. This tolerance overshoot can induce antigen-specific T cell and / or high-titer antibody responses, thereby inducing or eliciting an immune response directed against or reactive to the cancer or tumor expressing the antigen. In some embodiments, the induced or elicited immune response can be a cellular immune response, a humoral immune response, or both a cellular and humoral immune response. In some embodiments, the induced or elicited cellular immune response can include the induction or secretion of interferon-gamma (IFN-γ) and / or tumor necrosis factor alpha (TNF-α). In other embodiments, the induced or elicited immune response can reduce or inhibit one or more immunosuppressive factors that promote the growth of tumors or cancers that express the antigen, such as, but not limited to, factors that downregulate MHC presentation, antigen-specific regulatory T cells (Tregs), cytokines such as PD-L1, FasL, IL-10 and TFG-β, soluble factors produced by tumor-associated macrophages, tumor-associated fibroblasts, immunosuppressive cells, CTLA-4, PD-1, MDSC, MCP-1, and factors that upregulate immune checkpoint molecules.

[0046] In certain embodiments, the vaccine can mediate tumor elimination or prevent tumor cell growth by inducing: (1) a humoral immune response via a B cell response to generate antibodies that block the production of monocyte chemoattractant protein-1 (MCP-1), thereby inhibiting myeloid derived suppressor cells (MDSCs) and suppressing tumor growth; and (2) CD8 to attack and kill tumor cells. + (3) an increase in cytotoxic T lymphocytes (CTLs) such as IFN-γ and IFN-α, (4) an increase in helper T cell responses, and (5) an increase in inflammatory responses mediated by IFN-γ and IFN-α, or preferably all of the above. The vaccine can increase tumor-free survival by 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, and 45%. The vaccine can reduce tumor size by 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, and 60% after immunization. The vaccine can prevent and inhibit the increase in monocyte chemoattractant protein-1 (MCP-1), a cytokine secreted by myeloid-derived suppressor cells. The above vaccines can increase tumor survival rates by 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, and 60%.

[0047] The above vaccine can increase the cellular immune response in a subject administered the vaccine by about 50 to about 6,000 times, about 50 to about 5,500 times, about 50 to about 5,000 times, about 50 to about 4,500 times, about 100 to about 6,000 times, about 150 to about 6,000 times, about 200 to about 6,000 times, about 250 to about 6,000 times, or about 300 to about 6,000 times, compared to the cellular immune response in a subject not administered the vaccine. In some embodiments, the vaccine increases a cellular immune response in a subject administered the vaccine by about 50 fold, 100 fold, 150 fold, 200 fold, 250 fold, 300 fold, 350 fold, 400 fold, 450 fold, 500 fold, 550 fold, 600 fold, 650 fold, 700 fold, 750 fold, 800 fold, 850 fold, 900 fold, 950 fold, 1000 fold, 1100 fold, 1200 fold, 1300 fold, 1400 fold, 1500 fold, 1600 fold, 1700 fold, 1800 fold, 1900 fold, 2000 fold, 2100 fold, The increase may be 2200x, 2300x, 2400x, 2500x, 2600x, 2700x, 2800x, 2900x, 3000x, 3100x, 3200x, 3300x, 3400x, 3500x, 3600x, 3700x, 3800x, 3900x, 4000x, 4100x, 4200x, 4300x, 4400x, 4500x, 4600x, 4700x, 4800x, 4900x, 5000x, 5100x, 5200x, 5300x, 5400x, 5500x, 5600x, 5700x, 5800x, 5900x, or 6000x.

[0048] The above vaccine can increase the interferon-gamma (IFN-γ) concentration in a subject administered the vaccine by about 50 to about 6,000 times, about 50 to about 5,500 times, about 50 to about 5,000 times, about 50 to about 4,500 times, about 100 to about 6,000 times, about 150 to about 6,000 times, about 200 to about 6,000 times, about 250 to about 6,000 times, or about 300 to about 6,000 times compared to the IFN-γ concentration in a subject not administered the vaccine. In some embodiments, the vaccine increases IFN-γ levels in a subject administered the vaccine by about 50-fold, 100-fold, 150-fold, 200-fold, 250-fold, 300-fold, 350-fold, 400-fold, 450-fold, 500-fold, 550-fold, 600-fold, 650-fold, 700-fold, 750-fold, 800-fold, 850-fold, 900-fold, 950-fold, 1000-fold, 1100-fold, 1200-fold, 1300-fold, 1400-fold, 1500-fold, 1600-fold, 1700-fold, 1800-fold, 1900-fold, 2000-fold, 2100-fold, The increase may be 2200x, 2300x, 2400x, 2500x, 2600x, 2700x, 2800x, 2900x, 3000x, 3100x, 3200x, 3300x, 3400x, 3500x, 3600x, 3700x, 3800x, 3900x, 4000x, 4100x, 4200x, 4300x, 4400x, 4500x, 4600x, 4700x, 4800x, 4900x, 5000x, 5100x, 5200x, 5300x, 5400x, 5500x, 5600x, 5700x, 5800x, 5900x, or 6000x.

[0049] The vaccine can be a DNA vaccine. 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 entireties. The DNA vaccine can further include an element or agent that inhibits the vaccine from being integrated into a chromosome.

[0050] The vaccine can be RNA of one or more cancer antigens, and the RNA vaccine can be introduced into cells.

[0051] The vaccines described above are disclosed in, but not limited to, 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 , No. 174,993, No. 5,223,424, No. 5,225,336, No. 5,240,703, No. 5,242,829, No. 5,294,441 , No. 5,294,548, No. 5,310,668, No. 5,387,744, No. 5,389,368, No. 5,424,065, No. 5,451,49 The vaccines may be live attenuated vaccines, vaccines using recombinant vectors to deliver antigens, subunit vaccines, and glycoprotein vaccines, such as those described in US Pat. Nos. 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.

[0052] The vaccines of the present invention can have the characteristics required for an effective vaccine, such as being safe so that the vaccine itself does not cause illness or death; being protective against disease; inducing neutralizing antibodies; inducing protective T cell responses; and being easy to administer, with few side effects, biologically stable, and low cost per dose. The vaccine can achieve some or all of these characteristics by including the cancer antigens discussed below.

[0053] As described in more detail below, the vaccine can further comprise one or more inhibitors of one or more immune checkpoint molecules (i.e., immune checkpoint inhibitors). Immune checkpoint molecules are described in more detail below. An immune checkpoint inhibitor is any nucleic acid or protein that prevents the suppression of any element in the immune system, such as presentation by MHC classes, presentation by and / or differentiation of T cells, presentation by and / or differentiation of B cells, any cytokine, chemokine, or signaling for immune cell proliferation and / or differentiation. As also described in more detail below, the vaccine can be further combined with antibodies against checkpoint inhibitors, such as PD-1 and PDL-1, to increase stimulation of both cellular and humoral immune responses. Using an anti-PD-1 or anti-PDL-1 antibody prevents PD-1 or PDL-1 from suppressing T cell and / or B cell responses.

[0054] A cancer antigen The vaccine can comprise one or more cancer antigens. The cancer antigen can be a nucleic acid sequence, an amino acid sequence, or a combination thereof. The nucleic acid sequence can be DNA, RNA, cDNA, a variant thereof, a fragment thereof, or a combination thereof. The nucleic acid sequence can also comprise an additional sequence encoding a linker or tag sequence linked to the cancer antigen by a peptide bond. The amino acid sequence can be a protein, a peptide, a variant thereof, a fragment thereof, or a combination thereof. The cancer antigen can be a recombinant cancer antigen.

[0055] One method for designing the amino acid sequence of the nucleic acid and the recombinant cancer antigen encoded thereby is to introduce mutations that change specific amino acids in the entire amino acid sequence of the unmutated cancer antigen. The introduction of mutations does not significantly alter the cancer antigen so that it cannot be universally applied to mammalian subjects, preferably human or canine subjects, but the introduction of mutations alters the cancer antigen sufficiently that the resulting amino acid sequence exceeds the acceptable range, i.e., is considered a foreign antigen for eliciting an immune response. Alternatively, recombinant cancer antigens can be created that have at least 85% and up to 99% amino acid sequence identity, preferably at least 90% and up to 98%, more preferably at least 93% and up to 98%, or even more preferably at least 95% and up to 98% sequence identity, to the corresponding unmutated cancer antigen. In some examples, the recombinant cancer antigen has 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to the corresponding unmutated cancer antigen. The unmutated cancer antigen is an antigen normally associated with a particular cancer or cancer tumor. Depending on the cancer antigen, the consensus sequence for the cancer antigen can be across mammals, within a subtype of a species, or across viral strains or serotypes. Some cancer antigens do not vary significantly from their wild-type amino acid sequence. Some cancer antigens have nucleic acid / amino acid sequences that vary significantly between species, making it impossible to generate a consensus sequence. In these cases, a recombinant cancer antigen that will elicit an immune response beyond acceptable limits is generated when the recombinant cancer antigen has at least 85% and up to 99% amino acid sequence identity, preferably at least 90% and up to 98%, more preferably at least 93% and up to 98%, or even more preferably at least 95% and up to 98% sequence identity to its corresponding unmutated cancer antigen. In some examples, the recombinant cancer antigen has 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to its corresponding unmutated cancer antigen.The above approaches can be combined so that the final recombinant cancer antigen has a percent similarity to the amino acid sequence of the unmutated cancer antigen as discussed above.

[0056] The cancer antigen is one or more of the following antigens: tyrosinase (Tyr), tyrosinase-related protein 1 (TYRP1), tyrosinase-related protein 2 (TYRP2), melanoma-associated antigen 4 protein (MAGEA4), growth hormone-releasing hormone (GHRH) amino acid sequence, MART-1 / Melan-A antigen (MART-1 / Melan-A) amino acid sequence, cancer-testis antigen (NY-ESO-1), cancer-testis antigen II (NY-ESO-1), and PRAME. The vaccine may be a DNA vaccine containing a polynucleotide sequence encoding tyrosinase (Tyr), tyrosinase-related protein 1 (TYRP1), tyrosinase-related protein 2 (TYRP2), melanoma-associated antigen 4 (MAGEA4), growth hormone-releasing hormone (GHRH) amino acid sequence, MART-1 / Melan-A antigen (MART-1 / Melan-A) amino acid sequence, cancer-testis antigen (NY-ESO-1), cancer-testis antigen II (NY-ESO-1), PRAME, a viral antigen, or a combination thereof. The viral antigen may be derived from one or more of the following viruses: hepatitis B virus (e.g., core protein and surface protein), hepatitis C virus (e.g., nonstructural protein (NS) 34A (NS34A), NS5A, NS5B, NS4B), and human papilloma virus (HPV) 6, HPV11, HPV16, and HPV18.

[0057] (1) Tyrosinase (Tyr) The vaccines of the present invention can contain the cancer antigen tyrosinase (Tyr), a fragment thereof, or a variant thereof. Tyrosinase is a copper-containing enzyme with tyrosine hydroxylase and dopa oxidase catalytic activity that can be found in microbial, plant, and animal tissues. Specifically, tyrosinase catalyzes the production of melanin or other pigments by the oxidation of phenols such as tyrosine. Mutations in the TYR gene cause oculocutaneous albinism in mammals, and non-pathological genetic polymorphisms in the TYR gene contribute to changes in skin pigmentation.

[0058] Furthermore, in cancers or tumors such as melanoma, tyrosinase can become unregulated, leading to increased melanin synthesis. Thus, tyrosinase can be a cancer antigen associated with melanoma. In subjects with melanoma, tyrosinase can be a target for cytotoxic T cell recognition. However, in some instances, the immune response to cancers or tumors (including melanoma) can be suppressed, resulting in a microenvironment that supports tumor formation and / or growth, thus leading to disease progression.

[0059] Immune suppression can be facilitated by myeloid-derived suppressor cells (MDSCs), which are a mixed population of immature macrophages, granulocytes, dendritic cells, and myeloid cells. Myeloid cells can be a heterogeneous population of myeloid progenitor cells and immature myeloid cells (IMCs). Markers of MDSCs include expression of Gr-1 and CD11b (i.e., Gr-1 + and CD11b + Examples include cells.

[0060] Circulating MDSCs increase due to chronic infection, and expansion of MDSC numbers may be associated with autoimmunity and inflammation. In particular, MDSC proliferation (presence in tumors or cancerous tissues) may facilitate tumor growth and evasion from immune detection and / or immunomodulation; therefore, MDSCs may influence the immune response to anti-cancer vaccines.

[0061] MDSCs are regulated by Regulator of G-protein signaling 2 (Rgs2), which can be highly expressed in tumor-derived MDSCs. Rgs2 can also be widely expressed in various cells, such as bone marrow cells. MDSCs from tumor-bearing mice may exhibit different behaviors than MDSCs from tumor-free mice. One such difference may be the upregulation of the production of the chemokine MCP-1, which is secreted by MDSCs. MCP-1 can promote cell migration by signaling through CCR2, a G-protein coupled receptor (GPCR) present on monocytes, endothelial cells, and T cells. Thus, MCP-1 can induce endothelial cell migration and thereby promote angiogenesis. Blockade of MCP-1 via neutralizing antibodies can inhibit angiogenesis, thereby reducing tumor metastasis and increasing survival rates. Thus, MCP-1 can be considered an angiogenic factor. In addition to secreting MCP-1, MDSCs secrete growth factors, which may further contribute to tumor growth.

[0062] Tyr antigens can induce antigen-specific T cell and / or high-titer antibody responses, thereby inducing or eliciting an immune response directed against or reactive to cancers or tumors expressing the antigen. In some embodiments, the induced or elicited immune response can be a cellular immune response, a humoral immune response, or both a cellular and humoral immune response. In some embodiments, the induced or elicited immune response can include the induction or secretion of interferon-gamma (IFN-γ) and / or tumor necrosis factor alpha (TNF-α). In other embodiments, the induced or elicited immune response may reduce or inhibit one or more immunosuppressive factors that promote the growth of tumors or cancers that express the antigen, such as, but not limited to, factors that downregulate MHC presentation, antigen-specific regulatory T cells (Tregs), cytokines such as PD-L1, FasL, IL-10, and TFG-β, soluble factors produced by tumor-associated macrophages, tumor-associated fibroblasts, immunosuppressive cells, CTLA-4, PD-1, MDSC, MCP-1, and factors that upregulate immune checkpoint molecules, which are described in more detail below.

[0063] As demonstrated herein, Tyr antigens induce antigen-specific T cell and high-titer antibody responses against cancer or tumor cells (e.g., melanoma cells). Specifically, Tyr antigens induce (1) a humoral immune response via a B cell response to generate antibodies that block the production of monocyte chemoattractant protein-1 (MCP-1), thereby inhibiting myeloid-derived suppressor cells (MDSCs) and suppressing tumor growth; and (2) a CD8+ / CD8+ response to attack and kill tumor cells. +Tyr antigens are important targets for immune-mediated tumor elimination by inducing (1) an increase in cytotoxic T lymphocytes (CTLs) such as Tyrα, (2) an increase in helper T cell responses, and (3) an increase in inflammatory responses mediated by IFN-γ and TNF-α, or preferably all of the above. Thus, vaccines containing Tyr antigens (e.g., consensus Tyr antigens, described in more detail below) prevent immunosuppression by reducing the number of MDSCs present in cancer or tumor tissue, and inhibit angiogenesis in cancer or tumor tissue by reducing the production or secretion of MCP-1, thereby providing a protective immune response against tumor formation and tumor growth. Therefore, any user can design vaccines of the present invention to contain Tyr antigens that provide broad immunity against tumor formation, tumor metastasis, and tumor growth.

[0064] Tyr antigens can comprise protein epitopes that make them particularly effective as immunogens capable of inducing a counteracting anti-Tyr immune response. Tyr antigens can comprise full-length translation products, variants thereof, fragments thereof, or combinations thereof. Tyr antigens can comprise consensus proteins.

[0065] Nucleic acid sequences encoding consensus Tyr antigens can be optimized for codon usage and corresponding RNA transcripts. Nucleic acids encoding consensus Tyr antigens can be codon- and RNA-optimized for expression. In some embodiments, nucleic acid sequences encoding consensus Tyr antigens can include a Kozak sequence (e.g., GCC ACC) to improve translation efficiency. Nucleic acids encoding consensus Tyr antigens can include multiple stop codons (e.g., TGA TGA) to improve translation termination efficiency.

[0066] The nucleic acid encoding the consensus Tyr antigen can also encode an immunoglobulin E (IgE) leader sequence. The nucleic acid encoding the consensus Tyr antigen can further encode an IgE leader sequence, such that the amino acid sequence of the IgE leader sequence is linked to the amino acid sequence of the consensus Tyr antigen by a peptide bond. The nucleic acid encoding the consensus Tyr antigen can also include a nucleotide sequence encoding the IgE leader sequence. In some embodiments, the nucleic acid encoding the consensus Tyr antigen does not have, i.e., does not include, a nucleotide sequence encoding an IgE leader sequence.

[0067] The consensus Tyr antigen can be the nucleic acid sequence SEQ ID NO:1, which encodes the amino acid sequence SEQ ID NO:2. SEQ ID NO:1 encodes a consensus Tyr protein linked to an IgE leader sequence. The consensus Tyr protein can be linked to an IgE leader sequence and an HA tag. In other embodiments, the consensus Tyr protein may not have the IgE leader sequence and / or the HA tag, i.e., may not be linked to said sequence and / or tag.

[0068] In some embodiments, the consensus Tyr antigen can be a nucleic acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over the entire length of the nucleic acid sequence set forth in SEQ ID NO: 1. In other embodiments, the consensus Tyr antigen can be a nucleic acid sequence encoding an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over the entire length of the amino acid sequence set forth in SEQ ID NO: 2. The consensus Tyr antigen can be an amino acid sequence that has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over the entire length of the amino acid sequence set forth in SEQ ID NO:2.

[0069] Some embodiments relate to nucleic acid sequences encoding proteins homologous to the Tyr consensus protein, immunogenic fragments of the Tyr consensus protein, and immunogenic fragments of homologous proteins. Such nucleic acid molecules can be provided that encode immunogenic proteins having up to 95% homology to the consensus sequence, up to 96% homology to the consensus sequence, up to 97% homology to the consensus sequence, up to 98% homology to the consensus sequence, and up to 99% homology to the consensus sequence. Similarly, nucleic acid sequences encoding immunogenic fragments provided herein and immunogenic fragments of proteins homologous to the proteins provided herein are also provided.

[0070] Some embodiments relate to nucleic acid molecules encoding immunogenic proteins that are 95% homologous to the nucleic acid coding sequences herein. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins that are 96% homologous to the nucleic acid coding sequences herein. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins that are 97% homologous to the nucleic acid coding sequences herein. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins that are 98% homologous to the nucleic acid coding sequences herein. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins that are 99% homologous to the nucleic acid coding sequences herein. In some embodiments, a nucleic acid molecule having a coding sequence disclosed herein that is homologous to the coding sequence of a consensus protein disclosed herein comprises a sequence encoding an IgE leader sequence linked to the 5' end of the coding sequence encoding the homologous protein sequence disclosed herein.

[0071] Some embodiments relate to proteins having a particular identity, expressed as a percentage, to a full-length Tyr consensus protein, immunogenic fragments of the Tyr consensus protein, and nucleic acid sequences encoding immunogenic fragments of proteins having identity to the Tyr consensus protein. Such nucleic acid molecules can be provided that encode immunogenic proteins having up to 80% identity to the full-length Tyr consensus sequence, up to 85% identity to the full-length consensus sequence, up to 90% identity to the full-length Tyr consensus sequence, up to 91% identity to the full-length Tyr consensus sequence, up to 92% identity to the full-length Tyr consensus sequence, up to 93% identity to the full-length Tyr consensus sequence, up to 94% identity to the full-length Tyr consensus sequence, up to 95% identity to the full-length Tyr consensus sequence, up to 96% identity to the full-length Tyr consensus sequence, up to 97% identity to the full-length Tyr consensus sequence, up to 98% identity to the full-length Tyr consensus sequence, and up to 99% identity to the full-length Tyr consensus sequence. Similarly, nucleic acid sequences encoding immunogenic fragments of the immunogenic fragments set forth herein and of proteins having similar percentage identities as those set forth above for the Tyr proteins set forth herein are also provided.

[0072] In some embodiments, the nucleic acid sequence does not have a coding sequence that encodes a leader sequence. In some embodiments, the nucleic acid sequence does not have a coding sequence that encodes an IgE leader.

[0073] Some embodiments relate to fragments of SEQ ID NO: 1. The fragment can be at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1. The fragment can be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1. In some embodiments, the fragment includes a sequence encoding a leader sequence, e.g., an immunoglobulin leader, such as an IgE leader. In some embodiments, the fragment does not have a coding sequence encoding a leader sequence. In some embodiments, the fragment does not have a coding sequence encoding a leader sequence, e.g., an IgE leader.

[0074] Furthermore, the amino acid sequence of the consensus Tyr protein is SEQ ID NO: 2. The amino acid sequence of the consensus Tyr protein linked to an IgE leader is SEQ ID NO: 2. The amino acid sequence of the consensus Tyr protein linked to an IgE leader may be linked to an HA tag.

[0075] Some embodiments relate to proteins homologous to SEQ ID NO:2. Some embodiments relate to immunogenic proteins having 95% homology to the consensus protein sequence set forth in SEQ ID NO:2. Some embodiments relate to immunogenic proteins having 96% homology to the consensus protein sequence set forth in SEQ ID NO:2. Some embodiments relate to immunogenic proteins having 97% homology to the consensus protein sequence set forth in SEQ ID NO:2. Some embodiments relate to immunogenic proteins having 98% homology to the consensus protein sequence set forth in SEQ ID NO:2. Some embodiments relate to immunogenic proteins having 99% homology to the consensus protein sequence set forth in SEQ ID NO:2.

[0076] Some embodiments relate to a protein that is identical to SEQ ID NO:2. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 80% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO:2. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 85% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO:2. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 90% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO:2. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 91% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO:2. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 92% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO:2. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 93% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO:2. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 94% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO:2. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 95% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO:2. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 96% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 2. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 97% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 2. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 98% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 2. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 99% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 2.

[0077] In some embodiments, the protein does not have a leader sequence. In some embodiments, the protein does not have an IgE leader. A fragment of the consensus protein can comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the consensus protein. An immunogenic fragment of SEQ ID NO: 2 can be provided. An immunogenic fragment can comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of SEQ ID NO: 2. In some embodiments, the fragment includes a leader sequence, e.g., an immunoglobulin leader, such as an IgE leader. In some embodiments, the fragment lacks a leader sequence. In some embodiments, the fragment lacks a leader sequence, e.g., an IgE leader.

[0078] Immunogenic fragments of proteins having amino acid sequences homologous to immunogenic fragments of SEQ ID NO: 2 can be provided. Such immunogenic fragments can include at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of a protein that is 95% or more homologous to SEQ ID NO: 2. Some embodiments relate to immunogenic fragments having 96% homology to immunogenic fragments of the consensus protein sequences herein. Some embodiments relate to immunogenic fragments having 97% homology to immunogenic fragments of the consensus protein sequences herein. Some embodiments relate to immunogenic fragments having 98% homology to the immunogenic fragments of the consensus protein sequences herein. Some embodiments relate to immunogenic fragments having 99% homology to the immunogenic fragments of the consensus protein sequences herein. In some embodiments, the fragments include a leader sequence, e.g., an immunoglobulin leader, such as an IgE leader. In some embodiments, the fragments do not have a leader sequence. In some embodiments, the fragments do not have a leader sequence, e.g., an IgE leader.

[0079] Immunogenic fragments of proteins can be provided that have an amino acid sequence identical to an immunogenic fragment of SEQ ID NO: 2. Such immunogenic fragments can comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of a protein that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the fragments include a leader sequence, e.g., an immunoglobulin leader, such as an IgE leader. In some embodiments, the fragments lack a leader sequence. In some embodiments, the fragment does not have a leader sequence, for example an IgE leader.

[0080] As referred to herein with respect to the linkage of a signal peptide or leader sequence to the N-terminus of a protein, the signal peptide / leader sequence replaces the N-terminal methionine of the protein encoded by the start codon of a nucleic acid sequence encoding the protein without a signal peptide coding sequence.

[0081] (2) Tyrosinase-related protein 1 (TYRP1) The vaccines of the present invention can contain the cancer antigen tyrosinase-related protein 1 (TYRP1), a fragment thereof, or a variant thereof. TYRP1 is a 75-kDa transmembrane glycoprotein encoded by the TYRP1 gene and expressed in both normal and malignant melanocytes and melanoma cells. Like tyrosinase, TYRP1 contains a modified M-box that can bind to microphthalmia-associated transcription factor (MITF), which plays a central role in pigmentation, cell proliferation, and differentiation in melanocytes. TYRP1 can help stabilize tyrosinase and form heterodimers, which can prevent premature death of melanocytes by attenuating tyrosinase-mediated cytotoxicity.

