HK1216991B - Cancer vaccines and methods of treatment using the same - Google Patents

Description

Cancer vaccine and treatment method using the same

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent Application No. 61/799,952, filed March 15, 2013, which is hereby incorporated by reference in its entirety.

Technical Field

Disclosed herein are compositions and methods for treating cancer, and in particular vaccines that treat and provide protection against tumor growth.

background

Cancer is one of the main causes of death in the world, and is the second most common cause of death in the U.S., accounting for almost 1 in every 4 deaths.Cancer arises from the single cell that has been transformed into tumor cell from normal cell.Such conversion is normally a multi-stage process, progresses to malignant tumor from precancerous lesion.A plurality of factors facilitate this progress, comprise aging, hereditary contribution and to external agent such as physical carcinogen (for example, ultraviolet radiation and ionizing radiation), chemical carcinogen (for example, the component of asbestos, tobacco smoke etc.) and biological carcinogen (for example, some virus, bacterium and parasite) exposure.

The prevention, diagnosis and treatment of cancer can take many different forms. Prevention can include screening for predisposing factors (e.g., specific gene variants), changing behavior (e.g., smoking, diet and physical activity), and antiviral (e.g., human papillomavirus, hepatitis B virus) vaccination. Treatment can include chemotherapy, radiotherapy, and surgical resection of tumors or cancerous tissue. Although many preventive and therapeutic methods are available, such methods generally have limited success in effectively preventing and/or treating current cancers.

Therefore, there is a need for the identification and development of compositions and methods for the prevention and/or treatment of cancer to promote protection against disease and disease progression in the clinical management of cancer. In addition, there is a need for more effective treatments to delay disease progression and/or reduce mortality in subjects with cancer.

SUMMARY OF THE INVENTION

The present invention relates to vaccines comprising one or more nucleic acid or amino acid sequences that are no longer self-antigens and that stimulate an immune response against a specific cancer or a tumor associated with a specific cancer. The vaccine may also comprise immune checkpoint inhibitors that block any component of the immune system such as MHC class presentation, T cell presentation and/or differentiation, B cell presentation and/or differentiation, or inhibition of any cytokine, chemokine or signal transduction for immune cell proliferation and/or differentiation, such as anti-PD-1 and anti-PDL-1 antibodies. The one or more cancer antigens of the vaccine may be a nucleic acid encoding one or more amino acid sequences selected from the group consisting of: an amino acid sequence having 95% or greater identity with the amino acid sequence of tyrosinase (Tyr); an amino acid sequence having 95% or greater identity with the amino acid sequence of tyrosinase-related protein 1 (TYRP1); an amino acid sequence having 95% or greater identity with the amino acid sequence of tyrosinase-related protein 2 (TYRP2); an amino acid sequence having 95% or greater identity with the amino acid sequence of melanoma-associated antigen 4 protein (MAGEA4); an amino acid sequence having 95% or greater identity with the amino acid sequence of growth hormone releasing hormone (GHRH); an amino acid sequence having 95% or greater identity with the amino acid sequence of MART -1/melan-A antigen (MART-1/Melan-A); an amino acid sequence that is 95% or greater identical to the amino acid sequence of cancer-testis antigen (NY-ESO-1); an amino acid sequence that is 95% or greater identical to the amino acid sequence of cancer-testis antigen II (NY-ESO-2); an amino acid sequence that is 95% or greater identical to the amino acid sequence of PRAME; an amino acid sequence that is 95% or greater identical to the amino acid sequence of WT1; an amino acid sequence that is 95% or greater identical to the amino acid sequence of hTERT; or a combination thereof. The vaccine may also comprise a nucleic acid encoding one or more antigens selected from the group consisting of PSA, PSMA, STEAP, PSCA, MAGE A1, gp100, viral antigens, and combinations thereof.

The present invention further relates to a method for preventing or treating cancer in a subject in need thereof, the method comprising administering to a subject in need thereof a vaccine comprising a specific number of cancer antigens for treating or preventing a specific cancer. The method may include administering to a subject in need thereof a vaccine comprising a CMV cancer antigen for treating or preventing glioblastoma, or administering to a subject in need thereof a vaccine comprising a CMV cancer antigen in combination with any one or more of the cancer antigens hTERT, NY-ESO-1, MAGE-A1, or WT1 for treating or preventing glioblastoma; administering to a subject in need thereof a vaccine comprising one or more cancer antigens PSA, PSMA, or STEAP for treating or preventing prostate cancer, or administering to a subject in need thereof a vaccine comprising a CMV cancer antigen in combination with any one or more of the cancer antigens hTERT, NY-ESO-1, MAGE-A1, or WT1 for treating or preventing glioblastoma; administering to a subject in need thereof a vaccine comprising one or more cancer antigens PSA, PSMA, or STEAP for treating or preventing prostate cancer; or administering to a subject in need thereof a vaccine comprising a CMV cancer antigen in combination with any one or more of the cancer antigens hTERT, NY-ESO-1, MAGE-A1, or WT1. A vaccine comprising PSA, PSMA or STEAP in combination with any one or more of A1 or WT1 to treat or prevent prostate cancer; administering to a subject in need thereof a vaccine comprising one or more of the cancer antigens tyrosinase, PRAME or GP-100 to treat or prevent melanoma; administering to a subject in need thereof a vaccine comprising tyrosinase, PRAME or GP-100 in combination with any one or more of the cancer antigens hTERT, NY-ESO-1, MAGE-A1 or WT1 to treat or prevent melanoma; administering to a subject in need thereof a vaccine comprising one or more of the cancer antigens HPV administering to a subject in need thereof a vaccine comprising one or more cancer antigens tyrosinase, PRAME, or GP-100 to treat or prevent melanoma, or administering to a subject in need thereof a vaccine comprising tyrosinase, PRAME, or GP-100 in combination with any one or more cancer antigens hTERT, NY-ESO-1, MAGE-A1, or WT1 to treat or prevent melanoma; administering to a subject in need thereof a vaccine comprising one or more cancer antigens HPV6, HPV 11, or HPV 16 to treat or prevent anal cancer, or administering to a subject in need thereof a vaccine comprising one or more cancer antigens HPV 11, HPV 16 ... 6. HPV 11 or HPV 16 vaccine for the treatment or prevention of anal cancer; administering to a subject in need thereof a vaccine comprising one or more cancer antigens HBV core antigen, HBV surface antigen, HCV NS34A, HCV NS5A, HCV NS5B or HCV NS4B for the treatment or prevention of liver cancer; administering to a subject in need thereof a vaccine comprising HBV core antigen, HBV surface antigen, HCV NS34A, HCV NS5A, HCV NS5B or HCV NS4B in combination with any one or more cancer antigens hTERT, NY-ESO-1, MAGE-A1 or WT1 for the treatment or prevention of liver cancer; administering to a subject in need thereof a vaccine comprising one or more cancer antigens HPV 16 E6/E7 or HPV 18 E6/E7 for the treatment or prevention of cervical cancer; administering to a subject in need thereof a vaccine comprising one or more cancer antigens HPV 16 E6/E7 in combination with any one or more cancer antigens hTERT, NY-ESO-1, MAGE-A1 or WT1 E6/E7 or HPV 18 E6/E7 to treat or prevent cervical cancer; or administering to a subject in need thereof a vaccine comprising one or more cancer antigens PRAME, WT-1 or hTERT to treat or prevent blood cancer; or administering to a subject in need thereof a vaccine comprising PRAME, WT-1 or hTERT and any one or more of the cancer antigens NY-ESO-1 or MAGE-A1 to treat or prevent blood cancer, wherein the method may further comprise combining the administering steps of (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.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1A-E show the constructs of pTyr.