[0082] As mentioned above with respect to tyrosinase, tyrosinase-related protein 1 (TYRP-1) is also involved in the synthesis and pigmentation of melanin in melanocytes and can be recognized by the immune system in subjects with melanoma, and therefore TYRP-1 may be an antigen associated with melanoma.

[0083] TYRP-1 antigens can induce antigen-specific T cell and / or high-titer antibody responses, thereby inducing or eliciting an immune response directed against or reactive to cancers or tumors expressing the antigen. In some embodiments, the induced or elicited immune response can be a cellular immune response, a humoral immune response, or both a cellular and humoral immune response. In some embodiments, the induced or elicited cellular immune response can include the induction or secretion of interferon-gamma (IFN-γ) and / or tumor necrosis factor alpha (TNF-α). In other embodiments, the induced or elicited immune response may reduce or inhibit one or more immunosuppressive factors that promote the growth of tumors or cancers that express the antigen, such as, but not limited to, factors that downregulate MHC presentation, antigen-specific regulatory T cells (Tregs), cytokines such as PD-L1, FasL, IL-10, and TFG-β, soluble factors produced by tumor-associated macrophages, tumor-associated fibroblasts, immunosuppressive cells, CTLA-4, PD-1, MDSC, MCP-1, and factors that upregulate immune checkpoint molecules, which are described in more detail below.

[0084] TYRP-1 antigens can comprise protein epitopes that make them particularly effective as immunogens capable of inducing a counter-TYRP-1 immune response. TYRP-1 antigens can comprise full-length translation products, variants thereof, fragments thereof, or combinations thereof. TYRP-1 antigens can comprise consensus proteins.

[0085] Nucleic acid sequences encoding consensus TYRP-1 antigens can be optimized for codon usage and the corresponding RNA transcript. Nucleic acids encoding consensus TYRP-1 antigens can be codon- and RNA-optimized for expression. In some embodiments, nucleic acid sequences encoding consensus TYRP-1 antigens can include a Kozak sequence (e.g., GCC ACC) to improve translation efficiency. Nucleic acids encoding consensus TYRP-1 antigens can include multiple stop codons (e.g., TGA TGA) to improve translation termination efficiency.

[0086] The nucleic acid encoding the consensus TYRP-1 antigen can also encode an immunoglobulin E (IgE) leader sequence. The nucleic acid encoding the consensus TYRP-1 antigen can further encode an IgE leader sequence, such that the amino acid sequence of the IgE leader sequence is linked to the amino acid sequence of the consensus TYRP-1 antigen by a peptide bond. The nucleic acid encoding the consensus TYRP-1 antigen can also comprise a nucleotide sequence encoding the IgE leader sequence. In some embodiments, the nucleic acid encoding the consensus TYRP-1 antigen does not have, i.e., does not comprise, a nucleotide sequence encoding an IgE leader sequence.

[0087] The consensus TYRP-1 antigen may be the nucleic acid sequence SEQ ID NO:3, which encodes the amino acid sequence SEQ ID NO:4. SEQ ID NO:3 encodes a consensus TYRP-1 protein linked to an IgE leader sequence. The consensus TYRP-1 protein may be linked to an IgE leader sequence and an HA tag. In other embodiments, the consensus TYRP-1 protein may not have the IgE leader sequence and / or the HA tag, i.e., may not be linked to said sequence and / or tag.

[0088] In some embodiments, the consensus TYRP-1 antigen can be a nucleic acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over the entire length of the nucleic acid sequence set forth in SEQ ID NO: 3. In other embodiments, the consensus TYRP-1 antigen can be a nucleic acid sequence encoding an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over the entire length of the amino acid sequence set forth in SEQ ID NO: 4. The consensus TYRP-1 antigen can be an amino acid sequence that has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over the entire length of the amino acid sequence set forth in SEQ ID NO:4.

[0089] Some embodiments relate to nucleic acid sequences encoding proteins homologous to the TYRP-1 consensus protein, immunogenic fragments of the TYRP-1 consensus protein, and immunogenic fragments of homologous proteins. Such nucleic acid molecules can be provided that encode immunogenic proteins having up to 95% homology to the consensus sequence, up to 96% homology to the consensus sequence, up to 97% homology to the consensus sequence, up to 98% homology to the consensus sequence, and up to 99% homology to the consensus sequence. Similarly, nucleic acid sequences encoding immunogenic fragments set forth herein and immunogenic fragments of proteins homologous to the proteins set forth herein are also provided.

[0090] Some embodiments relate to nucleic acid molecules encoding immunogenic proteins that are 95% homologous to the nucleic acid coding sequences herein. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins that are 96% homologous to the nucleic acid coding sequences herein. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins that are 97% homologous to the nucleic acid coding sequences herein. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins that are 98% homologous to the nucleic acid coding sequences herein. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins that are 99% homologous to the nucleic acid coding sequences herein. In some embodiments, a nucleic acid molecule having a coding sequence disclosed herein that is homologous to the coding sequence of a consensus protein disclosed herein comprises a sequence encoding an IgE leader sequence linked to the 5' end of the coding sequence encoding the homologous protein sequence disclosed herein.

[0091] Some embodiments relate to proteins having a particular identity, expressed as a percentage, to the full-length TYRP-1 consensus protein, immunogenic fragments of the TYRP-1 consensus protein, and nucleic acid sequences encoding immunogenic fragments of proteins having identity to the TYRP-1 consensus protein. Such nucleic acid molecules can be provided which encode immunogenic proteins having up to 80% identity to the full-length TYRP-1 consensus sequence, up to 85% identity to the full-length TYRP-1 consensus sequence, up to 90% identity to the full-length TYRP-1 consensus sequence, up to 91% identity to the full-length TYRP-1 consensus sequence, up to 92% identity to the full-length TYRP-1 consensus sequence, up to 93% identity to the full-length TYRP-1 consensus sequence, up to 94% identity to the full-length TYRP-1 consensus sequence, up to 95% identity to the full-length TYRP-1 consensus sequence, up to 96% identity to the full-length TYRP-1 consensus sequence, up to 97% identity to the full-length TYRP-1 consensus sequence, up to 98% identity to the full-length TYRP-1 consensus sequence, and up to 99% identity to the full-length TYRP-1 consensus sequence. Similarly, nucleic acid sequences encoding immunogenic fragments of the proteins set forth herein and having similar percent identities as set forth above for the TYRP-1 proteins set forth herein are also provided.

[0092] In some embodiments, the nucleic acid sequence does not have a coding sequence that encodes a leader sequence. In some embodiments, the nucleic acid sequence does not have a coding sequence that encodes an IgE leader.

[0093] Some embodiments relate to fragments of SEQ ID NO:3. The fragment can be at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:3. The fragment can be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a fragment of SEQ ID NO:3. In some embodiments, the fragment includes a sequence encoding a leader sequence, e.g., an immunoglobulin leader, such as an IgE leader. In some embodiments, the fragment does not have a coding sequence encoding a leader sequence. In some embodiments, the fragment does not have a coding sequence encoding a leader sequence, e.g., an IgE leader.

[0094] Additionally, the amino acid sequence of the consensus TYRP-1 protein is SEQ ID NO: 4. The amino acid sequence of the consensus TYRP-1 protein linked to an IgE leader is SEQ ID NO: 4. The amino acid sequence of the consensus TYRP-1 protein linked to an IgE leader may be linked to an HA tag.

[0095] Some embodiments relate to proteins homologous to SEQ ID NO:4. Some embodiments relate to immunogenic proteins having 95% homology to the consensus protein sequence set forth in SEQ ID NO:4. Some embodiments relate to immunogenic proteins having 96% homology to the consensus protein sequence set forth in SEQ ID NO:4. Some embodiments relate to immunogenic proteins having 97% homology to the consensus protein sequence set forth in SEQ ID NO:4. Some embodiments relate to immunogenic proteins having 98% homology to the consensus protein sequence set forth in SEQ ID NO:4. Some embodiments relate to immunogenic proteins having 99% homology to the consensus protein sequence set forth in SEQ ID NO:4.

[0096] Some embodiments relate to a protein that is identical to SEQ ID NO:4. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 80% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO:4. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 85% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO:4. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 90% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO:4. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 91% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO:4. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 92% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO:4. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 93% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO:4. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 94% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO:4. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 95% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO:4. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 96% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 4. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 97% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 4. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 98% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 4. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 99% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 4.

[0097] In some embodiments, the protein does not have a leader sequence. In some embodiments, the protein does not have an IgE leader. Fragments of the consensus protein can comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the consensus TYRP-1 protein. Immunogenic fragments of SEQ ID NO: 4 can be provided. An immunogenic fragment can comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of SEQ ID NO: 4. In some embodiments, the fragment includes a leader sequence, e.g., an immunoglobulin leader, such as an IgE leader. In some embodiments, the fragment lacks a leader sequence. In some embodiments, the fragment lacks a leader sequence, e.g., an IgE leader.

[0098] Immunogenic fragments of proteins having amino acid sequences homologous to immunogenic fragments of SEQ ID NO: 4 can be provided. Such immunogenic fragments can include at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of a protein that is 95% or more homologous to SEQ ID NO: 4. Some embodiments relate to immunogenic fragments having 96% homology to immunogenic fragments of the consensus protein sequences herein. Some embodiments relate to immunogenic fragments having 97% homology to immunogenic fragments of the consensus protein sequences herein. Some embodiments relate to immunogenic fragments having 98% homology to the immunogenic fragments of the consensus protein sequences herein. Some embodiments relate to immunogenic fragments having 99% homology to the immunogenic fragments of the consensus protein sequences herein. In some embodiments, the fragments include a leader sequence, e.g., an immunoglobulin leader, such as an IgE leader. In some embodiments, the fragments do not have a leader sequence. In some embodiments, the fragments do not have a leader sequence, e.g., an IgE leader.

[0099] Immunogenic fragments of proteins can be provided that have an amino acid sequence identical to an immunogenic fragment of SEQ ID NO: 4. Such immunogenic fragments can comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of a protein that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the fragments include a leader sequence, e.g., an immunoglobulin leader, such as an IgE leader. In some embodiments, the fragments lack a leader sequence. In some embodiments, the fragment does not have a leader sequence, for example an IgE leader.

[0100] As referred to herein with respect to the linkage of a signal peptide or leader sequence to the N-terminus of a protein, the signal peptide / leader sequence replaces the N-terminal methionine of the protein encoded by the start codon of a nucleic acid sequence encoding the protein without a signal peptide coding sequence.

[0101] (3) Tyrosinase-related protein 2 (TYRP2) The vaccines of the present invention can contain the cancer antigen tyrosinase-related protein 2 (TYRP2, also known as dopachrome tautomerase (DCT)), a fragment thereof, or a variant thereof. TYRP2 / DCT is a 519-amino acid protein encoded by the TYRP2 / DCT gene and expressed in both normal and malignant melanocytes and melanoma cells. TYRP2 / DCT is a well-characterized melanocyte-specific enzyme that functions in conjunction with tyrosinase and TYRP1 in the conversion of L-tyrosine to melanin in melanocytes. DCT specifically catalyzes the tautomerization of the melanin precursor L-dopachrome to 5,6-dihydroindole-2-carboxylic acid (DHICA), which is subsequently oxidized by TYRP1 (as described above) to form eumelanin. Studies have shown that TYRP2 / DCT may be a mediator of drug resistance in melanoma cells with specificity for DNA-damaging agents. TYRP2 / DCT has been frequently reported to be highly expressed in melanoma, suggesting that this melanocyte-specific enzyme plays an important role in contributing to the intrinsic resistance phenotype of melanoma to various anticancer DNA-damaging agents.

[0102] As mentioned above with respect to tyrosinase, tyrosinase-related protein 2 (TYRP-2) is also involved in the synthesis of melanin and can be recognized by the immune system in subjects with melanoma. Furthermore, TYRP-2 can mediate drug resistance in melanoma cells. Therefore, TYRP-2 may be an antigen associated with melanoma.

[0103] TYRP-2 antigens can induce antigen-specific T cell and / or high-titer antibody responses, thereby inducing or eliciting an immune response directed against or reactive to cancers or tumors expressing the antigen. In some embodiments, the induced or elicited immune response can be a cellular immune response, a humoral immune response, or both a cellular and humoral immune response. In some embodiments, the induced or elicited cellular immune response can include the induction or secretion of interferon-gamma (IFN-γ) and / or tumor necrosis factor alpha (TNF-α). In other embodiments, the induced or elicited immune response may reduce or inhibit one or more immunosuppressive factors that promote the growth of tumors or cancers that express the antigen, such as, but not limited to, factors that downregulate MHC presentation, antigen-specific regulatory T cells (Tregs), cytokines such as PD-L1, FasL, IL-10, and TFG-β, soluble factors produced by tumor-associated macrophages, tumor-associated fibroblasts, immunosuppressive cells, CTLA-4, PD-1, MDSC, MCP-1, and factors that upregulate immune checkpoint molecules, which are described in more detail below.

[0104] A TYRP-2 antigen can comprise a protein epitope that makes the antigen particularly effective as an immunogen capable of inducing a counteracting anti-TYRP-2 immune response. A TYRP-2 antigen can comprise a full-length translation product, a variant thereof, a fragment thereof, or a combination thereof. A TYRP-2 antigen can comprise a consensus protein.

[0105] Nucleic acid sequences encoding consensus TYRP-2 antigens can be optimized for codon usage and the corresponding RNA transcript. Nucleic acids encoding consensus TYRP-2 antigens can be codon- and RNA-optimized for expression. In some embodiments, nucleic acid sequences encoding consensus TYRP-2 antigens can include a Kozak sequence (e.g., GCC ACC) to improve translation efficiency. Nucleic acids encoding consensus TYRP-2 antigens can include multiple stop codons (e.g., TGA TGA) to improve translation termination efficiency.

[0106] The nucleic acid encoding the consensus TYRP-2 antigen can also encode an immunoglobulin E (IgE) leader sequence. The nucleic acid encoding the consensus TYRP-2 antigen can further encode an IgE leader sequence, such that the amino acid sequence of the IgE leader sequence is linked to the amino acid sequence of the consensus TYRP-2 antigen by a peptide bond. The nucleic acid encoding the consensus TYRP-2 antigen can also include a nucleotide sequence encoding the IgE leader sequence. In some embodiments, the nucleic acid encoding the consensus TYRP-2 antigen does not have, i.e., does not include, a nucleotide sequence encoding an IgE leader sequence.

[0107] The consensus TYRP-2 antigen may be the nucleic acid sequence SEQ ID NO:5, which encodes the amino acid sequence SEQ ID NO:6. SEQ ID NO:5 encodes a consensus TYRP-2 protein linked to an IgE leader sequence. The consensus TYRP-2 protein may be linked to an IgE leader sequence and an HA tag. In other embodiments, the consensus TYRP-2 protein may not have the IgE leader sequence and / or the HA tag, i.e., may not be linked to said sequence and / or tag.

[0108] In some embodiments, the consensus TYRP-2 antigen can be a nucleic acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over the entire length of the nucleic acid sequence set forth in SEQ ID NO: 5. In other embodiments, the consensus TYRP-2 antigen can be a nucleic acid sequence encoding an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over the entire length of the amino acid sequence set forth in SEQ ID NO: 6. The consensus TYRP-2 antigen can be an amino acid sequence that has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over the entire length of the amino acid sequence set forth in SEQ ID NO:6.

[0109] Some embodiments relate to nucleic acid sequences encoding proteins homologous to the TYRP-2 consensus protein, immunogenic fragments of the TYRP-2 consensus protein, and immunogenic fragments of homologous proteins. Such nucleic acid molecules can be provided that encode immunogenic proteins having up to 95% homology to the consensus sequence, up to 96% homology to the consensus sequence, up to 97% homology to the consensus sequence, up to 98% homology to the consensus sequence, and up to 99% homology to the consensus sequence. Similarly, nucleic acid sequences encoding immunogenic fragments set forth herein and immunogenic fragments of proteins homologous to the proteins set forth herein are also provided.

[0110] Some embodiments relate to nucleic acid molecules encoding immunogenic proteins that are 95% homologous to the nucleic acid coding sequences herein. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins that are 96% homologous to the nucleic acid coding sequences herein. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins that are 97% homologous to the nucleic acid coding sequences herein. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins that are 98% homologous to the nucleic acid coding sequences herein. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins that are 99% homologous to the nucleic acid coding sequences herein. In some embodiments, a nucleic acid molecule having a coding sequence disclosed herein that is homologous to the coding sequence of a consensus protein disclosed herein comprises a sequence encoding an IgE leader sequence linked to the 5' end of the coding sequence encoding the homologous protein sequence disclosed herein.

[0111] Some embodiments relate to proteins having a particular percent identity to the full-length TYRP-2 consensus protein, immunogenic fragments of the TYRP-2 consensus protein, and nucleic acid sequences encoding immunogenic fragments of proteins having identity to the TYRP-2 consensus protein. Such nucleic acid molecules can be provided which encode immunogenic proteins having up to 80% identity to the full-length TYRP-2 consensus sequence, up to 85% identity to the full-length TYRP-2 consensus sequence, up to 90% identity to the full-length TYRP-2 consensus sequence, up to 91% identity to the full-length TYRP-2 consensus sequence, up to 92% identity to the full-length TYRP-2 consensus sequence, up to 93% identity to the full-length TYRP-2 consensus sequence, up to 94% identity to the full-length TYRP-2 consensus sequence, up to 95% identity to the full-length TYRP-2 consensus sequence, up to 96% identity to the full-length TYRP-2 consensus sequence, up to 97% identity to the full-length TYRP-2 consensus sequence, up to 98% identity to the full-length TYRP-2 consensus sequence, and up to 99% identity to the full-length TYRP-2 consensus sequence. Similarly, nucleic acid sequences encoding immunogenic fragments of the proteins set forth herein and having similar percent identities as those set forth above for the TYRP-2 protein set forth herein are also provided.

[0112] In some embodiments, the nucleic acid sequence does not have a coding sequence that encodes a leader sequence. In some embodiments, the nucleic acid sequence does not have a coding sequence that encodes an IgE leader.

[0113] Some embodiments relate to fragments of SEQ ID NO: 5. The fragment can be at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 5. The fragment can be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a fragment of SEQ ID NO: 5. In some embodiments, the fragment includes a sequence encoding a leader sequence, e.g., an immunoglobulin leader, such as an IgE leader. In some embodiments, the fragment does not have a coding sequence encoding a leader sequence. In some embodiments, the fragment does not have a coding sequence encoding a leader sequence, e.g., an IgE leader.

[0114] Furthermore, the amino acid sequence of the consensus TYRP-2 protein is SEQ ID NO: 6. The amino acid sequence of the consensus TYRP-2 protein linked to an IgE leader is SEQ ID NO: 6. The amino acid sequence of the consensus TYRP-2 protein linked to an IgE leader may be linked to an HA tag.

[0115] Some embodiments relate to proteins homologous to SEQ ID NO: 2. Some embodiments relate to immunogenic proteins having 95% homology to the consensus protein sequence set forth in SEQ ID NO: 6. Some embodiments relate to immunogenic proteins having 96% homology to the consensus protein sequence set forth in SEQ ID NO: 6. Some embodiments relate to immunogenic proteins having 97% homology to the consensus protein sequence set forth in SEQ ID NO: 6. Some embodiments relate to immunogenic proteins having 98% homology to the consensus protein sequence set forth in SEQ ID NO: 6. Some embodiments relate to immunogenic proteins having 99% homology to the consensus protein sequence set forth in SEQ ID NO: 6.

[0116] Some embodiments relate to a protein that is identical to SEQ ID NO:6. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 80% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO:6. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 85% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO:6. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 90% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO:6. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 91% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO:6. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 92% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO:6. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 93% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO:6. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 94% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO:6. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 95% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO:6. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 96% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 6. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 97% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 6. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 98% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 6. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 99% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 6.

[0117] In some embodiments, the protein does not have a leader sequence. In some embodiments, the protein does not have an IgE leader. A fragment of the consensus protein can comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the consensus protein. An immunogenic fragment of SEQ ID NO: 6 can be provided. An immunogenic fragment can comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of SEQ ID NO: 6. In some embodiments, the fragment includes a leader sequence, e.g., an immunoglobulin leader, such as an IgE leader. In some embodiments, the fragment lacks a leader sequence. In some embodiments, the fragment lacks a leader sequence, e.g., an IgE leader.

[0118] Immunogenic fragments of proteins having amino acid sequences homologous to immunogenic fragments of SEQ ID NO: 6 can be provided. Such immunogenic fragments can include at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of a protein that is 95% or more homologous to SEQ ID NO: 6. Some embodiments relate to immunogenic fragments having 96% homology to immunogenic fragments of the consensus protein sequences herein. Some embodiments relate to immunogenic fragments having 97% homology to immunogenic fragments of the consensus protein sequences herein. Some embodiments relate to immunogenic fragments having 98% homology to the immunogenic fragments of the consensus protein sequences herein. Some embodiments relate to immunogenic fragments having 99% homology to the immunogenic fragments of the consensus protein sequences herein. In some embodiments, the fragments include a leader sequence, e.g., an immunoglobulin leader, such as an IgE leader. In some embodiments, the fragments do not have a leader sequence. In some embodiments, the fragments do not have a leader sequence, e.g., an IgE leader.

[0119] Immunogenic fragments of proteins can be provided that have an amino acid sequence identical to an immunogenic fragment of SEQ ID NO: 6. Such immunogenic fragments can comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of a protein that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, the fragments include a leader sequence, e.g., an immunoglobulin leader, such as an IgE leader. In some embodiments, the fragments lack a leader sequence. In some embodiments, the fragment does not have a leader sequence, for example an IgE leader.

[0120] As referred to herein with respect to the linkage of a signal peptide or leader sequence to the N-terminus of a protein, the signal peptide / leader sequence replaces the N-terminal methionine of the protein encoded by the start codon of a nucleic acid sequence encoding the protein without a signal peptide coding sequence.

[0121] (4) Melanoma-associated Antigen 4 (MAGEA4) The vaccine of the present invention may contain the cancer antigen melanoma-associated antigen 4 (MAGEA4), its fragment, or a variant thereof. MAGE-A4 is a 317-amino acid protein encoded by the MAGE-A4 gene and expressed in male germ cells and tumor cells of various tissue types, including gastrointestinal cancer, esophageal cancer, and lung cancer. MAGE-A4 binds to the oncoprotein gankyrin. This specific binding to MAGE-A4 is mediated by its C-terminus. Studies have shown that exogenous MAGE-A4 can partially inhibit the anchorage-independent growth of gankyrin-overexpressing cells in vitro and inhibit the formation of tumors metastasized from these cells in nude mice. This inhibition depends on the binding between MAGE-A4 and gankyrin, suggesting that the interaction between gankyrin and MAGE-A4 inhibits gankyrin-mediated carcinogenesis. Expression of MAGE in tumor tissues is a consequence, rather than a cause, of tumor development, and MAGE genes appear to be involved in immune processes by targeting and destroying early tumor cells.

[0122] Melanoma-associated antigen 4 (MAGEA4) may be involved in embryonic development and tumor transformation and / or progression. MAGEA4 is normally expressed in the testis and placenta. However, MAGEA4 can be expressed in many different types of tumors, such as melanoma, head and neck squamous cell carcinoma, lung cancer, and breast cancer. Therefore, MAGEA4 may be an antigen associated with various tumors.

[0123] The MAGEA4 antigen can induce antigen-specific T cell and / or high-titer antibody responses, thereby inducing or eliciting an immune response directed against or reactive to cancers or tumors expressing the antigen. In some embodiments, the induced or elicited immune response can be a cellular immune response, a humoral immune response, or both a cellular and humoral immune response. In some embodiments, the induced or elicited cellular immune response can include the induction or secretion of interferon-gamma (IFN-γ) and / or tumor necrosis factor alpha (TNF-α). In other embodiments, the induced or elicited immune response may reduce or inhibit one or more immunosuppressive factors that promote the growth of tumors or cancers that express the antigen, such as, but not limited to, factors that downregulate MHC presentation, antigen-specific regulatory T cells (Tregs), cytokines such as PD-L1, FasL, IL-10, and TFG-β, soluble factors produced by tumor-associated macrophages, tumor-associated fibroblasts, immunosuppressive cells, CTLA-4, PD-1, MDSC, MCP-1, and factors that upregulate immune checkpoint molecules, which are described in more detail below.

[0124] The MAGEA4 antigen may comprise a protein epitope that makes the antigen particularly effective as an immunogen capable of inducing a counteracting anti-MAGEA4 immune response. The MAGEA4 antigen may comprise a full-length translation product, a variant thereof, a fragment thereof, or a combination thereof. The MAGEA4 antigen may comprise a consensus protein.

[0125] Nucleic acid sequences encoding consensus MAGEA4 antigens can be optimized for codon usage and corresponding RNA transcripts. Nucleic acids encoding consensus MAGEA4 antigens can be codon- and RNA-optimized for expression. In some embodiments, nucleic acid sequences encoding consensus MAGEA4 antigens can include a Kozak sequence (e.g., GCC ACC) to improve translation efficiency. Nucleic acids encoding consensus MAGEA4 antigens can include multiple stop codons (e.g., TGA TGA) to improve translation termination efficiency.

[0126] The nucleic acid encoding the consensus MAGEA4 antigen can also encode an immunoglobulin E (IgE) leader sequence. The nucleic acid encoding the consensus Tyr antigen can further encode an IgE leader sequence such that the amino acid sequence of the IgE leader sequence is linked to the amino acid sequence of the consensus MAGEA4 antigen by a peptide bond. The nucleic acid encoding the consensus MAGEA4 antigen can also comprise a nucleotide sequence encoding the IgE leader sequence. In some embodiments, the nucleic acid encoding the consensus MAGEA4 antigen does not have, i.e., does not comprise, a nucleotide sequence encoding an IgE leader sequence.

[0127] The consensus MAGEA4 antigen may be the nucleic acid sequence SEQ ID NO:7, which encodes the amino acid sequence SEQ ID NO:8. SEQ ID NO:7 encodes the consensus MAGEA4 protein linked to an IgE leader sequence. The consensus MAGEA4 protein may be linked to an IgE leader sequence and an HA tag. In other embodiments, the consensus MAGEA4 protein may not have the IgE leader sequence and / or the HA tag, i.e., may not be linked to said sequence and / or tag.

[0128] In some embodiments, the consensus MAGEA4 antigen can be a nucleic acid sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the entire length of the nucleic acid sequence set forth in SEQ ID NO: 7. In other embodiments, the consensus MAGEA4 antigen can be a nucleic acid sequence that encodes an amino acid sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the entire length of the amino acid sequence set forth in SEQ ID NO: 8. The consensus MAGEA4 antigen can be an amino acid sequence that has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over the entire length of the amino acid sequence set forth in SEQ ID NO:8.

[0129] Some embodiments relate to nucleic acid sequences encoding proteins homologous to the MAGEA4 consensus protein, immunogenic fragments of the MAGEA4 consensus protein, and immunogenic fragments of homologous proteins. Such nucleic acid molecules can be provided that encode immunogenic proteins having up to 95% homology to the consensus sequence, up to 96% homology to the consensus sequence, up to 97% homology to the consensus sequence, up to 98% homology to the consensus sequence, and up to 99% homology to the consensus sequence. Similarly, nucleic acid sequences encoding immunogenic fragments provided herein and immunogenic fragments of proteins homologous to the proteins provided herein are also provided.

[0130] Some embodiments relate to nucleic acid molecules encoding immunogenic proteins that are 95% homologous to the nucleic acid coding sequences herein. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins that are 96% homologous to the nucleic acid coding sequences herein. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins that are 97% homologous to the nucleic acid coding sequences herein. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins that are 98% homologous to the nucleic acid coding sequences herein. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins that are 99% homologous to the nucleic acid coding sequences herein. In some embodiments, a nucleic acid molecule having a coding sequence disclosed herein that is homologous to the coding sequence of a consensus protein disclosed herein comprises a sequence encoding an IgE leader sequence linked to the 5' end of the coding sequence encoding the homologous protein sequence disclosed herein.

[0131] Some embodiments relate to proteins having a particular identity expressed as a percentage to the full-length MAGEA4 consensus protein, immunogenic fragments of the MAGEA4 consensus protein, and nucleic acid sequences encoding immunogenic fragments of proteins having identity to the MAGEA4 consensus protein. Such nucleic acid molecules can be provided which encode immunogenic proteins having up to 80% identity to the full-length MAGEA4 consensus sequence, up to 85% identity to the full-length MAGEA4 consensus sequence, up to 90% identity to the full-length MAGEA4 consensus sequence, up to 91% identity to the full-length MAGEA4 consensus sequence, up to 92% identity to the full-length MAGEA4 consensus sequence, up to 93% identity to the full-length MAGEA4 consensus sequence, up to 94% identity to the full-length MAGEA4 consensus sequence, up to 95% identity to the full-length MAGEA4 consensus sequence, up to 96% identity to the full-length MAGEA4 consensus sequence, up to 97% identity to the full-length MAGEA4 consensus sequence, up to 98% identity to the full-length MAGEA4 consensus sequence, and up to 99% identity to the full-length MAGEA4 consensus sequence. Similarly, nucleic acid sequences encoding immunogenic fragments of the proteins shown herein and having similar percentage identities to those shown above for the MAGEA4 protein shown herein are also provided.

[0132] In some embodiments, the nucleic acid sequence does not have a coding sequence that encodes a leader sequence. In some embodiments, the nucleic acid sequence does not have a coding sequence that encodes an IgE leader.

[0133] Some embodiments relate to fragments of SEQ ID NO: 7. The fragment can be at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 7. The fragment can be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a fragment of SEQ ID NO: 7. In some embodiments, the fragment includes a sequence encoding a leader sequence, e.g., an immunoglobulin leader, such as an IgE leader. In some embodiments, the fragment does not have a coding sequence encoding a leader sequence. In some embodiments, the fragment does not have a coding sequence encoding a leader sequence, e.g., an IgE leader.

[0134] Furthermore, the amino acid sequence of the consensus MAGEA4 protein is SEQ ID NO: 8. The amino acid sequence of the consensus MAGEA4 protein linked to an IgE leader is SEQ ID NO: 8. The amino acid sequence of the consensus MAGEA4 protein linked to an IgE leader may be linked to an HA tag.

[0135] Some embodiments relate to proteins homologous to SEQ ID NO:8. Some embodiments relate to immunogenic proteins having 95% homology to the consensus protein sequence set forth in SEQ ID NO:8. Some embodiments relate to immunogenic proteins having 96% homology to the consensus protein sequence set forth in SEQ ID NO:8. Some embodiments relate to immunogenic proteins having 97% homology to the consensus protein sequence set forth in SEQ ID NO:8. Some embodiments relate to immunogenic proteins having 98% homology to the consensus protein sequence set forth in SEQ ID NO:8. Some embodiments relate to immunogenic proteins having 99% homology to the consensus protein sequence set forth in SEQ ID NO:8.

[0136] Some embodiments relate to a protein that is identical to SEQ ID NO:8. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 80% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO:8. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 85% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO:8. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 90% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO:8. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 91% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO:8. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 92% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO:8. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 93% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO:8. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 94% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO:8. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 95% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO:8. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 96% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 8. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 97% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 8. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 98% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 8. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 99% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 8.

[0137] In some embodiments, the protein does not have a leader sequence. In some embodiments, the protein does not have an IgE leader. A fragment of the consensus protein can comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the consensus protein. An immunogenic fragment of SEQ ID NO: 8 can be provided. An immunogenic fragment can comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of SEQ ID NO: 8. In some embodiments, the fragment includes a leader sequence, e.g., an immunoglobulin leader, such as an IgE leader. In some embodiments, the fragment lacks a leader sequence. In some embodiments, the fragment lacks a leader sequence, e.g., an IgE leader.

[0138] Immunogenic fragments of proteins having amino acid sequences homologous to immunogenic fragments of SEQ ID NO: 8 can be provided. Such immunogenic fragments can include at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of a protein that is 95% or more homologous to SEQ ID NO: 8. Some embodiments relate to immunogenic fragments having 96% homology to immunogenic fragments of the consensus protein sequences herein. Some embodiments relate to immunogenic fragments having 97% homology to immunogenic fragments of the consensus protein sequences herein. Some embodiments relate to immunogenic fragments having 98% homology to the immunogenic fragments of the consensus protein sequences herein. Some embodiments relate to immunogenic fragments having 99% homology to the immunogenic fragments of the consensus protein sequences herein. In some embodiments, the fragments include a leader sequence, e.g., an immunoglobulin leader, such as an IgE leader. In some embodiments, the fragments do not have a leader sequence. In some embodiments, the fragments do not have a leader sequence, e.g., an IgE leader.

[0139] Immunogenic fragments of proteins can be provided that have an amino acid sequence identical to an immunogenic fragment of SEQ ID NO: 8. Such immunogenic fragments can comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of a protein that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, the fragment includes a leader sequence, e.g., an immunoglobulin leader, such as an IgE leader. In some embodiments, the fragment lacks a leader sequence. In some embodiments, the fragment does not have a leader sequence, for example an IgE leader.

[0140] As referred to herein with respect to the linkage of a signal peptide or leader sequence to the N-terminus of a protein, the signal peptide / leader sequence replaces the N-terminal methionine of the protein encoded by the start codon of a nucleic acid sequence encoding the protein without a signal peptide coding sequence.

[0141] (5) Growth hormone-releasing hormone (GHRH) The vaccines of the present invention can include the cancer antigen growth hormone-releasing hormone (GHRH, also known as growth-hormone-releasing factor (GRF or GHRF) or somatocrinin), a fragment thereof, or a variant thereof. GHRH is a 44-amino acid peptide hormone produced in the arcuate nucleus of the hypothalamus. GHRH is secreted by the hypothalamus and stimulates the release of growth hormone from the pituitary gland, a regulator of growth, metabolism, and body structure. GHRH also stimulates the production of growth hormone. GHRH antagonists inhibit the growth of various cancers, such as osteosarcoma, glioblastoma, prostate cancer, kidney cancer, pancreatic cancer, colorectal cancer, and breast cancer. GHRH can therefore be an antigen associated with various tumors.

[0142] GHRH antigens can induce antigen-specific T cell and / or high-titer antibody responses, thereby inducing or eliciting an immune response directed against or reactive to cancers or tumors expressing the antigen. In some embodiments, the induced or elicited immune response can be a cellular immune response, a humoral immune response, or both a cellular and humoral immune response. In some embodiments, the induced or elicited cellular immune response can include the induction or secretion of interferon-gamma (IFN-γ) and / or tumor necrosis factor alpha (TNF-α). In other embodiments, the induced or elicited immune response may reduce or inhibit one or more immunosuppressive factors that promote the growth of tumors or cancers that express the antigen, such as, but not limited to, factors that downregulate MHC presentation, antigen-specific regulatory T cells (Tregs), cytokines such as PD-L1, FasL, IL-10, and TFG-β, soluble factors produced by tumor-associated macrophages, tumor-associated fibroblasts, immunosuppressive cells, CTLA-4, PD-1, MDSC, MCP-1, and factors that upregulate immune checkpoint molecules, which are described in more detail below.

[0143] The GHRH antigen may comprise a protein epitope that makes it particularly effective as an immunogen capable of inducing a counteracting anti-GHRH immune response. The GHRH antigen may comprise a full-length translation product, a variant thereof, a fragment thereof, or a combination thereof. The GHRH antigen may comprise a consensus protein.

[0144] The nucleic acid sequence encoding the consensus GHRH antigen can be optimized for codon usage and the corresponding RNA transcript. The nucleic acid encoding the consensus GHRH antigen can be codon- and RNA-optimized for expression. In some embodiments, the nucleic acid sequence encoding the consensus GHRH antigen can include a Kozak sequence (e.g., GCC ACC) to improve translation efficiency. The nucleic acid encoding the consensus GHRH antigen can include multiple stop codons (e.g., TGA TGA) to improve translation termination efficiency.

[0145] The nucleic acid encoding the consensus GHRH antigen can also encode an immunoglobulin E (IgE) leader sequence. The nucleic acid encoding the consensus GHRH antigen can further encode an IgE leader sequence such that the amino acid sequence of the IgE leader sequence is linked to the amino acid sequence of the consensus GHRH antigen by a peptide bond. The nucleic acid encoding the consensus GHRH antigen can also comprise a nucleotide sequence encoding the IgE leader sequence. In some embodiments, the nucleic acid encoding the consensus GHRH antigen does not have, i.e., does not comprise, a nucleotide sequence encoding an IgE leader sequence.

[0146] The consensus GHRH antigen may be the nucleic acid sequence SEQ ID NO: 9, which encodes the amino acid sequence SEQ ID NO: 10. SEQ ID NO: 9 encodes a consensus GHRH protein linked to an IgE leader sequence. The consensus GHRH protein may be linked to an IgE leader sequence and an HA tag. In other embodiments, the consensus GHRH protein may not have an IgE leader sequence and / or an HA tag, i.e., may not be linked to said sequence and / or tag.

[0147] In some embodiments, the consensus GHRH antigen can be a nucleic acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over the entire length of the nucleic acid sequence set forth in SEQ ID NO: 9. In other embodiments, the consensus GHRH antigen can be a nucleic acid sequence encoding an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over the entire length of the amino acid sequence set forth in SEQ ID NO: 10. The consensus GHRH antigen may be an amino acid sequence that has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over the entire length of the amino acid sequence set forth in SEQ ID NO: 10.

[0148] Some embodiments relate to nucleic acid sequences encoding proteins homologous to the GHRH consensus protein, immunogenic fragments of the GHRH consensus protein, and immunogenic fragments of homologous proteins. Such nucleic acid molecules can be provided that encode immunogenic proteins having up to 95% homology to the consensus sequence, up to 96% homology to the consensus sequence, up to 97% homology to the consensus sequence, up to 98% homology to the consensus sequence, and up to 99% homology to the consensus sequence. Similarly, nucleic acid sequences encoding immunogenic fragments and immunogenic fragments of proteins homologous to the proteins provided herein are also provided.

[0149] Some embodiments relate to nucleic acid molecules encoding immunogenic proteins that are 95% homologous to the nucleic acid coding sequences herein. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins that are 96% homologous to the nucleic acid coding sequences herein. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins that are 97% homologous to the nucleic acid coding sequences herein. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins that are 98% homologous to the nucleic acid coding sequences herein. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins that are 99% homologous to the nucleic acid coding sequences herein. In some embodiments, a nucleic acid molecule having a coding sequence disclosed herein that is homologous to the coding sequence of a consensus protein disclosed herein comprises a sequence encoding an IgE leader sequence linked to the 5' end of the coding sequence encoding the homologous protein sequence disclosed herein.

[0150] Some embodiments relate to proteins having a particular identity expressed as a percentage to the full-length GHRH consensus protein, immunogenic fragments of the GHRH consensus protein, and nucleic acid sequences encoding immunogenic fragments of proteins having identity to the GHRH consensus protein. Such nucleic acid molecules can be provided which encode immunogenic proteins having up to 80% identity to the full-length GHRH consensus sequence, up to 85% identity to the full-length GHRH consensus sequence, up to 90% identity to the full-length GHRH consensus sequence, up to 91% identity to the full-length GHRH consensus sequence, up to 92% identity to the full-length GHRH consensus sequence, up to 93% identity to the full-length GHRH consensus sequence, up to 94% identity to the full-length GHRH consensus sequence, up to 95% identity to the full-length GHRH consensus sequence, up to 96% identity to the full-length GHRH consensus sequence, up to 97% identity to the full-length GHRH consensus sequence, up to 98% identity to the full-length GHRH consensus sequence, and up to 99% identity to the full-length GHRH consensus sequence. Similarly, nucleic acid sequences encoding immunogenic fragments of the proteins shown herein and having similar percent identities as those shown above for the GHRH proteins shown herein are also provided.

[0151] In some embodiments, the nucleic acid sequence does not have a coding sequence that encodes a leader sequence. In some embodiments, the nucleic acid sequence does not have a coding sequence that encodes an IgE leader.

[0152] Some embodiments relate to fragments of SEQ ID NO:9. The fragment can be at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:9. The fragment can be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a fragment of SEQ ID NO:9. In some embodiments, the fragment includes a sequence encoding a leader sequence, e.g., an immunoglobulin leader, such as an IgE leader. In some embodiments, the fragment does not have a coding sequence encoding a leader sequence. In some embodiments, the fragment does not have a coding sequence encoding a leader sequence, e.g., an IgE leader.

[0153] Furthermore, the amino acid sequence of the consensus GHRH protein is SEQ ID NO: 10. The amino acid sequence of the consensus GHRH protein linked to an IgE leader is SEQ ID NO: 10. The amino acid sequence of the consensus GHRH protein linked to an IgE leader may be linked to an HA tag.

[0154] Some embodiments relate to proteins homologous to SEQ ID NO: 10. Some embodiments relate to immunogenic proteins having 95% homology to the consensus protein sequence set forth in SEQ ID NO: 10. Some embodiments relate to immunogenic proteins having 96% homology to the consensus protein sequence set forth in SEQ ID NO: 10. Some embodiments relate to immunogenic proteins having 97% homology to the consensus protein sequence set forth in SEQ ID NO: 10. Some embodiments relate to immunogenic proteins having 98% homology to the consensus protein sequence set forth in SEQ ID NO: 10. Some embodiments relate to immunogenic proteins having 99% homology to the consensus protein sequence set forth in SEQ ID NO: 10.

[0155] Some embodiments relate to a protein that is identical to SEQ ID NO: 10. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 80% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 10. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 85% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 10. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 90% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 10. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 91% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 10. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 92% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 10. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 93% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 10. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 94% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 10. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 95% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 10. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 96% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 10. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 97% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 10. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 98% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 10. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 99% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 10.

[0156] In some embodiments, the protein does not have a leader sequence. In some embodiments, the protein does not have an IgE leader. A fragment of the consensus protein can comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the consensus protein. An immunogenic fragment of SEQ ID NO: 10 can be provided. An immunogenic fragment can comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of SEQ ID NO: 10. In some embodiments, the fragment includes a leader sequence, e.g., an immunoglobulin leader, such as an IgE leader. In some embodiments, the fragment lacks a leader sequence. In some embodiments, the fragment lacks a leader sequence, e.g., an IgE leader.

[0157] Immunogenic fragments of proteins having amino acid sequences homologous to immunogenic fragments of SEQ ID NO: 10 can be provided. Such immunogenic fragments can include at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of a protein that is 95% or more homologous to SEQ ID NO: 10. Some embodiments relate to immunogenic fragments having 96% homology to immunogenic fragments of the consensus protein sequences herein. Some embodiments relate to immunogenic fragments having 97% homology to immunogenic fragments of the consensus protein sequences herein. Some embodiments relate to immunogenic fragments having 98% homology to the immunogenic fragments of the consensus protein sequences herein. Some embodiments relate to immunogenic fragments having 99% homology to the immunogenic fragments of the consensus protein sequences herein. In some embodiments, the fragments include a leader sequence, e.g., an immunoglobulin leader, such as an IgE leader. In some embodiments, the fragments do not have a leader sequence. In some embodiments, the fragments do not have a leader sequence, e.g., an IgE leader.

[0158] Immunogenic fragments of proteins can be provided that have an amino acid sequence identical to an immunogenic fragment of SEQ ID NO: 10. Such immunogenic fragments can comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of a protein that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the fragments include a leader sequence, e.g., an immunoglobulin leader, such as an IgE leader. In some embodiments, the fragments lack a leader sequence. In some embodiments, the fragment does not have a leader sequence, for example an IgE leader.

[0159] As referred to herein with respect to the linkage of a signal peptide or leader sequence to the N-terminus of a protein, the signal peptide / leader sequence replaces the N-terminal methionine of the protein encoded by the start codon of a nucleic acid sequence encoding the protein without a signal peptide coding sequence.

[0160] (6) MART-1 / Melan-A The vaccines of the present invention can include the cancer antigen MART-1 (also known as Melan-A), its fragments, or variants. MART-1 is encoded by the MLANA gene, a 118-amino acid protein containing a single transmembrane domain, and is expressed in most melanoma cells. MART-1 forms complexes with structural proteins and affects their expression, stability, transport, and processing required for melanosome structure and maturation. Thus, MART-1 is essential for regulating mammalian pigmentation. Defects in melanosome maturation have been linked to cancer susceptibility. MART-1 can be expressed in a number of cancers, including, but not limited to, melanoma.

[0161] Melan-A, also known as melanoma antigen recognized by T cells (MART-1), is a melanocyte differentiation antigen and can be present in normal skin, retina, and melanocytes. Melan-A can be associated with the endoplasmic reticulum and melanosomes. While melan-A can be recognized by cytotoxic T cells as an antigen on melanoma cells, it can also be associated with other tumors of melanocytic origin or differentiation (i.e., cells containing melanosomes), such as clear cell sarcoma and melanotic neurofibroma. Thus, melan-A can be an antigen associated with a variety of tumors derived from cells containing melanosomes.

[0162] The Melan-A antigen can induce antigen-specific T cell and / or high-titer antibody responses, thereby inducing or eliciting an immune response directed against or reactive to cancers or tumors expressing the antigen. In some embodiments, the induced or elicited immune response can be a cellular immune response, a humoral immune response, or both a cellular and humoral immune response. In some embodiments, the induced or elicited cellular immune response can include the induction or secretion of interferon-gamma (IFN-γ) and / or tumor necrosis factor alpha (TNF-α). In other embodiments, the induced or elicited immune response may reduce or inhibit one or more immunosuppressive factors that promote the growth of tumors or cancers that express the antigen, such as, but not limited to, factors that downregulate MHC presentation, antigen-specific regulatory T cells (Tregs), cytokines such as PD-L1, FasL, IL-10, and TFG-β, soluble factors produced by tumor-associated macrophages, tumor-associated fibroblasts, immunosuppressive cells, CTLA-4, PD-1, MDSC, MCP-1, and factors that upregulate immune checkpoint molecules, which are described in more detail below.

[0163] The Melan-A antigen may comprise a protein epitope that makes it particularly effective as an immunogen capable of inducing a counteracting anti-Melan-A immune response. The Melan-A antigen may comprise a full-length translation product, a variant thereof, a fragment thereof, or a combination thereof. The Melan-A antigen may comprise a consensus protein.

[0164] Nucleic acid sequences encoding consensus melan-A antigens can be optimized for codon usage and corresponding RNA transcripts. Nucleic acids encoding consensus melan-A antigens can be codon- and RNA-optimized for expression. In some embodiments, nucleic acid sequences encoding consensus melan-A antigens can include a Kozak sequence (e.g., GCC ACC) to improve translation efficiency. Nucleic acids encoding consensus melan-A antigens can include multiple stop codons (e.g., TGA TGA) to improve translation termination efficiency.

[0165] The nucleic acid encoding the consensus melan-A antigen can also encode an immunoglobulin E (IgE) leader sequence. The nucleic acid encoding the consensus melan-A antigen can further encode an IgE leader sequence, such that the amino acid sequence of the IgE leader sequence is linked to the amino acid sequence of the consensus melan-A antigen by a peptide bond. The nucleic acid encoding the consensus melan-A antigen can also include a nucleotide sequence encoding the IgE leader sequence. In some embodiments, the nucleic acid encoding the consensus melan-A antigen does not have, i.e., does not include, a nucleotide sequence encoding an IgE leader sequence.

[0166] The consensus melan-A antigen may be the nucleic acid sequence SEQ ID NO:11, which encodes the amino acid sequence SEQ ID NO:12. SEQ ID NO:11 encodes the consensus melan-A protein linked to an IgE leader sequence. The consensus melan-A protein may be linked to an IgE leader sequence and an HA tag. In other embodiments, the consensus melan-A protein may not have the IgE leader sequence and / or the HA tag, i.e., may not be linked to said sequence and / or tag.

[0167] In some embodiments, the consensus melan-A antigen can be a nucleic acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over the entire length of the nucleic acid sequence set forth in SEQ ID NO: 11. In other embodiments, the consensus melan-A antigen can be a nucleic acid sequence encoding an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over the entire length of the amino acid sequence set forth in SEQ ID NO: 12. The consensus Melan-A antigen may be an amino acid sequence that has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over the entire length of the amino acid sequence set forth in SEQ ID NO: 12.

[0168] Some embodiments relate to nucleic acid sequences encoding proteins homologous to the Melan-A consensus protein, immunogenic fragments of the Melan-A consensus protein, and immunogenic fragments of homologous proteins. Such nucleic acid molecules can be provided that encode immunogenic proteins having up to 95% homology to the consensus sequence, up to 96% homology to the consensus sequence, up to 97% homology to the consensus sequence, up to 98% homology to the consensus sequence, and up to 99% homology to the consensus sequence. Similarly, nucleic acid sequences encoding immunogenic fragments as set forth herein and immunogenic fragments of proteins homologous to the proteins set forth herein are also provided.