Figures 2A and 2B show the immunization strategy and induction of cell-mediated immune responses by Tyr DNA vaccination, respectively.

FIG3 shows flow cytometric fluorescence activated cell sorting (FACS) of control and immunized mice.

Figures 4A and 4B show the induction of tyrosinase-specific antibodies in immunized mice.

Figures 5A and 5B show Kaplan-Meier survival curves and tumor volume curves, respectively, after tumor challenge in control and immunized mice.

Figures 6A and 6B show MDSC cell populations in immunized and non-immunized mice.

FIG7 shows staining of MDSCs in mice immunized with pVax1 and pTyr.

Figures 8A and 8b show MCP-1 secretion by MDSCs.

FIG9 shows the phylogenetic relationships of Tyr nucleotide sequences among the indicated organisms.

Figure 10 shows (A) a schematic diagram illustrating the plasmid map of pPRAME (also referred to herein as pGX1411); (B) staining of nuclei of RD and 293T cells and staining for the consensus PRAME antigen using DAPI; and (C) immunoblots for the consensus PRAME antigen in lysates from non-transfected cells ("control"), cells transfected with pVAX ("pVAX"), and cells transfected with pPRAME ("PRAME-pVAX").

Figure 11 shows graphs in (A) and (B) plotting groups of mice versus plaque forming units (SFU)/ ¹⁰⁶ splenocytes to interferon gamma (IFN-γ).

Figure 12 shows (A) a schematic diagram illustrating the plasmid map of pNY-ESO-1 (also referred to herein as pGX1409); (B) staining of cell nuclei and consensus NY-ESO-1 antigen with DAPI; and (C) immunoblots of consensus NY-ESO-1 antigen in RD and 293T lysates from non-transfected cells ("control"), cells transfected with pVAX ("pVAX"), and cells transfected with pNY-ESO-1 ("pNY-ESO-1").

FIG13 shows a graph plotting groups of mice versus plaque forming units (SFU)/10 ⁶ splenocytes against interferon gamma (IFN-γ).

FIG14 shows a graph plotting groups of mice versus plaque forming units (SFU)/10 ⁶ splenocytes against interferon gamma (IFN-γ).

Figure 15 shows a schematic diagram illustrating various cancers with some of their associated cancer antigens.

Details

The present invention relates to vaccines that can be customized for specific cancers and tumors. Specific cancer-associated antigens to be used in vaccines, 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), have been designed to allow customized vaccines to prevent or treat specific cancers. For example, tyrosinase antigen can be used in vaccines to prevent or treat 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.

One way to design nucleic acids and their encoded amino acid sequences for recombinant cancer antigens is by introducing mutations that alter specific amino acids in the complete amino acid sequence of a natural cancer antigen. The introduction of mutations does not alter the cancer antigen so much that it cannot be universally administered across mammalian subjects (preferably human or dog subjects), but alters it enough to break tolerance or be used as an exogenous antigen to generate an immune response. Another approach is to generate a consensus recombinant cancer antigen that has at least 85% and up to 99% amino acid sequence identity, preferably at least 90% and up to 98% sequence identity, more preferably at least 93% and up to 98% sequence identity, or even more preferably at least 95% and up to 98% sequence identity with its corresponding natural cancer antigen. In some cases, the recombinant cancer antigen has 95%, 96%, 97%, 98% or 99% amino acid sequence identity with its corresponding natural cancer antigen. The natural cancer antigen is an antigen that is typically associated with a specific cancer or cancer tumor. Depending on the cancer antigen, the consensus sequence of the cancer antigen can span mammalian species or within a subtype of a species or across viral strains or serotypes. Some cancer antigens will not change greatly with the wild-type amino acid sequence of cancer antigens. Some cancer antigens have nucleic acid/amino acid sequences with such large differences that they cannot produce consensus sequences between species. In these cases, recombinant cancer antigens that can break tolerance and produce an immune response are produced, wherein the recombinant cancer antigens and their corresponding natural cancer antigens have at least 85% and up to 99% amino acid sequence identity, preferably at least 90% and up to 98% sequence identity, more preferably at least 93% and up to 98% sequence identity or even more preferably at least 95% and up to 98% sequence identity. In some cases, the recombinant cancer antigens and their corresponding natural cancer antigens have 95%, 96%, 97%, 98% or 99% amino acid sequence identity. The aforementioned methods can be combined so that the final recombinant cancer antigen has the percentage similarity to the natural cancer antigen amino acid sequence as discussed above.

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

Vaccines can be further combined with antibodies for checkpoint inhibitors such as PD-1 and PDL-1 to enhance stimulation of cellular and humoral immune responses. Anti-PD-1 or anti-PDL-1 antibodies are used to prevent PD-1 or PDL-1 from suppressing T cells and/or B cell responses. In general, by designing cancer antigens to be recognized by the immune system, it is possible to help overcome other forms of immunosuppression produced by tumor cells, and these vaccines can be used in combination with suppression or inhibitory therapies (such as anti-PD-1 and anti-PDL-1 antibody therapies) to further enhance T cells and/or B cell responses.

1. Definition

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those understood by those of ordinary skill in the art. In the event of conflict, this document (including definitions) shall prevail. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in the implementation or testing of the present invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods and examples disclosed herein are merely illustrative and are not intended to be limiting. The terms used herein are only for the purpose of describing specific embodiments and are not intended to be limiting.

As used herein, the terms "comprises," "includes," "has," "have," "may," "contain," and variations thereof are intended to be open transitional phrases, terms, or words that do not exclude the possibility of additional actions or structures. The singular forms "a," "and," and "the" include plural references unless the context clearly dictates otherwise. The present disclosure also encompasses other embodiments that "comprise," "comprises," and "consists of," and "consists essentially of," the embodiments or components set forth herein, whether explicitly indicated or not.

For the recitation of numerical ranges herein, every intervening number therebetween is expressly included with equal precision. For example, for the range 6-9, the numbers 7 and 8 are included in addition to 6 and 9, and for the range 6.0-7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are expressly included.

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

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

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

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

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

"Electroporation," "electro-permeabilization," or "electrokinetic enhancement" ("EP"), as used interchangeably herein, refers to the use of transmembrane electric field pulses to induce microscopic pathways (pores) in biological membranes; their presence allows the passage of biomolecules such as plasmids, oligonucleotides, siRNA, drugs, ions, and water from one side of the cell membrane to the other.

As used herein with respect to nucleic acid sequences, "fragment" means a nucleic acid sequence or portion thereof that encodes a polypeptide capable of eliciting an immune response in a mammal that cross-reacts with an antigen disclosed herein. The fragment can be a DNA fragment selected from at least one of the various nucleotide sequences encoding the protein fragments shown 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% or more of the nucleic acid sequence shown below. In some embodiments, a fragment may comprise 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 300 nucleotides or more, 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, or at least 1000 nucleotides or more of at least one of the nucleic acid sequences shown below.

With respect to polypeptide sequences, "fragment" or "immunogenic fragment" means a polypeptide that is capable of eliciting an immune response in a mammal that cross-reacts with an antigen disclosed herein. A fragment can be a polypeptide fragment selected from at least one of the various amino acid sequences described below. 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 may 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, at least 180 amino acids or more amino acids of a protein sequence disclosed herein.