[0169] Some embodiments relate to nucleic acid molecules encoding immunogenic proteins that are 95% homologous to the nucleic acid coding sequences herein. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins that are 96% homologous to the nucleic acid coding sequences herein. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins that are 97% homologous to the nucleic acid coding sequences herein. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins that are 98% homologous to the nucleic acid coding sequences herein. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins that are 99% homologous to the nucleic acid coding sequences herein. In some embodiments, a nucleic acid molecule having a coding sequence disclosed herein that is homologous to the coding sequence of a consensus protein disclosed herein comprises a sequence encoding an IgE leader sequence linked to the 5' end of the coding sequence encoding the homologous protein sequence disclosed herein.

[0170] Some embodiments relate to nucleic acid sequences encoding proteins having a particular identity expressed as a percentage to the full-length Melan-A consensus protein, immunogenic fragments of the Melan-A consensus protein, and immunogenic fragments of proteins having identity to the Melan-A consensus protein. Such nucleic acid molecules can be provided which encode immunogenic proteins having up to 80% identity to the full-length Melan-A consensus sequence, up to 85% identity to the full-length Melan-A consensus sequence, up to 90% identity to the full-length Melan-A consensus sequence, up to 91% identity to the full-length Melan-A consensus sequence, up to 92% identity to the full-length Melan-A consensus sequence, up to 93% identity to the full-length Melan-A consensus sequence, up to 94% identity to the full-length Melan-A consensus sequence, up to 95% identity to the full-length Melan-A consensus sequence, up to 96% identity to the full-length Melan-A consensus sequence, up to 97% identity to the full-length Melan-A consensus sequence, up to 98% identity to the full-length Melan-A consensus sequence, and up to 99% identity to the full-length Melan-A consensus sequence. Similarly, nucleic acid sequences encoding immunogenic fragments of the proteins shown herein and having similar percentage identities to those shown above for the Melan-A proteins shown herein are also provided.

[0171] In some embodiments, the nucleic acid sequence does not have a coding sequence that encodes a leader sequence. In some embodiments, the nucleic acid sequence does not have a coding sequence that encodes an IgE leader.

[0172] Some embodiments relate to fragments of SEQ ID NO: 11. The fragment can be at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 11. The fragment can be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a fragment of SEQ ID NO: 11. In some embodiments, the fragment includes a sequence encoding a leader sequence, e.g., an immunoglobulin leader, such as an IgE leader. In some embodiments, the fragment does not have a coding sequence encoding a leader sequence. In some embodiments, the fragment does not have a coding sequence encoding a leader sequence, e.g., an IgE leader.

[0173] Furthermore, the amino acid sequence of the consensus melan-A protein is SEQ ID NO: 12. The amino acid sequence of the consensus melan-A protein linked to an IgE leader is SEQ ID NO: 12. The amino acid sequence of the consensus melan-A protein linked to an IgE leader may be linked to an HA tag.

[0174] Some embodiments relate to proteins homologous to SEQ ID NO: 12. Some embodiments relate to immunogenic proteins having 95% homology to the consensus protein sequence set forth in SEQ ID NO: 12. Some embodiments relate to immunogenic proteins having 96% homology to the consensus protein sequence set forth in SEQ ID NO: 12. Some embodiments relate to immunogenic proteins having 97% homology to the consensus protein sequence set forth in SEQ ID NO: 12. Some embodiments relate to immunogenic proteins having 98% homology to the consensus protein sequence set forth in SEQ ID NO: 12. Some embodiments relate to immunogenic proteins having 99% homology to the consensus protein sequence set forth in SEQ ID NO: 12.

[0175] Some embodiments relate to a protein that is identical to SEQ ID NO: 12. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 80% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 12. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 85% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 12. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 90% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 12. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 91% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 12. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 92% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 12. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 93% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 12. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 94% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 12. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 95% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 12. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 96% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 12. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 97% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 12. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 98% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 12. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 99% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 12.

[0176] In some embodiments, the protein does not have a leader sequence. In some embodiments, the protein does not have an IgE leader. A fragment of the consensus protein can comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the consensus protein. An immunogenic fragment of SEQ ID NO: 12 can be provided. An immunogenic fragment can comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of SEQ ID NO: 12. In some embodiments, the fragment includes a leader sequence, e.g., an immunoglobulin leader, such as an IgE leader. In some embodiments, the fragment lacks a leader sequence. In some embodiments, the fragment lacks a leader sequence, e.g., an IgE leader.

[0177] Immunogenic fragments of proteins having amino acid sequences homologous to immunogenic fragments of SEQ ID NO: 12 can be provided. Such immunogenic fragments can include at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of a protein that is 95% or more homologous to SEQ ID NO: 12. Some embodiments relate to immunogenic fragments having 96% homology to immunogenic fragments of the consensus protein sequences herein. Some embodiments relate to immunogenic fragments having 97% homology to immunogenic fragments of the consensus protein sequences herein. Some embodiments relate to immunogenic fragments having 98% homology to the immunogenic fragments of the consensus protein sequences herein. Some embodiments relate to immunogenic fragments having 99% homology to the immunogenic fragments of the consensus protein sequences herein. In some embodiments, the fragments include a leader sequence, e.g., an immunoglobulin leader, such as an IgE leader. In some embodiments, the fragments do not have a leader sequence. In some embodiments, the fragments do not have a leader sequence, e.g., an IgE leader.

[0178] Immunogenic fragments of proteins can be provided that have an amino acid sequence identical to an immunogenic fragment of SEQ ID NO: 12. Such immunogenic fragments can comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of a protein that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the fragments include a leader sequence, e.g., an immunoglobulin leader, such as an IgE leader. In some embodiments, the fragments lack a leader sequence. In some embodiments, the fragment does not have a leader sequence, for example an IgE leader.

[0179] As referred to herein with respect to the linkage of a signal peptide or leader sequence to the N-terminus of a protein, the signal peptide / leader sequence replaces the N-terminal methionine of the protein encoded by the start codon of a nucleic acid sequence encoding the protein without a signal peptide coding sequence.

[0180] (7)NY-ESO-1 The vaccines of the present invention can contain the cancer antigen New York Esophageal Cancer-1 (NY-ESO-1, also known as CTAG1), a fragment thereof, or a variant thereof. NY-ESO-1 is encoded by the CTAG1B gene and is a 180-amino acid long protein with a glycine-rich N-terminal region and an extremely hydrophobic C-terminal region. NY-ESO-1 expression is restricted in normal tissues but occurs frequently in cancers. NY-ESO-1 can be expressed in a number of cancers, including, but not limited to, bladder cancer, colorectal cancer, esophageal cancer, gastric cancer, hepatocellular carcinoma, head and neck cancer, melanoma, non-small cell lung cancer, ovarian cancer, pancreatic cancer, synovial cancer, and prostate cancer.

[0181] The cancer-testis antigen (NY-ESO-1) can be expressed in the testis and ovary. NY-ESO-1 is associated with various cancers and can induce humoral immune responses. Subjects with cancer or tumors can become immunogenic to NY-ESO-1. Therefore, NY-ESO-1 may be an antigen associated with various tumors.

[0182] The NY-ESO-1 antigen can induce antigen-specific T cell and / or high-titer antibody responses, thereby inducing or eliciting an immune response directed against or reactive to cancers or tumors expressing the antigen. In some embodiments, the induced or elicited immune response can be a cellular immune response, a humoral immune response, or both a cellular and humoral immune response. In some embodiments, the induced or elicited cellular immune response can include the induction or secretion of interferon-gamma (IFN-γ) and / or tumor necrosis factor alpha (TNF-α). In other embodiments, the induced or elicited immune response may reduce or inhibit one or more immunosuppressive factors that promote the growth of tumors or cancers that express the antigen, such as, but not limited to, factors that downregulate MHC presentation, antigen-specific regulatory T cells (Tregs), cytokines such as PD-L1, FasL, IL-10, and TFG-β, soluble factors produced by tumor-associated macrophages, tumor-associated fibroblasts, immunosuppressive cells, CTLA-4, PD-1, MDSC, MCP-1, and factors that upregulate immune checkpoint molecules, which are described in more detail below.

[0183] The NY-ESO-1 antigen can increase the cellular immune response in a subject administered the NY-ESO-1 antigen by approximately 50 to approximately 6,000 times, approximately 50 to approximately 5,500 times, approximately 50 to approximately 5,000 times, approximately 50 to approximately 4,500 times, approximately 100 to approximately 6,000 times, approximately 150 to approximately 6,000 times, approximately 200 to approximately 6,000 times, approximately 250 to approximately 6,000 times, or approximately 300 to approximately 6,000 times, compared to the cellular immune response in a subject not administered the NY-ESO-1 antigen. In some embodiments, the NY-ESO-1 antigen enhances the cellular immune response in a subject administered the NY-ESO-1 antigen by about 50-fold, 100-fold, 150-fold, 200-fold, 250-fold, 300-fold, 350-fold, 400-fold, 450-fold, 500-fold, 550-fold, 600-fold, 650-fold, 700-fold, 750-fold, 800-fold, 850-fold, 900-fold, 950-fold, 1000-fold, 1100-fold, 1200-fold, 1300-fold, 1400-fold, 1500-fold, 1600-fold, 1700-fold, 1800-fold, 1900-fold, or 2000-fold compared to the cellular immune response in a subject not administered the NY-ESO-1 antigen. , 2100x, 2200x, 2300x, 2400x, 2500x, 2600x, 2700x, 2800x, 2900x, 3000x, 3100x, 3200x, 3300x, 3400x, 3500x, 3600x, 3700x, 3800x, 3900x, 4000x, 4100x, 4200x, 4300x, 4400x, 4500x, 4600x, 4700x, 4800x, 4900x, 5000x, 5100x, 5200x, 5300x, 5400x, 5500x, 5600x, 5700x, 5800x, 5900x, or 6000x.

[0184] The NY-ESO-1 antigen can increase the concentration of interferon-gamma (IFN-γ) in a subject administered the NY-ESO-1 antigen by approximately 50 to approximately 6,000 times, approximately 50 to approximately 5,500 times, approximately 50 to approximately 5,000 times, approximately 50 to approximately 4,500 times, approximately 100 to approximately 6,000 times, approximately 150 to approximately 6,000 times, approximately 200 to approximately 6,000 times, approximately 250 to approximately 6,000 times, or approximately 300 to approximately 6,000 times, compared to the IFN-γ concentration in a subject not administered the NY-ESO-1 antigen. In some embodiments, the NY-ESO-1 antigen increases IFN-γ levels in a subject administered the NY-ESO-1 antigen by about 50-fold, 100-fold, 150-fold, 200-fold, 250-fold, 300-fold, 350-fold, 400-fold, 450-fold, 500-fold, 550-fold, 600-fold, 650-fold, 700-fold, 750-fold, 800-fold, 850-fold, 900-fold, 950-fold, 1000-fold, 1100-fold, 1200-fold, 1300-fold, 1400-fold, 1500-fold, 1600-fold, 1700-fold, 1800-fold, 1900-fold, or 2000-fold compared to IFN-γ levels in a subject not administered the NY-ESO-1 antigen. , 2100x, 2200x, 2300x, 2400x, 2500x, 2600x, 2700x, 2800x, 2900x, 3000x, 3100x, 3200x, 3300x, 3400x, 3500x, 3600x, 3700x, 3800x, 3900x, 4000x, 4100x, 4200x, 4300x, 4400x, 4500x, 4600x, 4700x, 4800x, 4900x, 5000x, 5100x, 5200x, 5300x, 5400x, 5500x, 5600x, 5700x, 5800x, 5900x, or 6000x.

[0185] The NY-ESO-1 antigen can comprise a protein epitope that makes the antigen particularly effective as an immunogen capable of inducing a counter-NY-ESO-1 immune response. The NY-ESO-1 antigen can comprise a full-length translation product, a variant thereof, a fragment thereof, or a combination thereof. The NY-ESO-1 antigen can comprise a consensus protein.

[0186] Nucleic acid sequences encoding consensus NY-ESO-1 antigens can be optimized for codon usage and corresponding RNA transcripts. Nucleic acids encoding consensus NY-ESO-1 antigens can be codon- and RNA-optimized for expression. In some embodiments, nucleic acid sequences encoding consensus NY-ESO-1 antigens can include a Kozak sequence (e.g., GCC ACC) to improve translation efficiency. Nucleic acids encoding consensus NY-ESO-1 antigens can include multiple stop codons (e.g., TGA TGA) to improve translation termination efficiency.

[0187] The nucleic acid encoding the consensus NY-ESO-1 antigen can also encode an immunoglobulin E (IgE) leader sequence. The nucleic acid encoding the consensus NY-ESO-1 antigen can further encode an IgE leader sequence such that the amino acid sequence of the IgE leader sequence is linked to the amino acid sequence of the consensus NY-ESO-1 antigen by a peptide bond. The nucleic acid encoding the consensus NY-ESO-1 antigen can also include a nucleotide sequence encoding the IgE leader sequence. In some embodiments, the nucleic acid encoding the consensus NY-ESO-1 antigen does not have, i.e., does not include, a nucleotide sequence encoding an IgE leader sequence.

[0188] The consensus NY-ESO-1 antigen can be the nucleic acid sequence SEQ ID NO: 13, which encodes the amino acid sequence SEQ ID NO: 14. SEQ ID NO: 13 encodes the consensus NY-ESO-1 protein linked to an IgE leader sequence. The consensus NY-ESO-1 protein can be linked to an IgE leader sequence and an HA tag. In other embodiments, the consensus NY-ESO-1 protein may not have the IgE leader sequence and / or the HA tag, i.e., may not be linked to said sequence and / or tag.

[0189] In some embodiments, the consensus NY-ESO-1 antigen can be a nucleic acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over the entire length of the nucleic acid sequence set forth in SEQ ID NO:13. In other embodiments, the consensus NY-ESO-1 antigen can be a nucleic acid sequence encoding an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over the entire length of the amino acid sequence set forth in SEQ ID NO: 14. The consensus NY-ESO-1 antigen can be an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over the entire length of the amino acid sequence set forth in SEQ ID NO: 14.

[0190] Some embodiments relate to nucleic acid sequences encoding proteins homologous to the NY-ESO-1 consensus protein, immunogenic fragments of the NY-ESO-1 consensus protein, and immunogenic fragments of homologous proteins. Such nucleic acid molecules can be provided that encode immunogenic proteins having up to 95% homology to the consensus sequence, up to 96% homology to the consensus sequence, up to 97% homology to the consensus sequence, up to 98% homology to the consensus sequence, and up to 99% homology to the consensus sequence. Similarly, nucleic acid sequences encoding immunogenic fragments and immunogenic fragments of proteins homologous to the proteins provided herein are also provided.

[0191] Some embodiments relate to nucleic acid molecules encoding immunogenic proteins that are 95% homologous to the nucleic acid coding sequences herein. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins that are 96% homologous to the nucleic acid coding sequences herein. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins that are 97% homologous to the nucleic acid coding sequences herein. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins that are 98% homologous to the nucleic acid coding sequences herein. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins that are 99% homologous to the nucleic acid coding sequences herein. In some embodiments, a nucleic acid molecule having a coding sequence disclosed herein that is homologous to the coding sequence of a consensus protein disclosed herein comprises a sequence encoding an IgE leader sequence linked to the 5' end of the coding sequence encoding the homologous protein sequence disclosed herein.

[0192] Some embodiments relate to proteins having a particular percent identity to the full-length NY-ESO-1 consensus protein, immunogenic fragments of the NY-ESO-1 consensus protein, and nucleic acid sequences encoding immunogenic fragments of proteins having identity to the NY-ESO-1 consensus protein, including up to 80% identity to the full-length NY-ESO-1 consensus sequence, up to 85% identity to the full-length NY-ESO-1 consensus sequence, up to 90% identity to the full-length NY-ESO-1 consensus sequence, up to 91% identity to the full-length NY-ESO-1 consensus sequence, up to 92% identity to the full-length NY-ESO-1 consensus sequence, up to 93% identity to the full-length NY-ESO-1 consensus sequence, up to 94% identity to the full-length NY-ESO-1 consensus sequence, up to 95% identity to the full-length NY-ESO-1 consensus sequence, up to 96% identity to the full-length NY-ESO-1 consensus sequence, up to 97% identity to the full-length NY-ESO-1 consensus sequence, up to 98% identity to the full-length NY-ESO-1 consensus sequence, up to 99% identity to the full-length NY-ESO-1 consensus sequence, up to 100% identity to the full-length NY-ESO-1 consensus sequence, up to 101% identity to the full-length NY-ESO-1 consensus sequence, up to 102% identity to the full-length NY-ESO-1 consensus sequence, up to 103% identity to the full-length NY-ESO-1 consensus sequence, up to 104% identity to the full-length NY-ESO-1 consensus sequence, up to 105% identity to the full-length NY-ESO-1 consensus sequence, up to 106% identity to the full-length NY-ESO-1 consensus sequence, up to 107% identity to the full-length NY-ESO- Such nucleic acid molecules can be provided that encode immunogenic proteins having up to 94% identity to the full-length NY-ESO-1 consensus sequence, up to 95% identity to the full-length NY-ESO-1 consensus sequence, up to 96% identity to the full-length NY-ESO-1 consensus sequence, up to 97% identity to the full-length NY-ESO-1 consensus sequence, up to 98% identity to the full-length NY-ESO-1 consensus sequence, and up to 99% identity to the full-length NY-ESO-1 consensus sequence. Similarly, nucleic acid sequences encoding immunogenic fragments of the immunogenic fragments set forth herein and proteins having similar percentage identities as set forth above to the NY-ESO-1 proteins set forth herein are also provided.

[0193] In some embodiments, the nucleic acid sequence does not have a coding sequence that encodes a leader sequence. In some embodiments, the nucleic acid sequence does not have a coding sequence that encodes an IgE leader.

[0194] Some embodiments relate to fragments of SEQ ID NO: 13. The fragment can be at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 13. The fragment can be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a fragment of SEQ ID NO: 13. In some embodiments, the fragment includes a sequence encoding a leader sequence, e.g., an immunoglobulin leader, such as an IgE leader. In some embodiments, the fragment does not have a coding sequence encoding a leader sequence. In some embodiments, the fragment does not have a coding sequence encoding a leader sequence, e.g., an IgE leader.

[0195] Furthermore, the amino acid sequence of the consensus NY-ESO-1 protein is SEQ ID NO: 14. The amino acid sequence of the consensus NY-ESO-1 protein linked to an IgE leader is SEQ ID NO: 14. The amino acid sequence of the consensus NY-ESO-1 protein linked to an IgE leader may be linked to an HA tag.

[0196] Some embodiments relate to proteins homologous to SEQ ID NO: 14. Some embodiments relate to immunogenic proteins having 95% homology to the consensus protein sequence set forth in SEQ ID NO: 14. Some embodiments relate to immunogenic proteins having 96% homology to the consensus protein sequence set forth in SEQ ID NO: 14. Some embodiments relate to immunogenic proteins having 97% homology to the consensus protein sequence set forth in SEQ ID NO: 14. Some embodiments relate to immunogenic proteins having 98% homology to the consensus protein sequence set forth in SEQ ID NO: 14. Some embodiments relate to immunogenic proteins having 99% homology to the consensus protein sequence set forth in SEQ ID NO: 14.

[0197] Some embodiments relate to a protein that is identical to SEQ ID NO: 14. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 80% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 14. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 85% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 14. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 90% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 14. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 91% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 14. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 92% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 14. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 93% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 14. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 94% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 14. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 95% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 14. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 96% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 14. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 97% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 14. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 98% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 14. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 99% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 14.

[0198] In some embodiments, the protein does not have a leader sequence. In some embodiments, the protein does not have an IgE leader. A fragment of the consensus protein can comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the consensus protein. An immunogenic fragment of SEQ ID NO: 14 can be provided. An immunogenic fragment can comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of SEQ ID NO: 14. In some embodiments, the fragment includes a leader sequence, e.g., an immunoglobulin leader, such as an IgE leader. In some embodiments, the fragment lacks a leader sequence. In some embodiments, the fragment lacks a leader sequence, e.g., an IgE leader.

[0199] Immunogenic fragments of proteins having amino acid sequences homologous to immunogenic fragments of SEQ ID NO: 14 can be provided. Such immunogenic fragments can include at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of a protein that is 95% or more homologous to SEQ ID NO: 14. Some embodiments relate to immunogenic fragments having 96% homology to immunogenic fragments of the consensus protein sequences herein. Some embodiments relate to immunogenic fragments having 97% homology to immunogenic fragments of the consensus protein sequences herein. Some embodiments relate to immunogenic fragments having 98% homology to the immunogenic fragments of the consensus protein sequences herein. Some embodiments relate to immunogenic fragments having 99% homology to the immunogenic fragments of the consensus protein sequences herein. In some embodiments, the fragments include a leader sequence, e.g., an immunoglobulin leader, such as an IgE leader. In some embodiments, the fragments do not have a leader sequence. In some embodiments, the fragments do not have a leader sequence, e.g., an IgE leader.

[0200] Immunogenic fragments of proteins can be provided that have an amino acid sequence identical to an immunogenic fragment of SEQ ID NO: 14. Such immunogenic fragments can comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of a protein that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, the fragment includes a leader sequence, e.g., an immunoglobulin leader, such as an IgE leader. In some embodiments, the fragment lacks a leader sequence. In some embodiments, the fragment does not have a leader sequence, for example an IgE leader.

[0201] As referred to herein with respect to the linkage of a signal peptide or leader sequence to the N-terminus of a protein, the signal peptide / leader sequence replaces the N-terminal methionine of the protein encoded by the start codon of a nucleic acid sequence encoding the protein without a signal peptide coding sequence.

[0202] (8)NY-ESO-2 The vaccines of the present invention can contain the cancer antigen New York esophageal carcinoma-2 (NY-ESO-2; also known as cancer-testis antigen 2, ESO2, and LAGE1), a fragment thereof, or a variant thereof. NY-ESO-2 is an autoimmunogenic tumor antigen belonging to the ESO / LAGE family of cancer-testis antigens. NY-ESO-2 can be expressed in a number of cancers, including melanoma, breast cancer, bladder cancer, and prostate cancer, and is commonly expressed in the testis. In addition, NY-ESO-2 can be observed in 25-50% of tumor samples from melanoma, non-small cell lung cancer, bladder cancer, prostate cancer, and head and neck cancer. The gene encoding NY-ESO-2 also contains an additional open reading frame encoding a protein named CAMEL, a tumor antigen recognized by melanoma-specific cytotoxic T lymphocytes.

[0203] Similar to NY-ESO-1, NY-ESO-2 can be expressed in the testis and ovary. NY-ESO-2 is also associated with various cancers and can be immunogenic in subjects with cancer or tumors. Therefore, NY-ESO-2 may be an antigen associated with many tumors.

[0204] The NY-ESO-2 antigen can induce antigen-specific T cell and / or high-titer antibody responses, thereby inducing or eliciting an immune response directed against or reactive to cancers or tumors expressing the antigen. In some embodiments, the induced or elicited immune response can be a cellular immune response, a humoral immune response, or both a cellular and humoral immune response. In some embodiments, the induced or elicited cellular immune response can include the induction or secretion of interferon-gamma (IFN-γ) and / or tumor necrosis factor alpha (TNF-α). In other embodiments, the induced or elicited immune response may reduce or inhibit one or more immunosuppressive factors that promote the growth of tumors or cancers that express the antigen, such as, but not limited to, factors that downregulate MHC presentation, antigen-specific regulatory T cells (Tregs), cytokines such as PD-L1, FasL, IL-10, and TFG-β, soluble factors produced by tumor-associated macrophages, tumor-associated fibroblasts, immunosuppressive cells, CTLA-4, PD-1, MDSC, MCP-1, and factors that upregulate immune checkpoint molecules, which are described in more detail below.

[0205] The NY-ESO-2 antigen can increase the cellular immune response in a subject administered the NY-ESO-2 antigen by about 50 to about 6,000 times, about 50 to about 5,500 times, about 50 to about 5,000 times, about 50 to about 4,500 times, about 100 to about 6,000 times, about 150 to about 6,000 times, about 200 to about 6,000 times, about 250 to about 6,000 times, or about 300 to about 6,000 times, compared to the cellular immune response in a subject not administered the NY-ESO-2 antigen. In some embodiments, the NY-ESO-2 antigen enhances the cellular immune response in a subject administered the NY-ESO-2 antigen by about 50 fold, 100 fold, 150 fold, 200 fold, 250 fold, 300 fold, 350 fold, 400 fold, 450 fold, 500 fold, 550 fold, 600 fold, 650 fold, 700 fold, 750 fold, 800 fold, 850 fold, 900 fold, 950 fold, 1000 fold, 1100 fold, 1200 fold, 1300 fold, 1400 fold, 1500 fold, 1600 fold, 1700 fold, 1800 fold, 1900 fold, 2000 fold, or 3000 fold compared to the cellular immune response in a subject not administered the NY-ESO-2 antigen. , 2100x, 2200x, 2300x, 2400x, 2500x, 2600x, 2700x, 2800x, 2900x, 3000x, 3100x, 3200x, 3300x, 3400x, 3500x, 3600x, 3700x, 3800x, 3900x, 4000x, 4100x, 4200x, 4300x, 4400x, 4500x, 4600x, 4700x, 4800x, 4900x, 5000x, 5100x, 5200x, 5300x, 5400x, 5500x, 5600x, 5700x, 5800x, 5900x, or 6000x.