As used herein, "genetic construct" refers to a DNA or RNA molecule comprising a nucleotide sequence encoding a protein. The coding sequence includes start and stop signals operatively linked to regulatory elements (including a promoter and a polyadenylation signal) 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 containing the necessary regulatory elements operatively linked to a coding sequence encoding a protein so that when present in the cells of an individual, the coding sequence will be expressed.

As used herein, the term "homology" refers to the degree of complementarity. Partial homology or complete homology (i.e., identical) may exist. A partially complementary sequence that at least partially inhibits hybridization of a completely complementary sequence to a target sequence refers to the use of the functional term "substantially homologous." When used to refer to double-stranded nucleic acid sequences such as cDNA or genomic clones, as used herein, the term "substantially homologous" refers to probes that can hybridize to the chains of a double-stranded nucleic acid sequence under conditions of low stringency. When used to refer to single-stranded nucleic acid sequences, as used herein, the term "substantially homologous" refers to probes that can hybridize to (i.e., be complementary to) a single-stranded nucleic acid template sequence under conditions of low stringency.

As used herein in the context of two or more nucleic acid or polypeptide sequences, "identical" or "identity" means that the sequences have a specified percentage of residues that are identical over a specified region. Percentages can be calculated by optimally aligning the two sequences, comparing the two sequences over a specified region, determining the number of positions at which identical residues are present in the two sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the specified region, and multiplying the result by 100 to obtain the percentage of sequence identity. Where the two sequences have different lengths or the alignment produces one or more staggered ends and the specified comparison region includes only a single sequence, the residues of the single sequence are included in the denominator, rather than the numerator, of the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered to be equivalent. Identity can be obtained artificially or by using a computer sequence algorithm such as BLAST or BLAST2.0.

As used herein, "immune response" means the activation of a host's immune system, such as a mammal's immune system, in response to the introduction of an antigen. The immune response can be in the form of a cellular response or a humoral response, or both.

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

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

As used herein, "operably linked" means that the expression of a gene is under the control of a promoter to which it is spatially linked. A 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, variations in this distance are acceptable without loss of promoter function.

As used herein, "peptide," "protein," or "polypeptide" may mean a linked sequence of amino acids, and may be natural, synthetic, or modified, or a combination of natural and synthetic.

As used herein, "promoter" means a molecule of synthetic or natural origin that is capable of conferring, activating or enhancing the expression of a nucleic acid in a cell. A promoter may comprise one or more specific transcriptional regulatory sequences to further enhance the expression of the sequence and/or alter its spatial expression and/or temporal expression. A promoter may also comprise distal enhancer or repressor elements that are up to several thousand base pairs away from the transcription start site. Promoters may be derived from sources including viruses, bacteria, fungi, plants, insects and animals. A promoter may constitutively or differentially regulate the expression of a genomic component in response to a cell, tissue or organ in which expression occurs or in response to an external stimulus such as physiological stress, a pathogen, a metal ion or an inducer. Representative examples of promoters include bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operator-promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, SV40 early promoter or SV 40 late promoter and CMV IE promoter.

"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 as described herein. The signal peptide/leader sequence typically directs the localization of the protein. As used herein, the signal peptide/leader sequence preferably promotes secretion of the protein from the cell in which it is produced. Following secretion from the cell, the signal peptide/leader sequence is typically cleaved from the remainder of the protein (usually referred to as the mature protein). The signal peptide/leader sequence is attached to the amino terminus (i.e., the N terminus) of the protein.

As used herein, "stringent hybridization conditions" means conditions under which a first nucleic acid sequence (e.g., a probe) will hybridize to a second nucleic acid sequence (e.g., a target) (such as in a complex mixture of nucleic acids). Stringent conditions are sequence-dependent and are different in different situations. Stringent conditions may be selected to be about 5-10°C lower than the thermal melting point (Tm) of the specific 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 (due to an excess of target sequence, 50% of the probes are occupied at equilibrium at Tm). Stringent conditions can be those in which the salt concentration is less than about 1.0 M sodium ion, such as about 0.01-1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e.g., about 10-50 nucleotides) and at least about 60° C. for long probes (e.g., greater than about 50 nucleotides). Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal can be at least 2 to 10 times background hybridization. Exemplary stringent hybridization conditions include the following: incubation in 50% formamide, 5× SSC, and 1% SDS at 42° C., or incubation in 5× SSC, 1% SDS at 65° C., with a wash in 0.2× SSC and 0.1% SDS at 65° C.

As used herein, "subject" may refer to a mammal that is desired or in need of immunization with the vaccines described herein. The mammal may be a human, chimpanzee, dog, cat, horse, cow, mouse, or rat.

As used herein, "substantially complementary" means that a first sequence is substantially complementary to the complement of a second sequence at 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, 480, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1110, 1120, 1130, 1140, 1150, 1160, 1170, 1180, 1190, 220, 221 In some embodiments, the two sequences 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 over a region of 50, 540 or more nucleotides or amino acids, or the two sequences hybridize under stringent hybridization conditions.

As used herein, "substantially identical" means that if the first sequence is substantially complementary to the complement of the second sequence, then the first and second sequences are identical within 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, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100 93%, 94%, 95%, 96%, 97%, 98% or 99% identical over a region or with respect to a nucleic acid of at least 100, 180, 270, 360, 450, 540 or more nucleotides or amino acids.

As used herein, "treating" or "treating" may mean protecting an animal from a disease by preventing, inhibiting, suppressing, or completely eliminating the disease. Preventing a disease includes administering a vaccine of the present invention to an animal before the onset of the disease. Suppressing a disease includes administering a vaccine of the present invention to an animal after the disease has developed but before its clinical manifestation. Suppressing a disease includes administering a vaccine of the present invention to an animal after clinical manifestation of the disease.

As used herein, "variant" with respect to a nucleic acid 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 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 sequence substantially identical thereto.

For peptides or polypeptides, a "variant" refers to a peptide or polypeptide that differs in amino acid sequence by insertion, deletion, or conservative substitution of amino acids, but retains at least one biological activity. Variant can also refer to a protein having an amino acid sequence that is substantially identical to a reference protein having an amino acid sequence that retains at least one biological activity. Conservative substitution of amino acids, i.e., replacing an amino acid with a different amino acid having similar properties (e.g., hydrophobicity, degree and distribution of charged regions), is generally considered in the art to include minor changes. As understood in the art, these minor changes can be identified, in part, by considering the hydropathic index of the amino acids. Kyte et al., J. Mol. Biol. 157:105-132 (1982). The hydropathic index of an amino acid is based on considerations of its hydrophobicity and charge. It is known in the art that amino acids with similar hydropathic indices can be substituted and still retain protein function. In one aspect, amino acids with hydropathic indices of ±2 are substituted. The hydrophilicity of amino acids can also be used to indicate substitutions that result in proteins retaining biological function. Considering the hydrophilicity of amino acids in the context of peptides allows calculation of the maximum local average hydrophilicity of the peptide, which is a useful metric that has been reported to be closely related to antigenicity and immunogenicity. U.S. Patent No. 4,554,101 is fully incorporated herein by reference. As understood in the art, substitution of amino acids with similar hydrophilicity values can result in peptides retaining biological activity, such as immunogenicity. Amino acids with hydrophilicity values within ± 2 of each other can be used for substitution. The hydrophobicity index and hydrophilicity value of amino acids are affected by the specific side chains of the amino acids. Consistent with this observation, amino acid substitutions that are compatible with biological functions are considered to depend on the relative similarity of the amino acids, and specifically the side chains of those amino acids, as shown by hydrophobicity, hydrophilicity, charge, size, and other properties.