[0206] The NY-ESO-2 antigen can increase the concentration of interferon-gamma (IFN-γ) in a subject administered the NY-ESO-2 antigen by about 50 to about 6,000 times, about 50 to about 5,500 times, about 50 to about 5,000 times, about 50 to about 4,500 times, about 100 to about 6,000 times, about 150 to about 6,000 times, about 200 to about 6,000 times, about 250 to about 6,000 times, or about 300 to about 6,000 times compared to the IFN-γ concentration in a subject not administered the NY-ESO-2 antigen. In some embodiments, the NY-ESO-2 antigen increases IFN-γ levels in a subject administered the NY-ESO-2 antigen by about 50-fold, 100-fold, 150-fold, 200-fold, 250-fold, 300-fold, 350-fold, 400-fold, 450-fold, 500-fold, 550-fold, 600-fold, 650-fold, 700-fold, 750-fold, 800-fold, 850-fold, 900-fold, 950-fold, 1000-fold, 1100-fold, 1200-fold, 1300-fold, 1400-fold, 1500-fold, 1600-fold, 1700-fold, 1800-fold, 1900-fold, or 2000-fold compared to IFN-γ levels in a subject not administered the NY-ESO-2 antigen. , 2100x, 2200x, 2300x, 2400x, 2500x, 2600x, 2700x, 2800x, 2900x, 3000x, 3100x, 3200x, 3300x, 3400x, 3500x, 3600x, 3700x, 3800x, 3900x, 4000x, 4100x, 4200x, 4300x, 4400x, 4500x, 4600x, 4700x, 4800x, 4900x, 5000x, 5100x, 5200x, 5300x, 5400x, 5500x, 5600x, 5700x, 5800x, 5900x, or 6000x.

[0207] The NY-ESO-2 antigen can comprise a protein epitope that makes the antigen particularly effective as an immunogen capable of inducing a counter-NY-ESO-2 immune response. The NY-ESO-2 antigen can comprise a full-length translation product, a variant thereof, a fragment thereof, or a combination thereof. The NY-ESO-2 antigen can comprise a consensus protein.

[0208] Nucleic acid sequences encoding consensus NY-ESO-2 antigens can be optimized for codon usage and corresponding RNA transcripts. Nucleic acids encoding consensus NY-ESO-2 antigens can be codon- and RNA-optimized for expression. In some embodiments, nucleic acid sequences encoding consensus NY-ESO-2 antigens can include a Kozak sequence (e.g., GCC ACC) to improve translation efficiency. Nucleic acids encoding consensus NY-ESO-2 antigens can include multiple stop codons (e.g., TGA TGA) to improve translation termination efficiency.

[0209] The nucleic acid encoding the consensus NY-ESO-2 antigen can also encode an immunoglobulin E (IgE) leader sequence. The nucleic acid encoding the consensus NY-ESO-2 antigen can further encode an IgE leader sequence such that the amino acid sequence of the IgE leader sequence is linked to the amino acid sequence of the consensus NY-ESO-2 antigen by a peptide bond. The nucleic acid encoding the consensus NY-ESO-2 antigen can also include a nucleotide sequence encoding the IgE leader sequence. In some embodiments, the nucleic acid encoding the consensus NY-ESO-2 antigen does not have, i.e., does not include, a nucleotide sequence encoding an IgE leader sequence.

[0210] The consensus NY-ESO-2 antigen can be the nucleic acid sequence SEQ ID NO: 15, which encodes the amino acid sequence SEQ ID NO: 16. SEQ ID NO: 1 encodes the consensus NY-ESO-2 protein linked to an IgE leader sequence. The consensus NY-ESO-2 protein can be linked to an IgE leader sequence and an HA tag. In other embodiments, the consensus NY-ESO-2 protein may not have the IgE leader sequence and / or the HA tag, i.e., may not be linked to said sequence and / or tag.

[0211] In some embodiments, the consensus NY-ESO-2 antigen can be a nucleic acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over the entire length of the nucleic acid sequence set forth in SEQ ID NO: 15. In other embodiments, the consensus NY-ESO-2 antigen can be a nucleic acid sequence encoding an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over the entire length of the amino acid sequence set forth in SEQ ID NO: 16. The consensus NY-ESO-2 antigen can be an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over the entire length of the amino acid sequence set forth in SEQ ID NO: 16.

[0212] Some embodiments relate to nucleic acid sequences encoding proteins homologous to the NY-ESO-2 consensus protein, immunogenic fragments of the NY-ESO-2 consensus protein, and immunogenic fragments of homologous proteins. Such nucleic acid molecules can be provided that encode immunogenic proteins having up to 95% homology to the consensus sequence, up to 96% homology to the consensus sequence, up to 97% homology to the consensus sequence, up to 98% homology to the consensus sequence, and up to 99% homology to the consensus sequence. Similarly, nucleic acid sequences encoding immunogenic fragments and immunogenic fragments of proteins homologous to the proteins provided herein are also provided.

[0213] Some embodiments relate to nucleic acid molecules encoding immunogenic proteins that are 95% homologous to the nucleic acid coding sequences herein. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins that are 96% homologous to the nucleic acid coding sequences herein. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins that are 97% homologous to the nucleic acid coding sequences herein. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins that are 98% homologous to the nucleic acid coding sequences herein. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins that are 99% homologous to the nucleic acid coding sequences herein. In some embodiments, a nucleic acid molecule having a coding sequence disclosed herein that is homologous to the coding sequence of a consensus protein disclosed herein comprises a sequence encoding an IgE leader sequence linked to the 5' end of the coding sequence encoding the homologous protein sequence disclosed herein.

[0214] Some embodiments relate to proteins having a particular percent identity to the full-length NY-ESO-2 consensus protein, immunogenic fragments of the NY-ESO-2 consensus protein, and nucleic acid sequences encoding immunogenic fragments of proteins having identity to the NY-ESO-2 consensus protein, including up to 80% identity to the full-length NY-ESO-2 consensus sequence, up to 85% identity to the full-length NY-ESO-2 consensus sequence, up to 90% identity to the full-length NY-ESO-2 consensus sequence, up to 91% identity to the full-length NY-ESO-2 consensus sequence, up to 92% identity to the full-length NY-ESO-2 consensus sequence, up to 93% identity to the full-length NY-ESO-2 consensus sequence, up to 94% identity to the full-length NY-ESO-2 consensus sequence, up to 95% identity to the full-length NY-ESO-2 consensus sequence, up to 96% identity to the full-length NY-ESO-2 consensus sequence, up to 97% identity to the full-length NY-ESO-2 consensus sequence, up to 98% identity to the full-length NY-ESO-2 consensus sequence, up to 99% identity to the full-length NY-ESO-2 consensus sequence, up to 100% identity to the full-length NY-ESO-2 consensus sequence, up to 101% identity to the full-length NY-ESO-2 consensus sequence, up to 102% identity to the full-length NY-ESO-2 consensus sequence, up to 103% identity to the full-length NY-ESO-2 consensus sequence, up to 104% identity to the full-length NY-ESO-2 consensus sequence, up to 105% identity to the full-length NY-ESO-2 consensus sequence, up to 106% identity to the full-length NY-ESO-2 consensus sequence, up to 107% identity to the full-length NY-ESO- Such nucleic acid molecules can be provided that encode immunogenic proteins having up to 94% identity to the full-length NY-ESO-2 consensus sequence, up to 95% identity to the full-length NY-ESO-2 consensus sequence, up to 96% identity to the full-length NY-ESO-2 consensus sequence, up to 97% identity to the full-length NY-ESO-2 consensus sequence, up to 98% identity to the full-length NY-ESO-2 consensus sequence, and up to 99% identity to the full-length NY-ESO-2 consensus sequence. Similarly, nucleic acid sequences encoding immunogenic fragments of the immunogenic fragments set forth herein and proteins having similar percentage identities as set forth above to the NY-ESO-2 proteins set forth herein are also provided.

[0215] In some embodiments, the nucleic acid sequence does not have a coding sequence that encodes a leader sequence. In some embodiments, the nucleic acid sequence does not have a coding sequence that encodes an IgE leader.

[0216] Some embodiments relate to fragments of SEQ ID NO: 15. The fragment can be at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 15. The fragment can be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a fragment of SEQ ID NO: 15. In some embodiments, the fragment includes a sequence encoding a leader sequence, e.g., an immunoglobulin leader, such as an IgE leader. In some embodiments, the fragment does not have a coding sequence encoding a leader sequence. In some embodiments, the fragment does not have a coding sequence encoding a leader sequence, e.g., an IgE leader.

[0217] Furthermore, the amino acid sequence of the consensus NY-ESO-2 protein is SEQ ID NO: 16. The amino acid sequence of the consensus NY-ESO-2 protein linked to an IgE leader is SEQ ID NO: 16. The amino acid sequence of the consensus NY-ESO-2 protein linked to an IgE leader may be linked to an HA tag.

[0218] Some embodiments relate to proteins homologous to SEQ ID NO: 16. Some embodiments relate to immunogenic proteins having 95% homology to the consensus protein sequence set forth in SEQ ID NO: 16. Some embodiments relate to immunogenic proteins having 96% homology to the consensus protein sequence set forth in SEQ ID NO: 16. Some embodiments relate to immunogenic proteins having 97% homology to the consensus protein sequence set forth in SEQ ID NO: 16. Some embodiments relate to immunogenic proteins having 98% homology to the consensus protein sequence set forth in SEQ ID NO: 16. Some embodiments relate to immunogenic proteins having 99% homology to the consensus protein sequence set forth in SEQ ID NO: 16.

[0219] Some embodiments relate to a protein that is identical to SEQ ID NO: 16. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 80% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 16. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 85% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 16. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 90% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 16. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 91% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 16. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 92% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 16. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 93% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 16. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 94% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 16. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 95% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 16. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 96% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 16. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 97% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 16. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 98% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 16. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 99% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 16.

[0220] In some embodiments, the protein does not have a leader sequence. In some embodiments, the protein does not have an IgE leader. A fragment of the consensus protein can comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the consensus protein. An immunogenic fragment of SEQ ID NO: 16 can be provided. An immunogenic fragment can comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of SEQ ID NO: 16. In some embodiments, the fragment comprises a leader sequence, e.g., an immunoglobulin leader, such as an IgE leader. In some embodiments, the fragment lacks a leader sequence. In some embodiments, the fragment lacks a leader sequence, e.g., an IgE leader.

[0221] Immunogenic fragments of proteins having amino acid sequences homologous to immunogenic fragments of SEQ ID NO: 16 can be provided. Such immunogenic fragments can include at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of a protein that is 95% or more homologous to SEQ ID NO: 16. Some embodiments relate to immunogenic fragments that are 96% homologous to immunogenic fragments of the consensus protein sequences herein. Some embodiments relate to immunogenic fragments that are 97% homologous to immunogenic fragments of the consensus protein sequences herein. Some embodiments relate to immunogenic fragments having 98% homology to the immunogenic fragments of the consensus protein sequences herein. Some embodiments relate to immunogenic fragments having 99% homology to the immunogenic fragments of the consensus protein sequences herein. In some embodiments, the fragments include a leader sequence, e.g., an immunoglobulin leader, such as an IgE leader. In some embodiments, the fragments do not have a leader sequence. In some embodiments, the fragments do not have a leader sequence, e.g., an IgE leader.

[0222] Immunogenic fragments of proteins can be provided that have an amino acid sequence identical to an immunogenic fragment of SEQ ID NO: 16. Such immunogenic fragments can comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of a protein that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 16. In some embodiments, the fragment includes a leader sequence, e.g., an immunoglobulin leader, such as an IgE leader. In some embodiments, the fragment lacks a leader sequence. In some embodiments, the fragment does not have a leader sequence, for example an IgE leader.

[0223] As referred to herein with respect to the linkage of a signal peptide or leader sequence to the N-terminus of a protein, the signal peptide / leader sequence replaces the N-terminal methionine of the protein encoded by the start codon of a nucleic acid sequence encoding the protein without a signal peptide coding sequence.

[0224] (9) PRAME The vaccines of the present invention can contain the cancer antigen PRAME, its fragments, or variants. PRAME is a 509-amino acid protein encoded by the PRAME gene and expressed in the testis, placenta, endometrium, ovaries, and adrenal glands, as well as in tissues derived from melanoma, lung cancer, kidney cancer, and head and neck cancer. PRAME is also expressed in adult and pediatric acute leukemia and multiple myeloma. PRAME contains an immunogenic nanopeptide that can elicit a cytotoxic response when presented by HLA-A24. Studies have shown that overexpression of PRAME in cultured cells induces caspase-independent cell death, which is responsible for a slower proliferation rate. Other studies have demonstrated that overexpression of PRAME also benefits proliferation or survival by antagonizing retinoic acid receptor (RAR) signaling, contributing to the tumorigenesis process. Disruption of RAR signaling impairs the regulation of cell proliferation, development, and differentiation.

[0225] PRAME may have an expression pattern similar to that of the cancer-testis antigens MAGE, BAGE, and GAGE, i.e., expression in the testis. However, PRAME can be expressed in human melanoma and acute leukemia. PRAME can be recognized by cytolytic T lymphocytes. Therefore, PRAME may be an antigen associated with melanoma and leukemia.

[0226] A PRAME antigen can induce an antigen-specific T cell and / or high-titer antibody response, thereby inducing or eliciting an immune response directed against or reactive to a cancer or tumor expressing the antigen. In some embodiments, the induced or elicited immune response can be a cellular immune response, a humoral immune response, or both a cellular and humoral immune response. In some embodiments, the induced or elicited cellular immune response can include the induction or secretion of interferon-gamma (IFN-γ) and / or tumor necrosis factor alpha (TNF-α). In other embodiments, the induced or elicited immune response may reduce or inhibit one or more immunosuppressive factors that promote the growth of tumors or cancers that express the antigen, such as, but not limited to, factors that downregulate MHC presentation, antigen-specific regulatory T cells (Tregs), cytokines such as PD-L1, FasL, IL-10, and TFG-β, soluble factors produced by tumor-associated macrophages, tumor-associated fibroblasts, immunosuppressive cells, CTLA-4, PD-1, MDSC, MCP-1, and factors that upregulate immune checkpoint molecules, which are described in more detail below.

[0227] The PRAME antigen can increase the cellular immune response in a subject administered the PRAME antigen by about 50 to about 6,000 times, about 50 to about 5,500 times, about 50 to about 5,000 times, about 50 to about 4,500 times, about 100 to about 6,000 times, about 150 to about 6,000 times, about 200 to about 6,000 times, about 250 to about 6,000 times, or about 300 to about 6,000 times, compared to the cellular immune response in a subject not administered the PRAME antigen. In some embodiments, the PRAME antigen increases the cellular immune response in a subject administered the PRAME antigen by about 50 fold, 100 fold, 150 fold, 200 fold, 250 fold, 300 fold, 350 fold, 400 fold, 450 fold, 500 fold, 550 fold, 600 fold, 650 fold, 700 fold, 750 fold, 800 fold, 850 fold, 900 fold, 950 fold, 1000 fold, 1100 fold, 1200 fold, 1300 fold, 1400 fold, 1500 fold, 1600 fold, 1700 fold, 1800 fold, 1900 fold, 2000 fold, 2100 fold, or 2200 fold compared to the cellular immune response in a subject not administered the PRAME antigen. fold, 2200x, 2300x, 2400x, 2500x, 2600x, 2700x, 2800x, 2900x, 3000x, 3100x, 3200x, 3300x, 3400x, 3500x, 3600x, 3700x, 3800x, 3900x, 4000x, 4100x, 4200x, 4300x, 4400x, 4500x, 4600x, 4700x, 4800x, 4900x, 5000x, 5100x, 5200x, 5300x, 5400x, 5500x, 5600x, 5700x, 5800x, 5900x, or 6000x.

[0228] The PRAME antigen can increase the concentration of interferon-gamma (IFN-γ) in a subject administered the PRAME antigen by approximately 50 to approximately 6,000 times, approximately 50 to approximately 5,500 times, approximately 50 to approximately 5,000 times, approximately 50 to approximately 4,500 times, approximately 100 to approximately 6,000 times, approximately 150 to approximately 6,000 times, approximately 200 to approximately 6,000 times, approximately 250 to approximately 6,000 times, or approximately 300 to approximately 6,000 times compared to the IFN-γ concentration in a subject not administered the PRAME antigen. In some embodiments, the PRAME antigen increases the IFN-γ concentration in a subject administered the PRAME antigen by about 50-fold, 100-fold, 150-fold, 200-fold, 250-fold, 300-fold, 350-fold, 400-fold, 450-fold, 500-fold, 550-fold, 600-fold, 650-fold, 700-fold, 750-fold, 800-fold, 850-fold, 900-fold, 950-fold, 1000-fold, 1100-fold, 1200-fold, 1300-fold, 1400-fold, 1500-fold, 1600-fold, 1700-fold, 1800-fold, 1900-fold, 2000-fold, 2100-fold, or 2200-fold compared to the IFN-γ concentration in a subject not administered the PRAME antigen. fold, 2200x, 2300x, 2400x, 2500x, 2600x, 2700x, 2800x, 2900x, 3000x, 3100x, 3200x, 3300x, 3400x, 3500x, 3600x, 3700x, 3800x, 3900x, 4000x, 4100x, 4200x, 4300x, 4400x, 4500x, 4600x, 4700x, 4800x, 4900x, 5000x, 5100x, 5200x, 5300x, 5400x, 5500x, 5600x, 5700x, 5800x, 5900x, or 6000x.

[0229] The PRAME antigen may comprise a protein epitope that makes the antigen particularly effective as an immunogen capable of inducing a counteracting anti-PRAME immune response. The PRAME antigen may comprise a full-length translation product, a variant thereof, a fragment thereof, or a combination thereof. The PRAME antigen may comprise a consensus protein.

[0230] Nucleic acid sequences encoding consensus PRAME antigens can be optimized for codon usage and corresponding RNA transcripts. Nucleic acids encoding consensus PRAME antigens can be codon- and RNA-optimized for expression. In some embodiments, nucleic acid sequences encoding consensus PRAME antigens can include a Kozak sequence (e.g., GCC ACC) to improve translation efficiency. Nucleic acids encoding consensus PRAME antigens can include multiple stop codons (e.g., TGA TGA) to improve translation termination efficiency.

[0231] The nucleic acid encoding the consensus PRAME antigen can also encode an immunoglobulin E (IgE) leader sequence. The nucleic acid encoding the consensus PRAME antigen can further encode an IgE leader sequence such that the amino acid sequence of the IgE leader sequence is linked to the amino acid sequence of the consensus PRAME antigen by a peptide bond. The nucleic acid encoding the consensus PRAME antigen can also include a nucleotide sequence encoding the IgE leader sequence. In some embodiments, the nucleic acid encoding the consensus PRAME antigen does not have, i.e., does not include, a nucleotide sequence encoding an IgE leader sequence.

[0232] The consensus PRAME antigen can be the nucleic acid sequence SEQ ID NO: 17, which encodes the amino acid sequence SEQ ID NO: 18. SEQ ID NO: 17 encodes the consensus PRAME protein linked to an IgE leader sequence. The consensus PRAME protein can be linked to an IgE leader sequence and an HA tag. In other embodiments, the consensus PRAME protein may not have the IgE leader sequence and / or the HA tag, i.e., may not be linked to said sequence and / or tag.

[0233] In some embodiments, the consensus PRAME antigen can be a nucleic acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over the entire length of the nucleic acid sequence set forth in SEQ ID NO: 17. In other embodiments, the consensus PRAME antigen can be a nucleic acid sequence encoding an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over the entire length of the amino acid sequence set forth in SEQ ID NO: 18. The consensus PRAME antigen can be an amino acid sequence that has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over the entire length of the amino acid sequence set forth in SEQ ID NO: 18.

[0234] Some embodiments relate to nucleic acid sequences encoding proteins homologous to the PRAME consensus protein, immunogenic fragments of the PRAME consensus protein, and immunogenic fragments of homologous proteins. Such nucleic acid molecules can be provided that encode immunogenic proteins having up to 95% homology to the consensus sequence, up to 96% homology to the consensus sequence, up to 97% homology to the consensus sequence, up to 98% homology to the consensus sequence, and up to 99% homology to the consensus sequence. Similarly, nucleic acid sequences encoding immunogenic fragments set forth herein and immunogenic fragments of proteins homologous to the proteins set forth herein are also provided.

[0235] Some embodiments relate to nucleic acid molecules encoding immunogenic proteins that are 95% homologous to the nucleic acid coding sequences herein. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins that are 96% homologous to the nucleic acid coding sequences herein. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins that are 97% homologous to the nucleic acid coding sequences herein. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins that are 98% homologous to the nucleic acid coding sequences herein. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins that are 99% homologous to the nucleic acid coding sequences herein. In some embodiments, a nucleic acid molecule having a coding sequence disclosed herein that is homologous to the coding sequence of a consensus protein disclosed herein comprises a sequence encoding an IgE leader sequence linked to the 5' end of the coding sequence encoding the homologous protein sequence disclosed herein.

[0236] Some embodiments relate to proteins having a particular identity expressed as a percentage to the full-length PRAME consensus protein, immunogenic fragments of the PRAME consensus protein, and nucleic acid sequences encoding immunogenic fragments of proteins having identity to the PRAME consensus protein. Such nucleic acid molecules can be provided that encode immunogenic proteins having up to 80% identity to the full-length PRAME consensus sequence, up to 85% identity to the full-length PRAME consensus sequence, up to 90% identity to the full-length PRAME consensus sequence, up to 91% identity to the full-length PRAME consensus sequence, up to 92% identity to the full-length PRAME consensus sequence, up to 93% identity to the full-length PRAME consensus sequence, up to 94% identity to the full-length PRAME consensus sequence, up to 95% identity to the full-length PRAME consensus sequence, up to 96% identity to the full-length PRAME consensus sequence, up to 97% identity to the full-length PRAME consensus sequence, up to 98% identity to the full-length PRAME consensus sequence, and up to 99% identity to the full-length PRAME consensus sequence. Similarly, nucleic acid sequences encoding immunogenic fragments of the proteins shown herein and having similar percent identities as those shown above for the PRAME proteins shown herein are also provided.

[0237] In some embodiments, the nucleic acid sequence does not have a coding sequence that encodes a leader sequence. In some embodiments, the nucleic acid sequence does not have a coding sequence that encodes an IgE leader.

[0238] Some embodiments relate to fragments of SEQ ID NO: 17. The fragment can be at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 17. The fragment can be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a fragment of SEQ ID NO: 17. In some embodiments, the fragment includes a sequence encoding a leader sequence, e.g., an immunoglobulin leader, such as an IgE leader. In some embodiments, the fragment does not have a coding sequence encoding a leader sequence. In some embodiments, the fragment does not have a coding sequence encoding a leader sequence, e.g., an IgE leader.

[0239] Furthermore, the amino acid sequence of the consensus PRAME protein is SEQ ID NO: 18. The amino acid sequence of the consensus PRAME protein linked to an IgE leader is SEQ ID NO: 18. The amino acid sequence of the consensus PRAME protein linked to an IgE leader may be linked to an HA tag.

[0240] Some embodiments relate to proteins homologous to SEQ ID NO: 18. Some embodiments relate to immunogenic proteins having 95% homology to the consensus protein sequence set forth in SEQ ID NO: 18. Some embodiments relate to immunogenic proteins having 96% homology to the consensus protein sequence set forth in SEQ ID NO: 18. Some embodiments relate to immunogenic proteins having 97% homology to the consensus protein sequence set forth in SEQ ID NO: 18. Some embodiments relate to immunogenic proteins having 98% homology to the consensus protein sequence set forth in SEQ ID NO: 18. Some embodiments relate to immunogenic proteins having 99% homology to the consensus protein sequence set forth in SEQ ID NO: 18.

[0241] Some embodiments relate to a protein that is identical to SEQ ID NO: 18. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 80% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 18. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 85% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 18. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 90% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 18. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 91% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 18. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 92% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 18. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 93% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 18. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 94% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 18. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 95% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 18. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 96% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 18. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 97% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 18. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 98% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 18. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 99% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 18.