Variants can be substantially identical nucleotide sequences on the full-length full gene sequence or its fragment. The nucleotide sequence can have 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity on the full-length gene sequence or its fragment. Variants can be substantially identical amino acid sequences on the full-length amino acid sequence or its fragment. The amino acid sequence can have 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity on the full-length amino acid sequence or its fragment.

As used herein, "vector" means a nucleic acid sequence containing an origin of replication. A vector may be a viral vector, a bacteriophage, a bacterial artificial chromosome, or a yeast artificial chromosome. A vector may be a DNA or RNA vector. A vector may be an autonomously replicating extrachromosomal vector, and preferably, a DNA plasmid. The vector may contain or include one or more heterologous nucleic acid sequences.

2. Vaccines

The present invention relates to anti-cancer vaccines. Vaccines may contain one or more cancer antigens. Vaccines may prevent tumor growth. Vaccines may reduce tumor growth. Vaccines may prevent the metastasis of tumor cells. Depending on the cancer antigen, vaccines may be targeted to treat liver cancer, prostate cancer, melanoma, blood cancer, head and neck cancer, glioblastoma, recurrent respiratory papillomatosis, anal cancer, cervical cancer, and brain cancer.

The first step in vaccine development is to identify cancer antigens that are not recognized by the immune system and are self-antigens. The identified cancer antigens are converted 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 the recombinant cancer antigens from self-antigens to foreign antigens breaks the immune system's tolerance to the antigen. To break tolerance, several redesign measures can be applied to cancer antigens, as described below.

The recombinant cancer antigens of the vaccine are not recognized as self, thereby breaking tolerance. The breaking of tolerance can induce antigen-specific T cells and/or high-titer antibody responses, thereby inducing or triggering an immune response against the cancer or tumor expressing the antigen or reacting with the cancer or tumor. In some embodiments, the induced or triggered immune response can be a cellular immune response, a humoral immune response, or a cellular and humoral immune response. In some embodiments, the induced or triggered cellular immune response can include the induction or secretion of interferon-γ (IFN-γ) and/or tumor necrosis factor α (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 expressing the antigen, such as, but not limited to, factors that downregulate MHC presentation, factors that upregulate antigen-specific regulatory T cells (Tregs), PD-L1, FasL, cytokines such as IL-10 and TFG-β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by immunosuppressive cells, CTLA-4, PD-1, MDSC, MCP-1, and immune checkpoint molecules.

In certain embodiments, the vaccine can mediate the clearance of tumor cells or prevent their growth by inducing the following responses: (1) humoral immunity via a B cell response (to produce antibodies that block the production of monocyte chemoattractant protein-1 (MCP-1), thereby delaying the production of myeloid-derived suppressor cells (MDSC) and inhibiting tumor growth); (2) increasing cytotoxic T lymphocytes such as CD8 ⁺ (CTL) to attack and kill tumor cells; (3) increasing T helper cell responses; and (4) increasing inflammatory responses via IFN-γ and TFN-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 reduced tumor mass 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 prevented and blocked the increase of monocyte chemoattractant protein 1 (MCP-1), a cytokine secreted by myeloid-derived suppressor cells. The vaccine increased tumor survival 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%.

The vaccine can enhance the cellular immune response of a subject administered the vaccine by about 50-fold to about 6000-fold, about 50-fold to about 5500-fold, about 50-fold to about 5000-fold, about 50-fold to about 4500-fold, about 100-fold to about 6000-fold, about 150-fold to about 6000-fold, about 200-fold to about 6000-fold, about 250-fold to about 6000-fold, or about 300-fold to about 6000-fold, compared to the cellular immune response of a subject not administered the vaccine. In some embodiments, the vaccine can enhance the cellular immune response of 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, 2200-fold, 2400-fold, 2500-fold, 2600-fold, 2700-fold, 2800-fold, 2900-fold, 3000-fold, 3100-fold, 3200-fold, 3300-fold, 3400-fold, 3500-fold, 3600-fold, 3700-fold, 3800-fold, 3900-fold, 4000-fold, 4100-fold, 4200-fold, 4300-fold, 4400-fold 2300 times, 2400 times, 2500 times, 2600 times, 2700 times, 2800 times, 2900 times, 3000 times, 3100 times, 3200 times, 3300 times, 3400 times, 3500 times, 3600 times, 3700 times, 3800 times, 3900 times, 4000 times, 4100 times, 4200 times, 4300 times, 4400 times, 4500 times, 4600 times, 4700 times, 4800 times, 4900 times, 5000 times, 5100 times, 5200 times, 5300 times, 5400 times, 5500 times, 5600 times, 5700 times, 5800 times, 5900 times or 6000 times.

The vaccine may increase interferon gamma (IFN-γ) levels in a subject administered the vaccine by about 50-fold to about 6000-fold, about 50-fold to about 5500-fold, about 50-fold to about 5000-fold, about 50-fold to about 4500-fold, about 100-fold to about 6000-fold, about 150-fold to about 6000-fold, about 200-fold to about 6000-fold, about 250-fold to about 6000-fold, or about 300-fold to about 6000-fold, compared to IFN-γ levels in a subject not administered the vaccine. In some embodiments, the vaccine may increase 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, 2200-fold, , 2300 times, 2400 times, 2500 times, 2600 times, 2700 times, 2800 times, 2900 times, 3000 times, 3100 times, 3200 times, 3300 times, 3400 times, 3500 times, 3600 times, 3700 times, 3800 times, 3900 times, 4000 times, 4100 times, 4200 times, 4300 times, 4400 times, 4500 times, 4600 times, 4700 times, 4800 times, 4900 times, 5000 times, 5100 times, 5200 times, 5300 times, 5400 times, 5500 times, 5600 times, 5700 times, 5800 times, 5900 times or 6000 times.

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 (incorporated herein by reference in their entirety). DNA vaccines can also contain elements or agents that inhibit their integration into chromosomes.

The vaccine can be RNA of one or more cancer antigens. The RNA vaccine can be introduced into cells.

The vaccine can be a live attenuated vaccine, a vaccine using a recombinant vector to deliver the antigen, a subunit vaccine, and a glycoprotein vaccine, such as, 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, 5,174,993, 5,223,424, 5,225,336, 5,240,703, 5,242,829, 5,294,441, 5,294,548 , 5,310,668, 5,387,744, 5,389,368, 5,424,065, 5,451,499, 5,453,364, 5,462,734, 5,470,734, 5,474,935, 5,482,713, 5,591,439, 5,643,579, 5,650,309, 5,698,202, 5,955,088, 6,034,298, 6,042,836, 6,156,319, and 6,589,529 (each of which is incorporated herein by reference).

The vaccines of the present invention may have the properties required of an effective vaccine, such as safety, so that the vaccine itself does not cause disease or death; has a protective effect against disease; induces neutralizing antibodies; induces protective T cell responses; and provides ease of administration, few side effects, biostability, and low cost per dose. Vaccines may achieve some or all of these properties by containing cancer antigens as discussed below.

As described in more detail below, vaccine can also include one or more inhibitors (that is, immune checkpoint inhibitors) of one or more immune checkpoint molecules.Immune checkpoint molecules are described in more detail below.Immune checkpoint inhibitors are any nucleic acid or protein that prevents any component in the immune system such as MHC class presentation, presentation and/or differentiation in T cells, B cell presentation and/or differentiation, any cytokine, chemokine or signal transduction for immune cell proliferation and/or differentiation.As also described in more detail below, vaccine can be further combined with antibodies for checkpoint inhibitors such as PD-1 and PDL-1, to enhance the stimulation of cellular and humoral immune responses.Use anti-PD-1 or anti-PDL-1 antibodies to prevent PD-1 or PDL-1 from suppressing T cells and/or B cells to respond.

a. Cancer antigens

A vaccine may comprise one or more cancer antigens. The cancer antigen may be a nucleic acid sequence, an amino acid sequence, or a combination thereof. The nucleic acid sequence may be DNA, RNA, cDNA, variants thereof, fragments thereof, or combinations thereof. The nucleic acid sequence may also include additional sequences encoding a linker or tag sequence that is linked to the cancer antigen via a peptide bond. The amino acid sequence may be a protein, a peptide, variants thereof, fragments thereof, or combinations thereof. The cancer antigen may be a recombinant cancer antigen.

One way to design the nucleic acid and amino acid sequence of a recombinant cancer antigen is to introduce mutations that alter specific amino acids in the complete amino acid sequence of a natural cancer antigen. The introduction of mutations does not alter the cancer antigen so much that it cannot be universally administered across mammalian subjects (preferably human or dog subjects), but alters it enough to break tolerance or be used as an exogenous antigen to generate an immune response. Another approach is to generate a consensus recombinant cancer antigen that has at least 85% and up to 99% amino acid sequence identity, preferably at least 90% and up to 98% sequence identity, more preferably at least 93% and up to 98% sequence identity, or even more preferably at least 95% and up to 98% sequence identity with its corresponding natural cancer antigen. In some cases, the recombinant cancer antigen has 95%, 96%, 97%, 98% or 99% amino acid sequence identity with its corresponding natural cancer antigen. The natural cancer antigen is an antigen that is typically associated with a specific cancer or cancer tumor. Depending on the cancer antigen, the consensus sequence of the cancer antigen can span mammalian species or within a subtype of a species or across viral strains or serotypes. Some cancer antigens will not change greatly with the wild-type amino acid sequence of cancer antigens. Some cancer antigens have nucleic acid/amino acid sequences with such large differences that they cannot produce consensus sequences between species. In these cases, recombinant cancer antigens that can break tolerance and produce an immune response are produced, wherein the recombinant cancer antigens and their corresponding natural cancer antigens have at least 85% and up to 99% amino acid sequence identity, preferably at least 90% and up to 98% sequence identity, more preferably at least 93% and up to 98% sequence identity or even more preferably at least 95% and up to 98% sequence identity. In some cases, the recombinant cancer antigens and their corresponding natural cancer antigens have 95%, 96%, 97%, 98% or 99% amino acid sequence identity. The aforementioned methods can be combined so that the final recombinant cancer antigen has the percentage similarity to the natural cancer antigen amino acid sequence as discussed above.

The cancer antigen can be 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), the amino acid sequence of growth hormone-releasing hormone (GHRH), the amino acid sequence of MART-1/melan-A antigen (MART-1/Melan-A), cancer testis antigen (NY-ESO-1), cancer testis antigen II (NY-ESO-1) and PRAME. The vaccine may be a DNA vaccine comprising a polynucleotide sequence encoding tyrosinase (Tyr), tyrosinase-related protein 1 (TYRP1), tyrosinase-related protein 2 (TYRP2), melanoma-associated antigen 4 protein (MAGEA4), an amino acid sequence of growth hormone-releasing hormone (GHRH), an amino acid sequence of MART-1/melan-A antigen (MART-1/Melan-A), 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 one or more antigens from 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 papillomavirus (HPV) 6, HPV 11, HPV 16, and HPV 18.

(1) Tyrosinase (Tyr)

The vaccine of the present invention may comprise 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 activities that can be found in microorganisms as well as plant and animal tissues. Specifically, tyrosinase catalyzes the production of melanin and other pigments through the oxidation of phenols such as tyrosine. Mutations in the TYR gene lead to oculocutaneous albinism in mammals, and non-pathological polymorphisms of the TYR gene contribute to changes in skin pigmentation.

In addition, in cancer or tumors such as melanoma, tyrosinase can become dysregulated, thereby leading to increased melanin synthesis. Therefore, tyrosinase can be a cancer antigen associated with melanoma. In subjects suffering from melanoma, tyrosinase can be a target recognized by cytotoxic T cells. However, in some cases, the immune response for cancer or tumor (including melanoma) can be suppressed, thereby leading to a microenvironment that supports tumor formation and/or growth and thus disease progression.

Immunosuppression can be promoted by myeloid-derived suppressor cells (MDSC), which are a mixed population of immature macrophages, granulocytes, dendritic cells, and myeloid cells. The myeloid cells can be a heterogeneous population of bone marrow progenitor cells and immature myeloid cells (IMC). The markers of MDSC can include the expression of Gr-1 and CD11b (i.e., Gr-1 ⁺ and CD11b ⁺ cells).

The circulation of MDSCs can be increased by chronic infection, and the expansion of MDSC populations can be associated with autoimmunity and inflammation. In particular, MDSC expansion (or presence in tumors or cancerous tissues) can promote tumor growth and evade immune detection and/or regulation. Therefore, MDSCs can affect the immune response to anti-cancer vaccines.

MDSCs can be regulated by regulators of G-protein signaling 2 (Rgs2), and Rgs2 is highly expressed in tumor-derived MDSCs. Rgs2 is also widely expressed in a variety of cells, such as myeloid cells. MDSCs derived from tumor-bearing mice can function differently from MDSCs derived from non-tumor-bearing mice. One such difference can be seen in 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) found on monocytes, endothelial cells, and T cells. Therefore, MCP-1 can cause the migration of endothelial cells, thereby promoting angiogenesis. Blocking MCP-1 via neutralizing antibodies can inhibit angiogenesis, thereby leading to reduced tumor metastasis and increased survival. Therefore, MCP-1 can be regarded as an angiogenic factor. In addition to secreting MCP-1, MDSCs can also secrete growth factors, which further promote tumor growth.

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

As shown herein, Tyr antigens induce antigen-specific T cells and high titer antibody responses against cancer cells or tumor cells (e.g., melanoma cells). In particular, Tyr antigens are important targets for immune-mediated clearance by inducing the following responses: (1) humoral immunity via B cell responses (to produce antibodies that block monocyte chemoattractant protein-1 (MCP-1) production, thereby delaying myeloid-derived suppressor cells (MDSCs) and inhibiting tumor growth); (2) increasing cytotoxic T lymphocytes such as CD8 ⁺ (CTLs) to attack and kill tumor cells; (3) increasing T helper cell responses; and (4) increasing inflammatory responses via IFN-γ and TFN-α, or preferably all of the above responses. Thus, vaccines comprising Tyr antigens (e.g., shared Tyr antigens, which will be described in more detail below) provide a protective immune response against tumor formation and tumor growth because these vaccines prevent immunosuppression by reducing the population of MDSCs found in cancerous or tumor tissues and block angiogenesis of cancerous or tumor tissues by reducing the production or secretion of MCP-1. Thus, any user can design a vaccine of the present invention comprising a Tyr antigen to provide broad immunity against tumor formation, tumor metastasis, and tumor growth.