[0242] In some embodiments, the protein does not have a leader sequence. In some embodiments, the protein does not have an IgE leader. A fragment of the consensus protein can comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the consensus protein. An immunogenic fragment of SEQ ID NO: 18 can be provided. An immunogenic fragment can comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of SEQ ID NO: 18. In some embodiments, the fragment comprises a leader sequence, e.g., an immunoglobulin leader, such as an IgE leader. In some embodiments, the fragment lacks a leader sequence. In some embodiments, the fragment lacks a leader sequence, e.g., an IgE leader.

[0243] Immunogenic fragments of proteins having amino acid sequences homologous to immunogenic fragments of SEQ ID NO: 18 can be provided. Such immunogenic fragments can include at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of a protein that is 95% or more homologous to SEQ ID NO: 18. Some embodiments relate to immunogenic fragments having 96% homology to immunogenic fragments of the consensus protein sequences herein. Some embodiments relate to immunogenic fragments having 97% homology to immunogenic fragments of the consensus protein sequences herein. Some embodiments relate to immunogenic fragments having 98% homology to the immunogenic fragments of the consensus protein sequences herein. Some embodiments relate to immunogenic fragments having 99% homology to the immunogenic fragments of the consensus protein sequences herein. In some embodiments, the fragments include a leader sequence, e.g., an immunoglobulin leader, such as an IgE leader. In some embodiments, the fragments do not have a leader sequence. In some embodiments, the fragments do not have a leader sequence, e.g., an IgE leader.

[0244] Immunogenic fragments of proteins can be provided that have an amino acid sequence identical to an immunogenic fragment of SEQ ID NO: 18. Such immunogenic fragments can comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of a protein that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 18. In some embodiments, the fragment includes a leader sequence, e.g., an immunoglobulin leader, such as an IgE leader. In some embodiments, the fragment lacks a leader sequence. In some embodiments, the fragment does not have a leader sequence, for example an IgE leader.

[0245] As referred to herein with respect to the linkage of a signal peptide or leader sequence to the N-terminus of a protein, the signal peptide / leader sequence replaces the N-terminal methionine of the protein encoded by the start codon of a nucleic acid sequence encoding the protein without a signal peptide coding sequence.

[0246] (10) PSA The vaccine of the present invention may comprise the cancer antigen prostate-specific antigen (PSA; also known as gamma-seminoprotein or kallikrein-3 (KLK3)), a fragment thereof, or a variant thereof. PSA is an androgen-regulated serine protease produced by prostate epithelial cells and prostate cancer cells and is encoded by the KLK3 gene. PSA is often used as a serum marker for prostate cancer. PSA is a member of the tissue kallikrein family and, after cleavage of the proenzyme to release the active enzyme, cleaves semenogelin in semen clots, thereby lysing the semen and allowing sperm to swim freely. In addition, the enzymatic activity of PSA is regulated by zinc concentration; high zinc concentrations inhibit the enzymatic activity of PSA.

[0247] PSA antigens can induce antigen-specific T cell and / or high-titer antibody responses, thereby inducing or eliciting an immune response directed against or reactive to cancers or tumors expressing the antigen. In some embodiments, the induced or elicited immune response can be a cellular immune response, a humoral immune response, or both a cellular and humoral immune response. In some embodiments, the induced or elicited cellular immune response can include the induction or secretion of interferon-gamma (IFN-γ) and / or tumor necrosis factor alpha (TNF-α). In other embodiments, the induced or elicited immune response may reduce or inhibit one or more immunosuppressive factors that promote the growth of tumors or cancers that express the antigen, such as, but not limited to, factors that downregulate MHC presentation, antigen-specific regulatory T cells (Tregs), cytokines such as PD-L1, FasL, IL-10, and TFG-β, soluble factors produced by tumor-associated macrophages, tumor-associated fibroblasts, immunosuppressive cells, CTLA-4, PD-1, MDSC, MCP-1, and factors that upregulate immune checkpoint molecules, which are described in more detail below.

[0248] A PSA antigen can contain a protein epitope that makes the antigen particularly effective as an immunogen capable of inducing a counteracting anti-PSA immune response. A PSA antigen can include a full-length translation product, a variant thereof, a fragment thereof, or a combination thereof. A PSA antigen can include a consensus protein.

[0249] Nucleic acid sequences encoding consensus PSA antigens can be optimized for codon usage and corresponding RNA transcripts. Nucleic acids encoding consensus PSA antigens can be codon- and RNA-optimized for expression. In some embodiments, nucleic acid sequences encoding consensus PSA antigens can include a Kozak sequence (e.g., GCC ACC) to improve translation efficiency. Nucleic acids encoding consensus PSA antigens can include multiple stop codons (e.g., TGA TGA) to improve translation termination efficiency.

[0250] The nucleic acid encoding the consensus PSA antigen can also encode an immunoglobulin E (IgE) leader sequence. The nucleic acid encoding the consensus PSA antigen can further encode an IgE leader sequence such that the amino acid sequence of the IgE leader sequence is linked to the amino acid sequence of the consensus PSA antigen by a peptide bond. The nucleic acid encoding the consensus PSAP antigen can also comprise a nucleotide sequence encoding the IgE leader sequence. In some embodiments, the nucleic acid encoding the consensus PSA antigen does not have, i.e. does not comprise, a nucleotide sequence encoding an IgE leader sequence.

[0251] In some embodiments, the nucleic acid encoding the consensus PSA antigen can be a heterologous nucleic acid sequence and / or can include one or more heterologous nucleic acid sequences.

[0252] (11)PSMA The vaccines of the present invention may comprise the cancer antigen prostate-specific membrane antigen (PSMA; also known as glutamate carboxypeptidase II (GCPII), N-acetyl-L-aspartyl-L-glutamate peptidase I (NAALADase I), and NAAG peptidase), a fragment thereof, or a variant thereof. PSMA is encoded by the folate hydrolase 1 (FOLH1) gene. PSMA is a zinc metalloenzyme known to exist in membranes and the extracellular space. PSMA is highly expressed in the human prostate and is upregulated in prostate cancer. PSMA is also known to be overexpressed in other cancers, such as solid tumors of the kidney, breast, and colon.

[0253] The PSMA antigen can induce antigen-specific T cell and / or high-titer antibody responses, thereby inducing or eliciting an immune response directed against or reactive to cancers or tumors expressing the antigen. In some embodiments, the induced or elicited immune response can be a cellular immune response, a humoral immune response, or both a cellular and humoral immune response. In some embodiments, the induced or elicited cellular immune response can include the induction or secretion of interferon-gamma (IFN-γ) and / or tumor necrosis factor alpha (TNF-α). In other embodiments, the induced or elicited immune response may reduce or inhibit one or more immunosuppressive factors that promote the growth of tumors or cancers that express the antigen, such as, but not limited to, factors that downregulate MHC presentation, antigen-specific regulatory T cells (Tregs), cytokines such as PD-L1, FasL, IL-10, and TFG-β, soluble factors produced by tumor-associated macrophages, tumor-associated fibroblasts, immunosuppressive cells, CTLA-4, PD-1, MDSC, MCP-1, and factors that upregulate immune checkpoint molecules, which are described in more detail below.

[0254] The PSMA antigen can comprise a protein epitope that makes the antigen particularly effective as an immunogen capable of inducing a counteracting anti-PSMA immune response. The PSMA antigen can comprise a full-length translation product, a variant thereof, a fragment thereof, or a combination thereof. The PSMA antigen can comprise a consensus protein.

[0255] Nucleic acid sequences encoding consensus PSMA antigens can be optimized for codon usage and corresponding RNA transcripts. Nucleic acids encoding consensus PSMA antigens can be codon- and RNA-optimized for expression. In some embodiments, nucleic acid sequences encoding consensus PSMA antigens can include a Kozak sequence (e.g., GCC ACC) to improve translation efficiency. Nucleic acids encoding consensus PSMA antigens can include multiple stop codons (e.g., TGA TGA) to improve translation termination efficiency.

[0256] The nucleic acid encoding the consensus PSMA antigen can also encode an immunoglobulin E (IgE) leader sequence. The nucleic acid encoding the consensus PSMA antigen can further encode an IgE leader sequence such that the amino acid sequence of the IgE leader sequence is linked to the amino acid sequence of the consensus PSMA antigen by a peptide bond. The nucleic acid encoding the consensus PSMA antigen can also include a nucleotide sequence encoding the IgE leader sequence. In some embodiments, the nucleic acid encoding the consensus PSMA antigen does not have, i.e., does not include, a nucleotide sequence encoding an IgE leader sequence.

[0257] In some embodiments, the nucleic acid encoding the consensus PSMA antigen can be a heterologous nucleic acid sequence and / or can include one or more heterologous nucleic acid sequences.

[0258] (12) STEAP The vaccine of the present invention can contain the cancer antigen six-transmembrane epithelial antigen of the prostate (STEAP), its fragment, or its variant. STEAP is a metalloreductase encoded by the STEAP1 gene. STEAP is widely expressed in prostate tissue and is upregulated in cancer cells. STEAP is predicted to be a six-transmembrane protein and is a cell surface antigen present at cell-cell junctions.

[0259] STEAP antigens can induce antigen-specific T cell and / or high-titer antibody responses, thereby inducing or eliciting an immune response directed against or reactive to cancers or tumors expressing the antigen. In some embodiments, the induced or elicited immune response can be a cellular immune response, a humoral immune response, or both a cellular and humoral immune response. In some embodiments, the induced or elicited cellular immune response can include the induction or secretion of interferon-gamma (IFN-γ) and / or tumor necrosis factor alpha (TNF-α). In other embodiments, the induced or elicited immune response may reduce or inhibit one or more immunosuppressive factors that promote the growth of tumors or cancers that express the antigen, such as, but not limited to, factors that downregulate MHC presentation, antigen-specific regulatory T cells (Tregs), cytokines such as PD-L1, FasL, IL-10, and TFG-β, soluble factors produced by tumor-associated macrophages, tumor-associated fibroblasts, immunosuppressive cells, CTLA-4, PD-1, MDSC, MCP-1, and factors that upregulate immune checkpoint molecules, which are described in more detail below.

[0260] A STEAP antigen can comprise a protein epitope that makes the antigen particularly effective as an immunogen capable of inducing a counteracting anti-STEAP immune response. A STEAP antigen can comprise a full-length translation product, a variant thereof, a fragment thereof, or a combination thereof. A STEAP antigen can comprise a consensus protein.

[0261] Nucleic acid sequences encoding consensus STEAP antigens can be optimized for codon usage and corresponding RNA transcripts. Nucleic acids encoding consensus STEAP antigens can be codon- and RNA-optimized for expression. In some embodiments, nucleic acid sequences encoding consensus STEAP antigens can include a Kozak sequence (e.g., GCC ACC) to improve translation efficiency. Nucleic acids encoding consensus STEAP antigens can include multiple stop codons (e.g., TGA TGA) to improve translation termination efficiency.

[0262] The nucleic acid encoding the consensus STEAP antigen can also encode an immunoglobulin E (IgE) leader sequence. The nucleic acid encoding the consensus STEAP antigen can further encode an IgE leader sequence such that the amino acid sequence of the IgE leader sequence is linked to the amino acid sequence of the consensus STEAP antigen by a peptide bond. The nucleic acid encoding the consensus STEAP antigen can also include a nucleotide sequence encoding the IgE leader sequence. In some embodiments, the nucleic acid encoding the consensus STEAP antigen does not have, i.e., does not include, a nucleotide sequence encoding an IgE leader sequence.

[0263] In some embodiments, the nucleic acid encoding the consensus STEAP antigen can be a heterologous nucleic acid sequence and / or can include one or more heterologous nucleic acid sequences.

[0264] (13) PSCA The vaccines of the present invention can contain the cancer antigen prostate-specific stem cell antigen (PSCA), a fragment thereof, or a variant thereof. PSCA is a glycosylphosphatidylinositol (GPI)-anchored cell surface protein encoded by an androgen-responsive gene. PSCA is a member of the Thy-1 / Ly-6 family of GPI-anchored cell surface antigens. PSCA is upregulated in many cancers, including prostate, bladder, and pancreatic cancers.

[0265] A PSCA antigen can induce antigen-specific T cell and / or high-titer antibody responses, thereby inducing or eliciting an immune response directed against or reactive to cancers or tumors expressing the antigen. In some embodiments, the induced or elicited immune response can be a cellular immune response, a humoral immune response, or both a cellular and humoral immune response. In some embodiments, the induced or elicited cellular immune response can include the induction or secretion of interferon-gamma (IFN-γ) and / or tumor necrosis factor alpha (TNF-α). In other embodiments, the induced or elicited immune response may reduce or inhibit one or more immunosuppressive factors that promote the growth of tumors or cancers that express the antigen, such as, but not limited to, factors that downregulate MHC presentation, antigen-specific regulatory T cells (Tregs), cytokines such as PD-L1, FasL, IL-10, and TFG-β, soluble factors produced by tumor-associated macrophages, tumor-associated fibroblasts, immunosuppressive cells, CTLA-4, PD-1, MDSC, MCP-1, and factors that upregulate immune checkpoint molecules, which are described in more detail below.

[0266] A PSCA antigen can comprise a protein epitope that makes it particularly effective as an immunogen capable of inducing a counteracting anti-PSCA immune response. A PSCA antigen can comprise a full-length translation product, a variant thereof, a fragment thereof, or a combination thereof. A PSCA antigen can comprise a consensus protein.

[0267] Nucleic acid sequences encoding consensus PSCA antigens can be optimized for codon usage and corresponding RNA transcripts. Nucleic acids encoding consensus PSCA antigens can be codon- and RNA-optimized for expression. In some embodiments, nucleic acid sequences encoding consensus PSCA antigens can include a Kozak sequence (e.g., GCC ACC) to improve translation efficiency. Nucleic acids encoding consensus PSCA antigens can include multiple stop codons (e.g., TGA TGA) to improve translation termination efficiency.

[0268] The nucleic acid encoding the consensus PSCA antigen can also encode an immunoglobulin E (IgE) leader sequence. The nucleic acid encoding the consensus PSCA antigen can further encode an IgE leader sequence such that the amino acid sequence of the IgE leader sequence is linked to the amino acid sequence of the consensus PSCA antigen by a peptide bond. The nucleic acid encoding the consensus PSCA antigen can also comprise a nucleotide sequence encoding the IgE leader sequence. In some embodiments, the nucleic acid encoding the consensus PSCA antigen does not have, i.e., does not contain, a nucleotide sequence encoding an IgE leader sequence.

[0269] In some embodiments, the nucleic acid encoding the consensus PSCA antigen can be a heterologous nucleic acid sequence and / or can include one or more heterologous nucleic acid sequences.

[0270] (14)hTERT The vaccines of the present invention can include the cancer antigen hTERT, a fragment thereof, or a variant thereof. hTERT is a human telomerase reverse transcriptase that synthesizes TTAGGG tags at the ends of telomeres to prevent cell death due to chromosomal deletion. Hyperproliferative cells can have abnormally high expression of hTERT. Abnormally high expression of hTERT can also occur in hyperproliferative cells infected with HCV and HPV. Therefore, immunotherapy against both HCV and HPV can be enhanced by targeting cells expressing hTERT at abnormal levels. HCV and HPV antigens are discussed in more detail below. hTERT cancer antigens can be further defined in U.S. Patent Application No. 14 / 139,660, filed December 23, 2013, which is incorporated by reference in its entirety.

[0271] In addition, expression of hTERT in hTERT gene-transfected dendritic cells inhibits CD8 in an antigen-specific manner. + induces cytotoxic T cells and CD4 + Thus, the use of hTERT expression in ACPs to delay senescence and sustain the ability of antigen presenting cells (APCs) to present a selected antigen can be used in immunotherapeutic methods, such as those described herein.

[0272] The hTERT antigen may be associated with or expressed in any number of cancers, including, but not limited to, melanoma, prostate cancer, liver cancer, cervical cancer, recurrent respiratory papillomatosis (RRP), anal cancer, head and neck cancer, and hematological cancer. Thus, the vaccines, when comprising an hTERT antigen described herein, can be used to treat subjects with any number of cancers, including, but not limited to, melanoma, prostate cancer, liver cancer, cervical cancer, recurrent respiratory papillomatosis (RRP), anal cancer, head and neck cancer, and hematological cancer.

[0273] nTERT antigens can induce antigen-specific T cell and / or high-titer antibody responses, thereby inducing or eliciting an immune response directed against or reactive to cancers or tumors expressing the antigen. In some embodiments, the induced or elicited immune response can be a cellular immune response, a humoral immune response, or both a cellular and humoral immune response. In some embodiments, the induced or elicited cellular immune response can include the induction or secretion of interferon-gamma (IFN-γ) and / or tumor necrosis factor alpha (TNF-α). In other embodiments, the induced or elicited immune response may reduce or inhibit one or more immunosuppressive factors that promote the growth of tumors or cancers that express the antigen, such as, but not limited to, factors that downregulate MHC presentation, antigen-specific regulatory T cells (Tregs), cytokines such as PD-L1, FasL, IL-10, and TFG-β, soluble factors produced by tumor-associated macrophages, tumor-associated fibroblasts, immunosuppressive cells, CTLA-4, PD-1, MDSC, MCP-1, and factors that upregulate immune checkpoint molecules, which are described in more detail below.

[0274] The hTERT antigen can comprise a protein epitope that makes the antigen particularly effective as an immunogen capable of inducing a counter-hTERT immune response. The hTERT antigen can comprise a full-length translation product, a variant thereof, a fragment thereof, or a combination thereof. The hTERT antigen can comprise a consensus protein.

[0275] Nucleic acid sequences encoding hTERT antigens or consensus hTERT antigens can be optimized for codon usage and corresponding RNA transcripts. Nucleic acids encoding hTERT antigens or consensus hTERT antigens can be codon- and RNA-optimized for expression. In some embodiments, nucleic acid sequences encoding hTERT antigens or consensus hTERT antigens can include a Kozak sequence (e.g., GCC ACC) to improve translation efficiency. Nucleic acids encoding hTERT antigens or consensus hTERT antigens can include multiple stop codons (e.g., TGA TGA) to improve translation termination efficiency.

[0276] The nucleic acid encoding the hTERT antigen or consensus hTERT antigen can also encode an immunoglobulin E (IgE) leader sequence. The nucleic acid encoding the hTERT antigen or consensus hTERT antigen can further encode an IgE leader sequence such that the amino acid sequence of the IgE leader sequence is linked to the respective amino acid sequence of the hTERT antigen or consensus hTERT antigen by a peptide bond. The nucleic acid encoding the hTERT antigen or consensus hTERT antigen can also comprise a nucleotide sequence encoding the IgE leader sequence. In some embodiments, the nucleic acid encoding the hTERT antigen or consensus hTERT antigen does not have, i.e., does not comprise, a nucleotide sequence encoding an IgE leader sequence.

[0277] In some embodiments, the nucleic acid encoding the hTERT antigen or consensus hTERT antigen can be a heterologous nucleic acid sequence and / or can include one or more heterologous nucleic acid sequences. The nucleic acid encoding the hTERT antigen or consensus hTERT antigen can be mutated to replace, or substitute, one or more amino acids or residues in the amino acid sequence of the hTERT antigen or consensus hTERT antigen with a different amino acid or residue, respectively, compared to the wild-type hTERT antigen. The nucleic acid encoding the hTERT antigen or consensus hTERT antigen can be mutated to replace, or substitute, one or more residues in the amino acid sequence of the hTERT antigen or consensus hTERT antigen with a different residue, respectively, compared to the wild-type hTERT antigen, thereby causing the immune system of a mammal administered the nucleic acid encoding the hTERT antigen or consensus hTERT antigen, the hTERT antigen or consensus hTERT antigen, or a combination thereof, to no longer tolerate hTERT. The nucleic acid encoding the hTERT antigen or consensus hTERT antigen can be mutated to replace arginine 589, aspartic acid 1005, or both arginine 589 and aspartic acid 1005 in the amino acid sequence of the hTERT antigen or consensus hTERT antigen with a tyrosine residue, i.e., to substitute the tyrosine residue, relative to the wild-type hTERT antigen.

[0278] The hTERT antigen can be the nucleic acid sequence SEQ ID NO:23, which encodes the amino acid sequence SEQ ID NO:24. SEQ ID NO:23 encodes an hTERT protein linked to an IgE leader sequence. The hTERT protein can be linked to an IgE leader sequence and an HA tag. In other embodiments, the hTERT protein may not have the IgE leader sequence and / or the HA tag, i.e., may not be linked to said sequence and / or tag.

[0279] In some embodiments, the hTERT antigen can be a nucleic acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over the entire length of the nucleic acid sequence set forth in SEQ ID NO: 23. In other embodiments, the hTERT antigen can be a nucleic acid sequence encoding an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over the entire length of the amino acid sequence set forth in SEQ ID NO: 24. The hTERT antigen can be an amino acid sequence that has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over the entire length of the amino acid sequence set forth in SEQ ID NO:24.

[0280] Some embodiments relate to nucleic acid sequences encoding proteins homologous to the hTERT protein, immunogenic fragments of the hTERT protein, and immunogenic fragments of homologous proteins. Such nucleic acid molecules can be provided that encode immunogenic proteins having up to 95% homology to the sequence, up to 96% homology to the sequence, up to 97% homology to the sequence, up to 98% homology to the sequence, and up to 99% homology to the sequence. Similarly, nucleic acid sequences encoding immunogenic fragments and immunogenic fragments of proteins homologous to the proteins provided herein are also provided.

[0281] Some embodiments relate to nucleic acid molecules encoding immunogenic proteins that are 95% homologous to the nucleic acid coding sequences herein. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins that are 96% homologous to the nucleic acid coding sequences herein. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins that are 97% homologous to the nucleic acid coding sequences herein. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins that are 98% homologous to the nucleic acid coding sequences herein. Some embodiments relate to nucleic acid molecules encoding immunogenic proteins that are 99% homologous to the nucleic acid coding sequences herein. In some embodiments, a nucleic acid molecule having a coding sequence disclosed herein that is homologous to the coding sequence of a consensus protein disclosed herein comprises a sequence encoding an IgE leader sequence linked to the 5' end of the coding sequence encoding the homologous protein sequence disclosed herein.

[0282] Some embodiments relate to nucleic acid sequences encoding proteins having a particular percent identity to the full-length hTERT protein, immunogenic fragments of the hTERT protein, and immunogenic fragments of proteins having identity to the hTERT protein. Such nucleic acid molecules can be provided that encode immunogenic proteins having up to 80% identity to the full-length hTERT sequence, up to 85% identity to the full-length hTERT sequence, up to 90% identity to the full-length hTERT sequence, up to 91% identity to the full-length hTERT sequence, up to 92% identity to the full-length hTERT sequence, up to 93% identity to the full-length hTERT sequence, up to 94% identity to the full-length hTERT sequence, up to 95% identity to the full-length hTERT sequence, up to 96% identity to the full-length hTERT sequence, up to 97% identity to the full-length hTERT sequence, up to 98% identity to the full-length hTERT sequence, and up to 99% identity to the full-length hTERT sequence. Similarly, nucleic acid sequences encoding immunogenic fragments of the proteins shown herein and having similar percent identities as those shown above for the HTERT proteins shown herein are also provided.

[0283] In some embodiments, the nucleic acid sequence does not have a coding sequence that encodes a leader sequence. In some embodiments, the nucleic acid sequence does not have a coding sequence that encodes an IgE leader.

[0284] Some embodiments relate to fragments of SEQ ID NO: 23. The fragment can be at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 23. The fragment can be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a fragment of SEQ ID NO: 23. In some embodiments, the fragment includes a sequence encoding a leader sequence, e.g., an immunoglobulin leader, such as an IgE leader. In some embodiments, the fragment does not have a coding sequence encoding a leader sequence. In some embodiments, the fragment does not have a coding sequence encoding a leader sequence, e.g., an IgE leader.

[0285] Furthermore, the amino acid sequence of the hTERT protein is SEQ ID NO: 24. The amino acid sequence of the hTERT protein linked to an IgE leader is SEQ ID NO: 24. The amino acid sequence of the hTERT protein linked to an IgE leader may be linked to an HA tag.

[0286] Some embodiments relate to proteins homologous to SEQ ID NO: 24. Some embodiments relate to immunogenic proteins having 95% homology to the protein sequence set forth in SEQ ID NO: 24. Some embodiments relate to immunogenic proteins having 96% homology to the protein sequence set forth in SEQ ID NO: 24. Some embodiments relate to immunogenic proteins having 97% homology to the protein sequence set forth in SEQ ID NO: 24. Some embodiments relate to immunogenic proteins having 98% homology to the protein sequence set forth in SEQ ID NO: 24. Some embodiments relate to immunogenic proteins having 99% homology to the protein sequence set forth in SEQ ID NO: 24.

[0287] Some embodiments relate to a protein that is identical to SEQ ID NO:24. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 80% identical to the full-length amino acid sequence set forth in SEQ ID NO:24. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 85% identical to the full-length amino acid sequence set forth in SEQ ID NO:24. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 90% identical to the full-length amino acid sequence set forth in SEQ ID NO:24. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 91% identical to the full-length amino acid sequence set forth in SEQ ID NO:24. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 92% identical to the full-length amino acid sequence set forth in SEQ ID NO:24. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 93% identical to the full-length amino acid sequence set forth in SEQ ID NO:24. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 94% identical to the full-length amino acid sequence set forth in SEQ ID NO:24. Some embodiments relate to an immunogenic protein having an amino acid sequence that is 95% identical to the full-length amino acid sequence set forth in SEQ ID NO:24. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 96% identical to the full-length amino acid sequence set forth in SEQ ID NO: 24. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 97% identical to the full-length amino acid sequence set forth in SEQ ID NO: 24. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 98% identical to the full-length amino acid sequence set forth in SEQ ID NO: 24. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 99% identical to the full-length amino acid sequence set forth in SEQ ID NO: 24.

[0288] In some embodiments, the protein does not have a leader sequence. In some embodiments, the protein does not have an IgE leader. A fragment of a protein can comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the protein. An immunogenic fragment of SEQ ID NO: 24 can be provided. An immunogenic fragment can comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of SEQ ID NO: 24. In some embodiments, the fragment comprises a leader sequence, e.g., an immunoglobulin leader, such as an IgE leader. In some embodiments, the fragment lacks a leader sequence. In some embodiments, the fragment lacks a leader sequence, e.g., an IgE leader.

[0289] Immunogenic fragments of proteins having amino acid sequences homologous to immunogenic fragments of SEQ ID NO: 24 can be provided. Such immunogenic fragments can include at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of a protein that is 95% or more homologous to SEQ ID NO: 18. Some embodiments relate to immunogenic fragments having 96% homology to immunogenic fragments of the protein sequences herein. Some embodiments relate to immunogenic fragments having 97% homology to immunogenic fragments of the protein sequences herein. Some embodiments relate to immunogenic fragments having 98% homology to immunogenic fragments of the protein sequences herein. Some embodiments relate to immunogenic fragments having 99% homology to the immunogenic fragments of the protein sequences herein. In some embodiments, the fragments include a leader sequence, e.g., an immunoglobulin leader, such as an IgE leader. In some embodiments, the fragments do not have a leader sequence. In some embodiments, the fragments do not have a leader sequence, e.g., an IgE leader.

[0290] Immunogenic fragments of proteins can be provided that have an amino acid sequence identical to an immunogenic fragment of SEQ ID NO: 24. Such immunogenic fragments can comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of a protein that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 24. In some embodiments, the fragment comprises a leader sequence, e.g., an immunoglobulin leader, such as an IgE leader. In some embodiments, the fragment does not have a leader sequence. In some embodiments, the fragment does not have a leader sequence, such as an IgE leader.

[0291] As referred to herein with respect to the linkage of a signal peptide or leader sequence to the N-terminus of a protein, the signal peptide / leader sequence replaces the N-terminal methionine of the protein encoded by the start codon of a nucleic acid sequence encoding the protein without a signal peptide coding sequence.

[0292] A fragment of SEQ ID NO:23 can comprise 30 or more nucleotides, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:23 can comprise 45 or more nucleotides, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:23 can comprise 60 or more nucleotides, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:23 can comprise 75 or more nucleotides, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:23 can comprise 90 or more nucleotides, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:23 can comprise 120 or more nucleotides, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:23 can comprise 150 or more nucleotides, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:23 can comprise 180 or more nucleotides, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:23 can comprise 210 or more nucleotides, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:23 can comprise 240 or more nucleotides, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:23 can comprise 270 or more nucleotides, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:23 can comprise 300 or more nucleotides, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:23 can comprise 360 ​​or more nucleotides, preferably including a sequence encoding an immunodominant epitope.In some embodiments, a fragment of SEQ ID NO:23 can comprise 420 or more nucleotides, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:23 can comprise 480 or more nucleotides, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:23 can comprise 540 or more nucleotides, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:23 can comprise 600 or more nucleotides, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:23 can comprise 300 or more nucleotides, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:23 can comprise 660 or more nucleotides, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:23 can comprise 720 or more nucleotides, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:23 can comprise 780 or more nucleotides, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:23 can comprise 840 or more nucleotides, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:23 can comprise 900 or more nucleotides, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:23 can comprise 960 or more nucleotides, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:23 can comprise 1020 or more nucleotides, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:23 can comprise 1080 or more nucleotides, preferably including a sequence encoding an immunodominant epitope.In some embodiments, a fragment of SEQ ID NO:23 can comprise 1140 or more nucleotides, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:23 can comprise 1200 or more nucleotides, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:23 can comprise 1260 or more nucleotides, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:23 can comprise 1320 or more nucleotides, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:23 can comprise 1380 or more nucleotides, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:23 can comprise 1440 or more nucleotides, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:23 can comprise 1500 or more nucleotides, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:23 can comprise 1560 or more nucleotides, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:23 can comprise 1620 or more nucleotides, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:23 can comprise 1680 or more nucleotides, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:23 can comprise 1740 or more nucleotides, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:23 can comprise 1800 or more nucleotides, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:23 can comprise 1860 or more nucleotides, preferably including a sequence encoding an immunodominant epitope.In some embodiments, a fragment of SEQ ID NO:23 can comprise 1920 or more nucleotides, preferably comprising a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:23 can comprise 1980 or more nucleotides, preferably comprising a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:23 can comprise 2040 or more nucleotides, preferably comprising a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:23 can comprise 2100 or more nucleotides, preferably comprising a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:23 can comprise 2160 or more nucleotides, preferably comprising a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:23 can comprise 2220 or more nucleotides, preferably comprising a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:23 can comprise 2280 or more nucleotides, preferably comprising a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:23 can comprise 2340 or more nucleotides, preferably comprising a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:23 can comprise 2400 or more nucleotides, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:23 can comprise 2460 or more nucleotides, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:23 can comprise 2520 or more nucleotides, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:23 can comprise 2580 or more nucleotides, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:23 can comprise 2640 or more nucleotides, preferably including a sequence encoding an immunodominant epitope.In some embodiments, a fragment of SEQ ID NO:23 can comprise 2700 or more nucleotides, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:23 can comprise 2760 or more nucleotides, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:23 can comprise 2820 or more nucleotides, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:23 can comprise 2880 or more nucleotides, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:23 can comprise 2940 or more nucleotides, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:23 can comprise 3000 or more nucleotides, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:23 can comprise 3060 or more nucleotides, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:23 can comprise 3120 or more nucleotides, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:23 can comprise 3180 or more nucleotides, preferably including sequences encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:23 can comprise 3240 or more nucleotides, preferably including sequences encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:23 can comprise 3300 or more nucleotides, preferably including sequences encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:23 can comprise 3360 or more nucleotides, preferably including sequences encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:23 can comprise 3420 or more nucleotides, preferably including sequences encoding an immunodominant epitope.In some embodiments, a fragment of SEQ ID NO:23 can comprise 3480 or more nucleotides, preferably including sequences encoding immunodominant epitopes. In some embodiments, a fragment of SEQ ID NO:23 can comprise coding sequences for an IgE leader sequence. In some embodiments, a fragment of SEQ ID NO:23 does not comprise coding sequences for an IgE leader sequence.

[0293] Fragments may be less than 60 nucleotides, in some embodiments less than 75 nucleotides, in some embodiments less than 90 nucleotides, in some embodiments less than 120 nucleotides, in some embodiments less than 150 nucleotides, in some embodiments less than 180 nucleotides, in some embodiments less than 210 nucleotides, in some embodiments less than 240 nucleotides, in some embodiments less than 270 nucleotides, in some embodiments less than 300 nucleotides, in some embodiments less than 360 nucleotides, in some embodiments less than 420 nucleotides, in some embodiments less than 480 nucleotides, in some embodiments less than 540 nucleotides, in some embodiments less than 600 nucleotides, in some embodiments less than 660 nucleotides, in some embodiments less than 720 nucleotides, in some embodiments less than 780 nucleotides, in some embodiments less than 840 nucleotides, in some embodiments less than 900 nucleotides, In some embodiments, the amino acid sequence is less than 1000 nucleotides, in some embodiments less than 960 nucleotides, in some embodiments less than 1020 nucleotides, in some embodiments less than 1080 nucleotides, in some embodiments less than 1140 nucleotides, in some embodiments less than 1200 nucleotides, in some embodiments less than 1260 nucleotides, in some embodiments less than 1320 nucleotides, in some embodiments less than 1380 nucleotides, in some embodiments less than 1440 nucleotides, in some embodiments less than 1500 nucleotides, in some embodiments less than 1560 nucleotides, in some embodiments less than 1620 nucleotides, in some embodiments less than 1680 nucleotides, in some embodiments less than 1740 nucleotides, in some embodiments less than 1800 nucleotides, in some embodiments less than 1860 nucleotides, in some embodiments less than 1920 nucleotides, in some embodiments less than 1980 nucleotides.In some embodiments, it may comprise fewer than 2040 nucleotides, in some embodiments fewer than 2100 nucleotides, in some embodiments fewer than 2160 nucleotides, in some embodiments fewer than 2220 nucleotides, in some embodiments fewer than 2280 nucleotides, in some embodiments fewer than 2340 nucleotides, in some embodiments fewer than 2400 nucleotides, in some embodiments fewer than 2460 nucleotides, in some embodiments fewer than 2520 nucleotides, in some embodiments fewer than 2580 nucleotides, in some embodiments fewer than 2640 nucleotides, in some embodiments fewer than 2700 nucleotides, in some embodiments fewer than 2760 nucleotides, in some embodiments fewer than 2820 nucleotides, in some embodiments fewer than 2860 nucleotides, in some embodiments fewer than 2940 nucleotides, in some embodiments fewer than 3000 nucleotides, in some embodiments fewer than 3060 nucleotides, in some embodiments fewer than 3120 nucleotides, in some embodiments fewer than 3180 nucleotides, in some embodiments fewer than 3240 nucleotides, in some embodiments fewer than 3300 nucleotides, in some embodiments fewer than 3360 nucleotides, in some embodiments fewer than 3420 nucleotides, in some embodiments fewer than 3480 nucleotides, and in some embodiments fewer than 3510 nucleotides.

[0294] A fragment of SEQ ID NO:24 can comprise 15 or more amino acids, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:24 can comprise 18 or more amino acids, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:24 can comprise 21 or more amino acids, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:24 can comprise 24 or more amino acids, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:24 can comprise 30 or more amino acids, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:24 can comprise 36 or more amino acids, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:24 can comprise 42 or more amino acids, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:24 can comprise 48 or more amino acids, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:24 can comprise 54 or more amino acids, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:24 can comprise 60 or more amino acids, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:24 can comprise 66 or more amino acids, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:24 can comprise 72 or more amino acids, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:24 can comprise 90 or more amino acids, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:24 can comprise 120 or more amino acids, preferably including a sequence encoding an immunodominant epitope.In some embodiments, a fragment of SEQ ID NO:24 can comprise 150 or more amino acids, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:24 can comprise 180 or more amino acids, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:24 can comprise 210 or more amino acids, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:24 can comprise 240 or more amino acids, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:24 can comprise 270 or more amino acids, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:24 can comprise 300 or more amino acids, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:24 can comprise 330 or more amino acids, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:24 can comprise 360 ​​or more amino acids, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:24 can comprise 390 or more amino acids, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:24 can comprise 420 or more amino acids, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:24 can comprise 450 or more amino acids, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:24 can comprise 480 or more amino acids, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:24 can comprise 510 or more amino acids, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:24 can comprise 540 or more amino acids, preferably including a sequence encoding an immunodominant epitope.In some embodiments, a fragment of SEQ ID NO:24 can comprise 570 or more amino acids, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:24 can comprise 600 or more amino acids, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:24 can comprise 630 or more amino acids, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:24 can comprise 660 or more amino acids, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:24 can comprise 690 or more amino acids, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:24 can comprise 720 or more amino acids, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:24 can comprise 750 or more amino acids, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:24 can comprise 780 or more amino acids, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:24 can comprise 810 or more amino acids, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:24 can comprise 840 or more amino acids, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:24 can comprise 870 or more amino acids, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:24 can comprise 900 or more amino acids, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:24 can comprise 930 or more amino acids, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:24 can comprise 960 or more amino acids, preferably including a sequence encoding an immunodominant epitope.In some embodiments, a fragment of SEQ ID NO:24 can comprise 990 or more amino acids, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:24 can comprise 1020 or more amino acids, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:24 can comprise 1050 or more amino acids, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:24 can comprise 1080 or more amino acids, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:24 can comprise 1110 or more amino acids, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:24 can comprise 1140 or more amino acids, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:24 can comprise 1170 or more amino acids, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:24 can comprise 1200 or more amino acids, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:24 can comprise 1230 or more amino acids, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:24 can comprise 1260 or more amino acids, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:24 can comprise 1290 or more amino acids, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:24 can comprise 1320 or more amino acids, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:24 can comprise 1350 or more amino acids, preferably including a sequence encoding an immunodominant epitope.In some embodiments, a fragment of SEQ ID NO:24 can comprise 1380 or more amino acids, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:24 can comprise 1410 or more amino acids, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:24 can comprise 1440 or more amino acids, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:24 can comprise 1470 or more amino acids, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:24 can comprise 1500 or more amino acids, preferably including a sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO:24 can comprise a coding sequence for an IgE leader sequence. In some embodiments, a fragment of SEQ ID NO:24 does not comprise a coding sequence for an IgE leader sequence.

[0295] Fragments may be fewer than 24 amino acids, in some embodiments fewer than 30 amino acids, in some embodiments fewer than 36 amino acids, in some embodiments fewer than 42 amino acids, in some embodiments fewer than 48 amino acids, in some embodiments fewer than 54 amino acids, in some embodiments fewer than 60 amino acids, in some embodiments fewer than 72 amino acids, in some embodiments fewer than 90 amino acids, in some embodiments fewer than 120 amino acids, in some embodiments fewer than 150 amino acids, in some embodiments fewer than 180 amino acids, in some embodiments fewer than 210 amino acids, in some embodiments fewer than 240 amino acids, in some embodiments fewer than 260 amino acids, in some embodiments fewer than 290 amino acids, in some embodiments fewer than 320 amino acids, in some embodiments fewer than 350 amino acids, in some embodiments fewer than 380 amino acids, in some embodiments fewer than 410 amino acids, in some embodiments fewer than 440 amino acids, In some embodiments, the amino acid sequence is less than 470 amino acids, in some embodiments less than 500 amino acids, in some embodiments less than 530 amino acids, in some embodiments less than 560 amino acids, in some embodiments less than 590 amino acids, in some embodiments less than 620 amino acids, in some embodiments less than 650 amino acids, in some embodiments less than 680 amino acids, in some embodiments less than 710 amino acids, in some embodiments less than 740 amino acids, in some embodiments less than 770 amino acids, in some embodiments less than 800 amino acids, in some embodiments less than 830 amino acids, in some embodiments less than 860 amino acids, in some embodiments less than 890 amino acids, in some embodiments less than 920 amino acids, in some embodiments less than 950 amino acids, in some embodiments less than 980 amino acids, in some embodiments less than 1010 amino acids, in some embodiments less than 1040 amino acids, in some embodiments less than 1070 amino acids.In some embodiments, it may comprise fewer than 1200 amino acids, in some embodiments, fewer than 1230 amino acids, in some embodiments, fewer than 1260 amino acids, in some embodiments, fewer than 1290 amino acids, in some embodiments, fewer than 1320 amino acids, in some embodiments, fewer than 1350 amino acids, in some embodiments, fewer than 1380 amino acids, in some embodiments, fewer than 1410 amino acids, in some embodiments, fewer than 1440 amino acids, in some embodiments, fewer than 1470 amino acids, and in some embodiments, fewer than 1500 amino acids.

[0296] (15) MAGE A1 The vaccine of the present invention can contain the cancer antigen melanoma-associated antigen 1 (MAGE A1), its fragment, or a variant thereof. MAGE A1 is a 280-amino acid protein encoded by the MAGE A1 gene and has been found to be expressed only by tumor cells and germ cells. The suppression of MAGE A1 in normal tissues is based on DNA methylation. These genes become active in many types of tumors during a genome-wide demethylation process, which often accompanies tumor development. Specifically, during malignant transformation, these genes become activated and expressed, potentially serving as antigenic targets recognized and attacked by the immune system. Thus, MAGE genes participate in immune processes by targeting and destroying some early tumor cells. MAGE A1 can be expressed in many cancers, including, but not limited to, melanoma, lung cancer, and esophageal squamous cell carcinoma.

[0297] The MAGE Al antigen can induce antigen-specific T cell and / or high titer antibody responses, thereby inducing or eliciting an immune response directed against or reactive to cancers or tumors expressing the antigen. In some embodiments, the induced or elicited immune response can be a cellular immune response, a humoral immune response, or both a cellular and humoral immune response. In some embodiments, the induced or elicited cellular immune response can include the induction or secretion of interferon-gamma (IFN-γ) and / or tumor necrosis factor alpha (TNF-α). In other embodiments, the induced or elicited immune response may reduce or inhibit one or more immunosuppressive factors that promote the growth of tumors or cancers that express the antigen, such as, but not limited to, factors that downregulate MHC presentation, antigen-specific regulatory T cells (Tregs), cytokines such as PD-L1, FasL, IL-10, and TFG-β, soluble factors produced by tumor-associated macrophages, tumor-associated fibroblasts, immunosuppressive cells, CTLA-4, PD-1, MDSC, MCP-1, and factors that upregulate immune checkpoint molecules, which are described in more detail below.

[0298] (16)WT1 The vaccine of the present invention can contain the cancer antigen Wilms' tumor 1 (WT1), its fragments, or its variants. WT1 is a transcription factor that contains a proline / glutamine-rich DNA-binding domain at its N-terminus and four zinc finger motifs at its C-terminus. WT1 is involved in the normal development of the urogenital system and interacts with many factors, including the tumor suppressor p53 and the serine protease HtrA2, which cleaves WT1 at multiple sites after treatment with cytotoxic drugs.

[0299] Mutations in WT1 can lead to tumorigenesis or cancer formation, such as Wilms' tumor or WT1-expressing tumors. Wilms' tumor often forms in one or both kidneys before metastasizing to other tissues, including, but not limited to, liver tissue, urinary tract tissue, lymphatic tissue, and lung tissue. Therefore, Wilms' tumor can be considered a metastatic tumor. Wilms' tumor usually occurs in young children (e.g., under 5 years of age) in both sporadic and hereditary forms. The WT1 cancer antigen is further defined in PCT / US13 / 75141, filed December 23, 2013, which is hereby incorporated by reference in its entirety.

[0300] The WT-1 antigen can induce antigen-specific T cell and / or high-titer antibody responses, thereby inducing or eliciting an immune response directed against or reactive to cancers or tumors expressing the antigen. In some embodiments, the induced or elicited immune response can be a cellular immune response, a humoral immune response, or both a cellular and humoral immune response. In some embodiments, the induced or elicited cellular immune response can include the induction or secretion of interferon-gamma (IFN-γ) and / or tumor necrosis factor alpha (TNF-α). In other embodiments, the induced or elicited immune response may reduce or inhibit one or more immunosuppressive factors that promote the growth of tumors or cancers that express the antigen, such as, but not limited to, factors that downregulate MHC presentation, antigen-specific regulatory T cells (Tregs), cytokines such as PD-L1, FasL, IL-10, and TFG-β, soluble factors produced by tumor-associated macrophages, tumor-associated fibroblasts, immunosuppressive cells, CTLA-4, PD-1, MDSC, MCP-1, and factors that upregulate immune checkpoint molecules, which are described in more detail below.

[0301] Thus, the vaccine can be used to treat subjects suffering from Wilms' tumor. The vaccine can be used to treat subjects suffering from any number of cancers, including, but not limited to, melanoma, prostate cancer, liver cancer, cervical cancer, recurrent respiratory papillomatosis (RRP), anal cancer, head and neck cancer, and hematological cancer. The vaccine can also be used to treat subjects with WT1-expressing cancers or tumors, and to prevent the development of such tumors in the subject. The WT1 antigen can differ from the native "normal" WT1 gene, thereby providing treatment or prevention for WT1 antigen-expressing tumors. Thus, provided herein are WT1 antigen sequences (i.e., mutant WT1 genes or sequences) that differ from the native WT1 gene.

[0302] The transcript of the native WT1 gene is processed into various mRNAs, and the resulting proteins do not all have the same value in inducing immune responses. The mutant WT1 genes described herein avoid selective processing, produce a single full-length transcript, and induce stronger effector T and B cell responses. The first mutant WT1 sequence is designated CON WT1 with modified zinc fingers, i.e., ConWT1-L. SEQ ID NO: 19 is the nucleic acid sequence encoding the WT1 antigen CON WT1 with modified zinc fingers. SEQ ID NO: 20 is the amino acid sequence of the WT1 antigen CON WT1 with modified zinc fingers. The second mutant WT1 sequence is designated CON WT1 without zinc fingers, i.e., ConWT1-S. SEQ ID NO: 21 is the nucleic acid sequence encoding the WT1 antigen CON WT1 without zinc fingers. SEQ ID NO: 22 is the amino acid sequence of the WT1 antigen CON WT1 without modified zinc fingers.

[0303] The WT1 antigen may be a consensus antigen (or immunogen) sequence derived from two or more species. The WT1 antigen may include a consensus sequence and / or modifications for improved expression. Modifications may include codon optimization, RNA optimization, addition of a Kozak sequence (e.g., GCC ACC) to enhance translation initiation, and / or addition of an immunoglobulin leader sequence to enhance the immunogenicity of the WT1 antigen. The WT1 antigen may include a signal peptide, such as, but not limited to, an immunoglobulin signal peptide, for example, an immunoglobulin E (IgE) signal peptide or an immunoglobulin G (IgG) signal peptide. In some embodiments, the WT1 consensus antigen may include a hemagglutinin (HA) tag. The WT1 consensus antigen may be designed to elicit stronger and broader cellular and / or humoral immune responses than the corresponding codon-optimized WT1 antigen.

[0304] The WT1 consensus antigen contains one or more mutations in one or more zinc fingers, which can induce stronger and broader cellular and / or humoral immune responses than the corresponding codon-optimized WT1 antigen. The one or more mutations can be substitutions of one or more amino acids that coordinate the zinc ion in one or more zinc fingers. The one or more amino acids that coordinate the zinc ion can be a CCHH motif. Thus, in some embodiments, the one or more mutations can be substitutions of one, two, three, or all four amino acids in the CCHH motif.

[0305] In other embodiments, the one or more mutations are such that residues 312, 317, 342, and 347 of SEQ ID NO: 20 are any residue other than cysteine ​​(C), and residues 330, 334, 360, and 364 of SEQ ID NO: 20 are any residue other than histidine (H). In particular, the one or more mutations are such that residues 312, 317, 330, 334, 342, 347, 360, and 364 of SEQ ID NO: 20 are glycine (G).

[0306] In other embodiments, one or more zinc fingers can be removed from the WT1 consensus antigen. One, two, three, or all four zinc fingers can be removed from the WT1 consensus antigen.

[0307] The WT1 consensus antigen can be the nucleic acid sequence SEQ ID NO: 19 that encodes SEQ ID NO: 20. In some embodiments, the WT1 consensus antigen can be a nucleic acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity over the entire length of the nucleic acid sequence set forth in SEQ ID NO: 19. In other embodiments, the WT1 consensus antigen can be a nucleic acid sequence encoding an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity over the entire length of the nucleic acid sequence set forth in SEQ ID NO: 20.

[0308] In still another embodiment, the WT1 consensus antigen may be a nucleic acid sequence encoding an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity over the entire length of the nucleic acid sequence set forth in SEQ ID NO: 20, with the proviso that residues 312, 317, 342, and 347 of SEQ ID NO: 20 are any residues other than cysteine ​​(C), and residues 330, 334, 360, and 364 of SEQ ID NO: 20 are any residues other than histidine (H). In another embodiment, the WT1 consensus antigen may be a nucleic acid sequence encoding an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity over the entire length of the nucleic acid sequence set forth in SEQ ID NO: 20, with the proviso that residues 312, 317, 330, 334, 342, 347, 360, and 364 of SEQ ID NO: 20 are glycine (G).