Tyr antigens may comprise protein epitopes that make them particularly effective as immunogens that can induce anti-Tyr immune responses against them. Tyr antigens may comprise full-length translation products, variants thereof, fragments thereof, or combinations thereof. Tyr antigens may comprise common proteins.

The nucleic acid sequence encoding the consensus Tyr antigen can be optimized for codon usage and corresponding RNA transcripts. The nucleic acid encoding the consensus Tyr antigen can be codons and RNA optimized for expression. In some embodiments, the nucleic acid sequence encoding the consensus Tyr antigen can include a Kozak sequence (e.g., GCC ACC) to enhance translation efficiency. The nucleic acid encoding the consensus Tyr antigen can include multiple stop codons (e.g., TGA TGA) to enhance translation efficiency.

The nucleic acid encoding the consensus Tyr antigen may also encode an immunoglobulin E (IgE) leader sequence. The nucleic acid encoding the consensus Tyr antigen may 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 via a peptide bond. The nucleic acid encoding the consensus Tyr antigen may also include a nucleotide sequence encoding the IgE leader sequence. In some embodiments, the nucleic acid encoding the consensus Tyr antigen does not contain or does not contain a nucleotide sequence encoding an IgE leader sequence.

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 the 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 contain or be linked to an IgE leader sequence and/or an HA tag.

In some embodiments, the consensus Tyr 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 shown in SEQ ID NO: 1. In other embodiments, the consensus Tyr 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 shown in SEQ ID NO: 2. The consensus Tyr antigen can be 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 shown in SEQ ID NO:2.

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 encoding 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 can be provided. Similarly, nucleic acid sequences encoding immunogenic fragments as described herein and immunogenic fragments of proteins homologous to the proteins described herein are also provided.

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

Some embodiments relate to nucleic acid sequences encoding proteins having a specified percentage identity to a full-length Tyr consensus protein, immunogenic fragments of a Tyr consensus protein, and immunogenic fragments of a protein having identity to a 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 Tyr 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. Likewise, nucleic acid sequences encoding immunogenic fragments set forth herein and immunogenic fragments of proteins having similar percentage identities to the Tyr proteins set forth herein as specified above are also provided.

In some embodiments, the nucleic acid sequence does not contain a coding sequence that encodes a leader sequence. In some embodiments, the nucleic acid sequence does not contain a coding sequence that encodes an IgE leader sequence.

Some embodiments relate to fragments of SEQ ID NO: 1. The fragments 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% of SEQ ID NO: 1. The fragments can have at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology to the fragments of SEQ ID NO: 1. The fragments can have 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% identity to the fragments of SEQ ID NO: 1. In some embodiments, the fragment includes a sequence encoding a leader sequence, such as, for example, an immunoglobulin leader sequence, such as an IgE leader sequence. In some embodiments, the fragment does not contain a coding sequence encoding a leader sequence. In some embodiments, the fragment does not contain a coding sequence encoding a leader sequence, such as, for example, an IgE leader sequence.

In addition, 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 the IgE leader sequence is SEQ ID NO: 2. The amino acid sequence of the consensus Tyr protein linked to the IgE leader sequence can be linked to an HA tag.

Some embodiments relate to proteins that are homologous to SEQ ID NO: 2. Some embodiments relate to immunogenic proteins that are 95% homologous to the consensus protein sequence shown in SEQ ID NO: 2. Some embodiments relate to immunogenic proteins that are 96% homologous to the consensus protein sequence shown in SEQ ID NO: 2. Some embodiments relate to immunogenic proteins that are 97% homologous to the consensus protein sequence shown in SEQ ID NO: 2. Some embodiments relate to immunogenic proteins that are 98% homologous to the consensus protein sequence shown in SEQ ID NO: 2. Some embodiments relate to immunogenic proteins that are 99% homologous to the consensus protein sequence shown in SEQ ID NO: 2.

Some embodiments relate to proteins identical to SEQ ID NO: 2. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 80% identical to the full-length consensus amino acid sequence as shown in SEQ ID NO: 2. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 85% identical to the full-length consensus amino acid sequence as shown in SEQ ID NO: 2. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 90% identical to the full-length consensus amino acid sequence as shown in SEQ ID NO: 2. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 91% identical to the full-length consensus amino acid sequence as shown in SEQ ID NO: 2. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 92% identical to the full-length consensus amino acid sequence as shown in SEQ ID NO: 2. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 93% identical to the full-length consensus amino acid sequence as shown in SEQ ID NO: 2. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 94% identical to the full-length consensus amino acid sequence as shown in SEQ ID NO: 2. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 95% identical to the full-length consensus amino acid sequence as shown 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 as shown 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 as shown 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 as shown 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 as shown in SEQ ID NO: 2.

In some embodiments, the protein does not contain a leader sequence. In some embodiments, the protein does not contain an IgE leader sequence. A fragment of the consensus protein may 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 may be provided. The immunogenic fragment may 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, such as, for example, an immunoglobulin leader sequence, such as an IgE leader sequence. In some embodiments, the fragment does not contain a leader sequence. In some embodiments, the fragment does not contain a leader sequence, such as, for example, an IgE leader sequence.

Immunogenic fragments of proteins having amino acid sequences homologous to immunogenic fragments of SEQ ID NO: 2 can be provided. 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 having 95% or greater homology 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 immunogenic fragments of the consensus protein sequences herein. Some embodiments relate to immunogenic fragments having 99% homology to immunogenic fragments of the consensus protein sequences herein. In some embodiments, the fragments include a leader sequence, such as, for example, an immunoglobulin leader sequence, such as an IgE leader sequence. In some embodiments, the fragments do not contain a leader sequence. In some embodiments, the fragments do not contain a leader sequence, such as, for example, an IgE leader sequence.

Immunogenic fragments of proteins having an amino acid sequence identical to an immunogenic fragment of SEQ ID NO: 2 can be provided. 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 having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the fragments include a leader sequence, such as, for example, an immunoglobulin leader sequence, such as an IgE leader sequence. In some embodiments, the fragments do not contain a leader sequence. In some embodiments, the fragment does not contain a leader sequence, such as, for example, an IgE leader sequence.

As indicated herein with respect to linking 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 that is encoded by the start codon of the nucleic acid sequence encoding the protein without the signal peptide coding sequence.

(2) Tyrosinase-related protein 1 (TYRP1)

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

As described above for tyrosinase, tyrosinase-related protein 1 (TYRP-1) is also involved in the synthesis of melanin and pigment machinery in melanocytes and can be recognized by the immune system of subjects with melanoma. Therefore, TYRP-1 can be an antigen associated with melanoma.

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

TYRP-1 antigens may comprise protein epitopes that make them particularly effective as immunogens against which an anti-TYRP-1 immune response can be induced. TYRP-1 antigens may comprise full-length translation products, variants thereof, fragments thereof, or combinations thereof. TYRP-1 antigens may comprise consensus proteins.

The nucleic acid sequence encoding the consensus TYRP-1 antigen can be optimized for codon usage and corresponding RNA transcripts. The nucleic acid encoding the consensus TYRP-1 antigen can be codon and RNA optimized for expression. In some embodiments, the nucleic acid sequence encoding the consensus TYRP-1 antigen may include a Kozak sequence (e.g., GCC ACC) to enhance the efficiency of translation. The nucleic acid encoding the consensus TYRP-1 antigen may include multiple stop codons (e.g., TGA TGA) to enhance the efficiency of translation termination.