[0309] The WT1 consensus antigen can be the amino acid sequence of SEQ ID NO: 20. In some embodiments, the WT1 consensus antigen can be an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity over the entire length of the nucleic acid sequence set forth in SEQ ID NO: 20. The WT1 consensus antigen may be an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity over the entire length of the nucleic acid sequence set forth in SEQ ID NO: 20, with the proviso that residues 312, 317, 342, and 347 of SEQ ID NO: 20 are any residues other than cysteine ​​(C), and residues 330, 334, 360, and 364 of SEQ ID NO: 20 are any residues other than histidine (H). In some embodiments, the WT1 consensus antigen may be an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity over the entire length of the nucleic acid sequence set forth in SEQ ID NO: 20, with the proviso that residues 312, 317, 330, 334, 342, 347, 360, and 364 of SEQ ID NO: 20 are glycine (G).

[0310] The WT1 consensus antigen can be the nucleic acid sequence SEQ ID NO: 21 that encodes SEQ ID NO: 22. In some embodiments, the WT1 consensus antigen can be a nucleic acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity over the entire length of the nucleic acid sequence set forth in SEQ ID NO: 21. In other embodiments, the WT1 consensus antigen can be a nucleic acid sequence encoding an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity over the entire length of the nucleic acid sequence set forth in SEQ ID NO: 22.

[0311] The WT1 consensus antigen can be the amino acid sequence of SEQ ID NO: 22. In some embodiments, the WT1 consensus antigen can be an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity over the entire length of the nucleic acid sequence set forth in SEQ ID NO: 22.

[0312] Immunogenic fragments of SEQ ID NO: 20 and SEQ ID NO: 22 are obtained. Immunogenic fragments may comprise at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of SEQ ID NO: 20 and / or SEQ ID NO: 22. In some embodiments, immunogenic fragments may comprise at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of SEQ ID NO: 20. with the proviso that, when residues 312, 317, 342, and 347 of SEQ ID NO:20 are present in the immunogenic fragment, they are any residue other than cysteine ​​(C), and when residues 330, 334, 360, and 364 of SEQ ID NO:20 are present in the immunogenic fragment, they are any residue other than histidine (H). In other embodiments, the immunogenic fragment may comprise at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of SEQ ID NO:20, with the proviso that, when residues 312, 317, 330, 334, 342, 347, 360, and 364 of SEQ ID NO:20 are present in the immunogenic fragment, they are glycine (G).

[0313] In some embodiments, an immunogenic fragment comprises a leader sequence, e.g., an immunoglobulin leader sequence, such as an immunoglobulin E (IgE) leader sequence. In some embodiments, an immunogenic fragment does not comprise a leader sequence.

[0314] Immunogenic fragments of proteins having amino acid sequences identical to immunogenic fragments of SEQ ID NOs: 20 and 22 are obtained. Such fragments may comprise at least 60%, at least 65%, at least 70%, at least 75%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of a protein having 95% or greater identity to SEQ ID NO: 20 and / or SEQ ID NO: 22. Some embodiments relate to immunogenic fragments having 96% or greater identity to immunogenic fragments of WT1 protein sequences described herein. Some embodiments relate to immunogenic fragments having 97% or greater identity to immunogenic fragments of WT1 protein sequences described herein. Some embodiments relate to immunogenic fragments having 98% or greater identity to immunogenic fragments of WT1 protein sequences described herein. Some embodiments relate to immunogenic fragments having 99% or greater identity to immunogenic fragments of WT1 protein sequences described herein. In some embodiments, the immunogenic fragment comprises a leader sequence, e.g., an immunoglobulin leader sequence, such as an IgE leader sequence. In some embodiments, the immunogenic fragment does not comprise a leader sequence.

[0315] Some embodiments relate to immunogenic fragments of SEQ ID NO: 19 and SEQ ID NO: 21. Immunogenic fragments can comprise at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of SEQ ID NO: 19 and / or SEQ ID NO: 21. In some embodiments, immunogenic fragments comprise sequences encoding a leader sequence, e.g., an immunoglobulin leader sequence, such as an IgE leader sequence. In some embodiments, immunogenic fragments do not comprise coding sequences encoding a leader sequence.

[0316] Immunogenic fragments of nucleic acids having nucleotide sequences identical to immunogenic fragments of SEQ ID NO: 19 and SEQ ID NO: 21 are obtained. Such fragments may comprise at least 60%, at least 65%, at least 70%, at least 75%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of a nucleic acid having 95% or greater identity to SEQ ID NO: 19 and / or SEQ ID NO: 21. Some embodiments relate to immunogenic fragments having 96% or greater identity to immunogenic fragments of WT1 nucleic acid sequences described herein. Some embodiments relate to immunogenic fragments having 97% or greater identity to immunogenic fragments of WT1 nucleic acid sequences described herein. Some embodiments relate to immunogenic fragments having 98% or greater identity to immunogenic fragments of WT1 nucleic acid sequences described herein. Some embodiments relate to immunogenic fragments having 99% or greater identity to immunogenic fragments of WT1 nucleic acid sequences described herein. In some embodiments, an immunogenic fragment includes a sequence encoding a leader sequence, e.g., an immunoglobulin leader sequence, such as an IgE leader sequence. In some embodiments, an immunogenic fragment does not have a coding sequence encoding a leader sequence.

[0317] (17) gp100 The vaccines of the present invention can include the cancer antigen glycoprotein 100 (gp100; also known as Trp2 and premelanosome protein (PMEL)), a fragment thereof, or a variant thereof. gp100 is encoded by the PMEL gene. gp100 is a 70-kDa type 1 transmembrane glycoprotein composed of 661 amino acids that plays a central role in melanosome biogenesis by participating in melanosome maturation from stage I to stage II. gp100 mediates the formation of striae within multivesicular bodies and is directly involved in premelanosome biogenesis. gp100 is enriched in premelanosomes relative to mature melanosomes, but is overexpressed by proliferating neoplastic melanocytes and during tumor growth. The gp100 protein contains various immunogenic epitopes recognized by cytotoxic T lymphocytes derived from peripheral blood and tumor-infiltrating lymphocytes of melanoma patients.

[0318] The gp100 antigen can induce antigen-specific T cell and / or high-titer antibody responses, thereby inducing or eliciting an immune response directed against or reactive to cancers or tumors expressing the antigen. In some embodiments, the induced or elicited immune response can be a cellular immune response, a humoral immune response, or both a cellular and humoral immune response. In some embodiments, the induced or elicited cellular immune response can include the induction or secretion of interferon-gamma (IFN-γ) and / or tumor necrosis factor alpha (TNF-α). In other embodiments, the induced or elicited immune response may reduce or inhibit one or more immunosuppressive factors that promote the growth of tumors or cancers that express the antigen, such as, but not limited to, factors that downregulate MHC presentation, antigen-specific regulatory T cells (Tregs), cytokines such as PD-L1, FasL, IL-10, and TFG-β, soluble factors produced by tumor-associated macrophages, tumor-associated fibroblasts, immunosuppressive cells, CTLA-4, PD-1, MDSC, MCP-1, and factors that upregulate immune checkpoint molecules, which are described in more detail below.

[0319] (18) Viral antigens The cancer antigen may be a viral antigen, a fragment thereof, or a variant thereof. The viral antigen may be an antigen derived from hepatitis B virus, hepatitis C virus, or human papillomavirus (HPV). The HPV may be HPV6, HPV11, HPV16, or HPV18, as described below.

[0320] The viral antigens can induce antigen-specific T cell and / or high-titer antibody responses, thereby inducing or eliciting an immune response directed against or reactive to cancers or tumors expressing the antigen. In some embodiments, the induced or elicited immune response can be a cellular immune response, a humoral immune response, or both a cellular and humoral immune response. In some embodiments, the induced or elicited cellular immune response can include the induction or secretion of interferon-gamma (IFN-γ) and / or tumor necrosis factor alpha (TNF-α). In other embodiments, the induced or elicited immune response may reduce or inhibit one or more immunosuppressive factors that promote the growth of tumors or cancers that express the antigen, such as, but not limited to, factors that downregulate MHC presentation, antigen-specific regulatory T cells (Tregs), cytokines such as PD-L1, FasL, IL-10, and TFG-β, soluble factors produced by tumor-associated macrophages, tumor-associated fibroblasts, immunosuppressive cells, CTLA-4, PD-1, MDSC, MCP-1, and factors that upregulate immune checkpoint molecules, which are described in more detail below.

[0321] (a) Hepatitis B virus antigen The viral antigen can be an antigen derived from Hepatitis B virus (HBV), a fragment thereof, or a variant thereof. HBV antigens can be associated with or cause liver cancer. In some embodiments, the HBV antigen can be a heterologous nucleic acid molecule(s), such as a plasmid(s), encoding one or more antigens from HBV. The HBV antigen can be full-length or an immunogenic fragment of the full-length protein.

[0322] The HBV antigen can include one or more modifications to improve consensus sequence and / or expression. The modified consensus sequence can include genetic modifications, including codon optimization, RNA optimization, and the addition of a highly efficient immunoglobulin leader sequence, to increase the immunogenicity of the construct. The consensus HBV antigen can also include a signal peptide, such as an immunoglobulin signal peptide, such as an IgE or IgG signal peptide, and in some embodiments, an HA tag. The immunogen can be designed to elicit a stronger and broader cellular immune response than the corresponding codon-optimized immunogen.

[0323] The HBV antigen can be an HBV core protein, an HBV surface protein, an HBV DNA polymerase, an HBV protein encoded by gene X, a fragment thereof, a variant thereof, or a combination thereof. The HBV antigen can be an HBV genotype A core protein, an HBV genotype B core protein, an HBV genotype C core protein, an HBV genotype D core protein, an HBV genotype E core protein, an HBV genotype F core protein, an HBV genotype G core protein, an HBV genotype H core protein, an HBV genotype A surface protein, an HBV genotype B surface protein, an HBV genotype C surface protein, an HBV genotype D surface protein, an HBV genotype E surface protein, an HBV genotype F surface protein, an HBV genotype G surface protein, an HBV genotype H surface protein, a fragment thereof, a variant thereof, or a combination thereof. The HBV antigen can be a consensus HBV core protein or a consensus HBV surface protein.

[0324] In some embodiments, the HBV antigen can be an HBV genotype A consensus core DNA sequence construct, an IgE leader sequence linked to a consensus sequence for HBV genotype A core protein, or an HBV genotype A consensus core protein sequence.

[0325] In other embodiments, the HBV antigen can be an HBV genotype B consensus core DNA sequence construct, an IgE leader sequence linked to a consensus sequence for HBV genotype B core protein, or an HBV genotype B consensus core protein sequence.

[0326] In yet other embodiments, the HBV antigen can be an HBV genotype C consensus core DNA sequence construct, an IgE leader sequence linked to a consensus sequence for the HBV genotype C core protein, or an HBV genotype C consensus core protein sequence.

[0327] In some embodiments, the HBV antigen can be an HBV genotype D consensus core DNA sequence construct, an IgE leader sequence linked to a consensus sequence for the HBV genotype D core protein, or an HBV genotype D consensus core protein sequence.

[0328] In other embodiments, the HBV antigen can be an HBV genotype E consensus core DNA sequence construct, an IgE leader sequence linked to a consensus sequence for the HBV genotype E core protein, or an HBV genotype E consensus core protein sequence.

[0329] In some embodiments, the HBV antigen can be an HBV genotype F consensus core DNA sequence construct, an IgE leader sequence linked to a consensus sequence for the HBV genotype F core protein, or an HBV genotype F consensus core protein sequence.

[0330] In other embodiments, the HBV antigen can be an HBV genotype G consensus core DNA sequence construct, an IgE leader sequence linked to a consensus sequence for the HBV genotype G core protein, or an HBV genotype G consensus core protein sequence.

[0331] In some embodiments, the HBV antigen can be an HBV genotype H consensus core DNA sequence construct, an IgE leader sequence linked to a consensus sequence for HBV genotype H core protein, or an HBV genotype H consensus core protein sequence.

[0332] In yet other embodiments, the HBV antigen can be an HBV genotype A consensus surface DNA sequence construct, an IgE leader sequence linked to a consensus sequence for an HBV genotype A surface protein, or an HBV genotype A consensus surface protein sequence.

[0333] In some embodiments, the HBV antigen can be an HBV genotype B consensus surface DNA sequence construct, an IgE leader sequence linked to a consensus sequence for an HBV genotype B surface protein, or an HBV genotype B consensus surface protein sequence.

[0334] In other embodiments, the HBV antigen can be an HBV genotype C consensus surface DNA sequence construct, an IgE leader sequence linked to a consensus sequence for an HBV genotype C surface protein, or an HBV genotype C consensus surface protein sequence.

[0335] In yet...

Claims

1. (a) a nucleic acid encoding one or more amino acid sequences selected from the group consisting of: (i) the amino acid sequence of tyrosinase (Tyr) (SEQ ID NO: 2); (ii) the amino acid sequence of tyrosinase-related protein 1 (TYRP1) (SEQ ID NO: 4); (iii) the amino acid sequence of tyrosinase-related protein 2 (TYRP2) (SEQ ID NO: 6); (iv) the amino acid sequence of melanoma-associated antigen 4 protein (MAGEA4) (SEQ ID NO: 8); (v) the amino acid sequence of growth hormone-releasing hormone (GHRH) (SEQ ID NO: 10); (vi) the amino acid sequence of MART-1 / Melan-A antigen (MART-1 / Melan-A) (SEQ ID NO: 12); (vii) the amino acid sequence of cancer-testis antigen (NY-ESO-1) (SEQ ID NO: 14); (viii) the amino acid sequence of cancer-testis antigen II (NY-ESO-2) (SEQ ID NO: 16); (ix) the amino acid sequence of PRAME (SEQ ID NO: 18); (x) the amino acid sequence of WT1 (SEQ ID NO: 20); (xi) the amino acid sequence of WTI (SEQ ID NO: 22), and (xii) the amino acid sequence of hTERT (SEQ ID NO: 24); (b) a nucleic acid encoding one or more amino acid sequences selected from the group consisting of: (i) an amino acid sequence that is 95% or more identical to the amino acid sequence of tyrosinase (Tyr) (SEQ ID NO: 2); (ii) an amino acid sequence that is 95% or more identical to the amino acid sequence of tyrosinase-related protein 1 (TYRP1) (SEQ ID NO: 4); (iii) an amino acid sequence that is 95% or more identical to the amino acid sequence of tyrosinase-related protein 2 (TYRP2) (SEQ ID NO: 6); (iv) an amino acid sequence that is 95% or more identical to the amino acid sequence of melanoma-associated antigen 4 protein (MAGEA4) (SEQ ID NO: 8); (v) an amino acid sequence that is 95% or more identical to the amino acid sequence of growth hormone-releasing hormone (GHRH) (SEQ ID NO: 10); (vi) an amino acid sequence that is 95% or more identical to the amino acid sequence of MART-1 / Melan-A antigen (MART-1 / Melan-A) (SEQ ID NO: 12); (vii) an amino acid sequence that is 95% or more identical to the amino acid sequence of the cancer / testis antigen (NY-ESO-1) (SEQ ID NO: 14); (viii) an amino acid sequence that is 95% or more identical to the amino acid sequence of cancer-testis antigen II (NY-ESO-2) (SEQ ID NO: 16); (ix) an amino acid sequence that is 95% or more identical to the amino acid sequence of PRAME (SEQ ID NO: 18); (x) an amino acid sequence that is 95% or more identical to the amino acid sequence of WT1 (SEQ ID NO: 20); (xi) an amino acid sequence that is 95% or more identical to the amino acid sequence of WT1 (SEQ ID NO: 22); and (xii) an amino acid sequence that is 95% or more identical to the amino acid sequence of hTERT (SEQ ID NO: 24); or (c) A combination of (a) and (b) A vaccine containing

2. 10. The vaccine of claim 1, further comprising a nucleic acid encoding one or more antigens selected from the group consisting of PSA, PSMA, STEAP, PSCA, MAGE A1, gp100, a viral antigen, and combinations thereof.

3. 3. The vaccine of claim 2, wherein the viral antigen is an antigen derived from hepatitis B virus (HBV), hepatitis C virus (HCV), or human papilloma virus (HPV).

4. 4. The vaccine of claim 3, wherein the HBV antigen is an HBV core antigen or an HBV surface antigen, or a combination thereof.

5. 4. The vaccine of claim 3, wherein the HCV antigen is an HCV NS34A antigen, an HCV NS5A antigen, an HCV NS5B antigen, an HCV NS4B antigen, or a combination thereof.

6. 4. The vaccine of claim 3, wherein the HPV antigen is an HPV 6 E6 antigen, an HPV 6 E7 antigen, an HPV 11 E6 antigen, an HPV 11 E7 antigen, an HPV 16 E6 antigen, an HPV 16 E7 antigen, an HPV 18 E6 antigen, an HPV 18 E7 antigen, or a combination thereof.

7. 2. The vaccine of claim 1, further comprising an immune checkpoint inhibitor selected from the group consisting of an anti-PD-1 antibody, an anti-PD-L1 antibody, and a combination thereof.

8. 2. The vaccine of claim 1, wherein the nucleic acid molecule comprises one or more nucleotide sequences selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, and SEQ ID NO:

23.

9. The vaccine of claim 1 , wherein the nucleic acid is a plasmid.

10. 2. The vaccine of claim 1, wherein the nucleic acid is one or more plasmids.

11. 10. The vaccine of claim 1, further comprising an adjuvant.

12. 12. The vaccine of claim 11, wherein the adjuvant is IL-12, IL-15, IL-28, or RANTES.

13. 10. A method of treating cancer in a subject in need thereof, comprising administering to said subject the vaccine of claim 1.

14. 14. The method of claim 13, wherein the administering comprises an electroporation step.

15. 14. The method of claim 13, further comprising administering to the subject an immune checkpoint inhibitor.

16. 16. The method of claim 15, wherein the immune checkpoint inhibitor is selected from the group consisting of an anti-PD-1 antibody, an anti-PD-L1 antibody, and a combination thereof.

17. 16. The method of claim 15, wherein the vaccine and immune checkpoint inhibitor are administered to the subject in a single formulation.

18. 16. The method of claim 15, wherein the vaccine and the immune checkpoint inhibitor are administered to the subject separately.

19. 14. The method of claim 13, wherein the cancer is selected from the group consisting of melanoma, head and neck cancer, prostate cancer, liver cancer, cervical cancer, recurrent respiratory papillomatosis (RRP), anal cancer, blood cancer, and combinations thereof.

20. A nucleic acid molecule comprising one or more nucleotide sequences selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, a nucleotide sequence that is 95% or more identical to SEQ ID NO:1, a nucleotide sequence that is 95% or more identical to SEQ ID NO:3, a nucleotide sequence that is 95% or more identical to SEQ ID NO:5, a nucleotide sequence that is 95% or more identical to SEQ ID NO:7, a nucleotide sequence that is 95% or more identical to SEQ ID NO:9, a nucleotide sequence that is 95% or more identical to SEQ ID NO:11, a nucleotide sequence that is 95% or more identical to SEQ ID NO:13, a nucleotide sequence that is 95% or more identical to SEQ ID NO:15, a nucleotide sequence that is 95% or more identical to SEQ ID NO:17, a nucleotide sequence that is 95% or more identical to SEQ ID NO:19, a nucleotide sequence that is 95% or more identical to SEQ ID NO:21, a nucleotide sequence that is 95% or more identical to SEQ ID NO:23, and combinations thereof.

21. 21. The nucleic acid molecule of claim 20, wherein the nucleotide sequence is a plasmid.

22. 22. The nucleic acid molecule of claim 21, wherein the nucleotide sequence is one or more plasmids.

23. An amino acid molecule comprising one or more amino acid sequences 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, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, an amino acid sequence that is 95% or more identical to SEQ ID NO:2, an amino acid sequence that is 95% or more identical to SEQ ID NO:4, an amino acid sequence that is 95% or more identical to SEQ ID NO:6, an amino acid sequence that is 95% or more identical to SEQ ID NO:8, an amino acid sequence that is 95% or more identical to SEQ ID NO:10, an amino acid sequence that is 95% or more identical to SEQ ID NO:12, an amino acid sequence that is 95% or more identical to SEQ ID NO:14, an amino acid sequence that is 95% or more identical to SEQ ID NO:16, an amino acid sequence that is 95% or more identical to SEQ ID NO:18, an amino acid sequence that is 95% or more identical to SEQ ID NO:20, an amino acid sequence that is 95% or more identical to SEQ ID NO:22, an amino acid sequence that is 95% or more identical to SEQ ID NO:24, and combinations thereof.

24. 1. A method for preventing or treating cancer in a subject in need thereof, comprising: (a) administering to a subject in need of said preventive or therapeutic method a vaccine comprising a CMV cancer antigen to treat or prevent glioblastoma, or administering to a subject in need of said preventive or therapeutic method a vaccine comprising a CMV cancer antigen in combination with one or more of the cancer antigens hTERT, NY-ESO-1, MAGE-A1, or WT1; (b) administering to a subject in need of said preventive or therapeutic method a vaccine comprising one or more of the cancer antigens PSA, PSMA, or STEAP to treat or prevent prostate cancer, or administering to a subject in need of said preventive or therapeutic method a vaccine comprising PSA, PSMA, or STEAP in combination with one or more of the cancer antigens hTERT, NY-ESO-1, MAGE-A1, or WT1 to treat or prevent prostate cancer; (c) administering to a subject in need of said preventive or therapeutic method a vaccine comprising one or more of the cancer antigens tyrosinase, PRAME, or GP-100 to treat or prevent melanoma, or administering to a subject in need of said preventive or therapeutic method a vaccine comprising tyrosinase, PRAME, or GP-100 in combination with one or more of the cancer antigens hTERT, NY-ESO-1, MAGE-A1, or WT1 to treat or prevent melanoma; (d) administering to a subject in need thereof a vaccine comprising one or more of the cancer antigens HPV 16 E6 or HPV 16 E7 to treat or prevent head and neck cancer, or administering to a subject in need thereof a vaccine comprising HPV 16 E6 or HPV 16 E7 in combination with one or more of the cancer antigens hTERT, NY-ESO-1, MAGE-A1, or WT1 to treat or prevent head and neck cancer; (e) administering to a subject in need of said preventive or therapeutic method a vaccine comprising one or more of the cancer antigens tyrosinase, PRAME, or GP-100 to treat or prevent melanoma, or administering to a subject in need of said preventive or therapeutic method a vaccine comprising tyrosinase, PRAME, or GP-100 in combination with one or more of the cancer antigens hTERT, NY-ESO-1, MAGE-A1, or WT1 to treat or prevent melanoma; (f) administering to a subject in need of said preventive or therapeutic method a vaccine comprising one or more of the cancer antigens HPV 6, HPV 11, or HPV 16 to treat or prevent anal cancer, or administering to a subject in need of said preventive or therapeutic method a vaccine comprising HPV 6, HPV 11, or HPV 16 in combination with one or more of the cancer antigens hTERT, NY-ESO-1, MAGE-A1, or WT1 to treat or prevent anal cancer; (g) administering to a subject in need of said preventive or therapeutic method a vaccine comprising one or more of the cancer antigens HBV core antigen, HBV surface antigen, HCV NS34A, HCV NS5A, HCV NS5B, or HCV NS4B to treat or prevent liver disease, or administering to a subject in need of said preventive or therapeutic method a vaccine comprising HBV core antigen, HBV surface antigen, HCV NS34A, HCV NS5A, HCV NS5B, or HCV NS4B in combination with one or more of the cancer antigens hTERT, NY-ESO-1, MAGE-A1, or WT1 to treat or prevent liver disease; (h) administering to a subject in need thereof a vaccine comprising one or more of the cancer antigens HPV 16 E6 / E7 or HPV 18 E6 / E7 to treat or prevent cervical cancer, or administering to a subject in need thereof a vaccine comprising HPV 16 E6 / E7 or HPV 18 E6 / E7 in combination with one or more of the cancer antigens hTERT, NY-ESO-1, MAGE-A1, or WT1 to treat or prevent cervical cancer; or (i) administering to a subject in need of the preventive or therapeutic method a vaccine comprising one or more of the cancer antigens PRAME, WT-1, or hTERT to treat or prevent blood cancer, or administering to a subject in need of the preventive or therapeutic method a vaccine comprising PRAME, WT-1, or hTERT in combination with one or more of the cancer antigens NY-ESO-1 or MAGE-A1 to treat or prevent blood cancer; Including, The method of treatment may further comprise combining the administering steps (a) to (i) with an immune checkpoint inhibitor selected from the group consisting of an anti-PD-1 antibody, an anti-PD-L1 antibody, and a combination thereof.

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