The nucleic acid encoding the consensus TYRP-1 antigen may also encode an immunoglobulin E (IgE) leader sequence. The nucleic acid encoding the consensus TYRP-1 antigen may 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 may also include a nucleotide sequence encoding the IgE leader sequence. In some embodiments, the nucleic acid encoding the consensus TYRP-1 antigen does not contain or contains a nucleotide sequence encoding an IgE leader sequence.

The consensus TYRP-1 antigen can be the nucleic acid sequence of SEQ ID NO:3, which encodes the amino acid sequence of SEQ ID NO:4. SEQ ID NO:3 encodes the consensus TYRP-1 protein linked to an IgE leader sequence. The consensus TYRP-1 protein can be linked to an IgE leader sequence and an HA tag. In other embodiments, the consensus TYRP-1 protein may not contain or be linked to an IgE leader sequence and/or an HA tag.

In some embodiments, the consensus TYRP-1 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 shown in SEQ ID NO: 3. In other embodiments, the consensus TYRP-1 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 shown in SEQ ID NO: 4. The consensus TYRP-1 antigen can be 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 shown in SEQ ID NO:4.

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 encoding 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 can be provided. Similarly, nucleic acid sequences encoding immunogenic fragments as described herein and immunogenic fragments of proteins homologous to the proteins described herein are also provided.

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

Some embodiments relate to nucleic acid sequences encoding proteins having a specified percentage identity to a full-length TYRP-1 consensus protein, immunogenic fragments of a TYRP-1 consensus protein, and immunogenic fragments of a protein having identity to a TYRP-1 consensus protein. Such nucleic acid molecules can be provided that 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. Likewise, proteins encoding the immunogenic fragments set forth herein and immunogenic fragments of proteins having similar percent identities to the TYRP-1 proteins set forth herein as specified above are also provided.

In some embodiments, the nucleic acid sequence does not contain a coding sequence that encodes a leader sequence. In some embodiments, the nucleic acid sequence does not contain a coding sequence that encodes an IgE leader sequence.

Some embodiments relate to fragments of SEQ ID NO: 3. The fragments 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% of SEQ ID NO: 3. The fragments can have at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology to the fragments of SEQ ID NO: 3. The fragments can have 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% identity to the fragments of SEQ ID NO: 3. In some embodiments, the fragment includes a sequence encoding a leader sequence, such as, for example, an immunoglobulin leader sequence, such as an IgE leader sequence. In some embodiments, the fragment does not contain a coding sequence encoding a leader sequence. In some embodiments, the fragment does not contain a coding sequence encoding a leader sequence, such as, for example, an IgE leader sequence.

In addition, 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 the IgE leader sequence is SEQ ID NO: 4. The amino acid sequence of the consensus TYRP-1 protein linked to the IgE leader sequence can be linked to an HA tag.

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 shown in SEQ ID NO: 4. Some embodiments relate to immunogenic proteins having 96% homology to the consensus protein sequence shown in SEQ ID NO: 4. Some embodiments relate to immunogenic proteins having 97% homology to the consensus protein sequence shown in SEQ ID NO: 4. Some embodiments relate to immunogenic proteins having 98% homology to the consensus protein sequence shown in SEQ ID NO: 4. Some embodiments relate to immunogenic proteins having 99% homology to the consensus protein sequence shown in SEQ ID NO: 4.

Some embodiments relate to a protein 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 as shown 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 as shown 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 as shown 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 as shown 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 as shown 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 as shown in SEQ ID NO: 4. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 94% identical to the full-length consensus amino acid sequence as shown in SEQ ID NO: 4. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 95% identical to the full-length consensus amino acid sequence as shown 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 as shown 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 as shown 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 as shown 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 as shown in SEQ ID NO: 4.

In some embodiments, the protein does not contain a leader sequence. In some embodiments, the protein does not contain an IgE leader sequence. A fragment of the consensus protein may 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. An immunogenic fragment of SEQ ID NO: 4 may be provided. The immunogenic fragment may 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, such as, for example, an immunoglobulin leader sequence, such as an IgE leader sequence. In some embodiments, the fragment does not contain a leader sequence. In some embodiments, the fragment does not contain a leader sequence, such as, for example, an IgE leader sequence.

Immunogenic fragments of proteins having amino acid sequences homologous to immunogenic fragments of SEQ ID NO: 4 can be provided. 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 having 95% or greater homology 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 immunogenic fragments of the consensus protein sequences herein. Some embodiments relate to immunogenic fragments having 99% homology to immunogenic fragments of the consensus protein sequences herein. In some embodiments, the fragments include a leader sequence, such as, for example, an immunoglobulin leader sequence, such as an IgE leader sequence. In some embodiments, the fragments do not contain a leader sequence. In some embodiments, the fragments do not contain a leader sequence, such as, for example, an IgE leader sequence.

Immunogenic fragments of proteins having an amino acid sequence identical to an immunogenic fragment of SEQ ID NO: 4 can be provided. 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 having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence shown in SEQ ID NO: 4. In some embodiments, the fragments include a leader sequence, such as, for example, an immunoglobulin leader sequence, such as an IgE leader sequence. In some embodiments, the fragments do not contain a leader sequence. In some embodiments, the fragment does not contain a leader sequence, such as, for example, an IgE leader sequence.

As indicated herein with respect to linking 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 that is encoded by the start codon of the nucleic acid sequence encoding the protein without the signal peptide coding sequence.

(3) Tyrosinase-related protein 2 (TYRP2)

The vaccine of the present invention may comprise the cancer antigen tyrosinase-related protein 2 (TYRP2; also known as dopachrome tautomerase (DCT)), a fragment thereof, or a variant thereof. TYRP2/DCT, encoded by the TYRP2/DCT gene, is a protein composed of 519 amino acids and is expressed in normal and malignant melanocytes and melanoma cells. TYRP2/DCT is a well-characterized melanocyte-specific enzyme that binds to tyrosinase and TYRP1 in melanocytes and plays a role in the conversion of L-tyrosine to melanin. 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 discussed 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. As TYRP2/DCT is frequently reported to be highly expressed in melanoma, this melanocyte-specific enzyme plays an important role in contributing to the intrinsic resistance phenotype of melanoma to various anticancer DNA-damaging drugs.

As described above for tyrosinase, tyrosinase-related protein 2 (TYRP-2) is also involved in the synthesis of melanin and can be recognized by the immune system of subjects with melanoma. In addition, TYRP-2 can mediate drug resistance in melanoma cells. Therefore, TYRP-2 may be an antigen associated with melanoma.

The TRYP-2 antigen can induce antigen-specific T cells and/or high-titer antibody responses, thereby inducing or triggering an immune response to a cancer or tumor expressing the antigen or to the cancer or tumor reaction. In some embodiments, the induced or triggered immune response can be a cellular immune response, a humoral immune response, or a cellular and humoral immune response. In some embodiments, the induced or triggered cellular immune response can include the induction or secretion of interferon-γ (IFN-γ) and/or tumor necrosis factor α (TNF-α). In other embodiments, the induced or triggered immune response can reduce or inhibit one or more immunosuppressive factors that promote the growth of tumors or cancers expressing the antigen, for example, but not limited to factors that downregulate MHC presentation, factors that upregulate antigen-specific regulatory T cells (Treg), PD-L1, FasL, cytokines such as IL-10 and TFG-β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by immunosuppressive cells, CTLA-4, PD-1, MDSC, MCP-1, and immune checkpoint molecules, which are described in more detail below.

TYRP2 antigens may comprise protein epitopes that make them particularly effective as immunogens against which an anti-TYRP2 immune response can be induced. TYRP2 antigens may comprise full-length translation products, variants thereof, fragments thereof, or combinations thereof. TYRP2 antigens may comprise a consensus protein.

The nucleic acid sequence encoding the consensus TYRP2 antigen can be optimized for codon usage and corresponding RNA transcripts. The nucleic acid encoding the consensus TYRP2 antigen can be codons and RNA optimized for expression. In some embodiments, the nucleic acid sequence encoding the consensus TYRP2 antigen may include a Kozak sequence (e.g., GCC ACC) to enhance the efficiency of translation. The nucleic acid encoding the consensus TYRP2 antigen may include multiple stop codons (e.g., TGA TGA) to enhance the efficiency of translation termination.

The nucleic acid encoding the consensus TYRP2 antigen may also encode an immunoglobulin E (IgE) leader sequence. The nucleic acid encoding the consensus TYRP2 antigen may 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 TYRP2 antigen by a peptide bond. The nucleic acid encoding the consensus TYRP2 antigen may also include a nucleotide sequence encoding the IgE leader sequence. In some embodiments, the nucleic acid encoding the consensus TYRP2 antigen does not contain or does not contain a nucleotide sequence encoding an IgE leader sequence.

The consensus TYRP2 antigen can be the nucleic acid sequence SEQ ID NO: 5, which encodes the amino acid sequence SEQ ID NO: 6. SEQ ID NO: 5 encodes the consensus TYRP2 protein linked to an IgE leader sequence. The consensus TYRP2 protein can be linked to an IgE leader sequence and an HA tag. In other embodiments, the consensus TYRP2 protein may not contain or be linked to an IgE leader sequence and/or an HA tag.

In some embodiments, the consensus TYRP2 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 shown in SEQ ID NO: 5. In other embodiments, the consensus TYRP2 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 shown in SEQ ID NO: 6. The consensus TYRP2 antigen can be 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 shown in SEQ ID NO:6.

Some embodiments relate to nucleic acid sequences encoding proteins homologous to the TYRP2 consensus protein, immunogenic fragments of the TYRP2 consensus protein, and immunogenic fragments of homologous proteins. Such nucleic acid molecules encoding 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 can be provided. Similarly, nucleic acid sequences encoding immunogenic fragments as described herein and immunogenic fragments of proteins homologous to the proteins described herein are also provided.

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

Some embodiments relate to nucleic acid sequences encoding proteins having a specified percentage identity to a full-length TYRP2 consensus protein, immunogenic fragments of a TYRP2 consensus protein, and immunogenic fragments of a protein having identity to a TYRP2 consensus protein. Such nucleic acid molecules can be provided that encode immunogenic proteins having up to 80% identity to the full-length TYRP2 consensus sequence, up to 85% identity to the full-length consensus sequence, up to 90% identity to the full-length TYRP2 consensus sequence, up to 91% identity to the full-length TYRP2 consensus sequence, up to 92% identity to the full-length TYRP2 consensus sequence, up to 93% identity to the full-length TYRP2 consensus sequence, up to 94% identity to the full-length TYRP2 consensus sequence, up to 95% identity to the full-length TYRP2 consensus sequence, up to 96% identity to the full-length TYRP2 consensus sequence, up to 97% identity to the full-length TYRP2 consensus sequence, up to 98% identity to the full-length TYRP2 consensus sequence, and up to 99% identity to the full-length TYRP2 consensus sequence. Likewise, nucleic acid sequences encoding immunogenic fragments set forth herein and immunogenic fragments of proteins having similar percentage identities to the TYRP2 proteins set forth herein as specified above are also provided.

In some embodiments, the nucleic acid sequence does not contain a coding sequence that encodes a leader sequence. In some embodiments, the nucleic acid sequence does not contain a coding sequence that encodes an IgE leader sequence.

Some embodiments relate to fragments of SEQ ID NO: 5. The fragments 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% of SEQ ID NO: 5. The fragments can have at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology to the fragments of SEQ ID NO: 5. The fragments can have 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% identity to the fragments of SEQ ID NO: 5. In some embodiments, the fragment includes a sequence encoding a leader sequence, such as, for example, an immunoglobulin leader sequence, such as an IgE leader sequence. In some embodiments, the fragment does not contain a coding sequence encoding a leader sequence. In some embodiments, the fragment does not contain a coding sequence encoding a leader sequence, such as, for example, an IgE leader sequence.

In addition, the amino acid sequence of the consensus TYRP2 protein is SEQ ID NO: 6. The amino acid sequence of the consensus TYRP2 protein linked to the IgE leader sequence is SEQ ID NO: 6. The amino acid sequence of the consensus TYRP2 protein linked to the IgE leader sequence can be linked to an HA tag.

Some embodiments relate to proteins that are homologous to SEQ ID NO: 2. Some embodiments relate to immunogenic proteins that are 95% homologous to the consensus protein sequence shown in SEQ ID NO: 6. Some embodiments relate to immunogenic proteins that are 96% homologous to the consensus protein sequence shown in SEQ ID NO: 6. Some embodiments relate to immunogenic proteins that are 97% homologous to the consensus protein sequence shown in SEQ ID NO: 6. Some embodiments relate to immunogenic proteins that are 98% homologous to the consensus protein sequence shown in SEQ ID NO: 6. Some embodiments relate to immunogenic proteins that are 99% homologous to the consensus protein sequence shown in SEQ ID NO: 6.

Some embodiments relate to proteins identical to SEQ ID NO: 6. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 80% identical to the full-length consensus amino acid sequence as shown in SEQ ID NO: 6. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 85% identical to the full-length consensus amino acid sequence as shown in SEQ ID NO: 6. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 90% identical to the full-length consensus amino acid sequence as shown in SEQ ID NO: 6. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 91% identical to the full-length consensus amino acid sequence as shown in SEQ ID NO: 6. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 92% identical to the full-length consensus amino acid sequence as shown in SEQ ID NO: 6. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 93% identical to the full-length consensus amino acid sequence as shown in SEQ ID NO: 6. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 94% identical to the full-length consensus amino acid sequence as shown in SEQ ID NO: 6. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 95% identical to the full-length consensus amino acid sequence as shown 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 as shown 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 as shown 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 as shown 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 as shown in SEQ ID NO: 6.

In some embodiments, the protein does not contain a leader sequence. In some embodiments, the protein does not contain an IgE leader sequence. 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. The immunogenic fragment may 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, such as, for example, an immunoglobulin leader sequence, such as an IgE leader sequence. In some embodiments, the fragment does not contain a leader sequence. In some embodiments, the fragment does not contain a leader sequence, such as, for example, an IgE leader sequence.

Immunogenic fragments of proteins having amino acid sequences homologous to immunogenic fragments of SEQ ID NO: 6 can be provided. 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 having 95% or greater homology 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 immunogenic fragments of the consensus protein sequences herein. Some embodiments relate to immunogenic fragments having 99% homology to immunogenic fragments of the consensus protein sequences herein. In some embodiments, the fragments include a leader sequence, such as, for example, an immunoglobulin leader sequence, such as an IgE leader sequence. In some embodiments, the fragments do not contain a leader sequence. In some embodiments, the fragments do not contain a leader sequence, such as, for example, an IgE leader sequence.

Immunogenic fragments of proteins having an amino acid sequence identical to an immunogenic fragment of SEQ ID NO: 6 can be provided. 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 having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence shown in SEQ ID NO: 6. In some embodiments, the fragments include a leader sequence, such as, for example, an immunoglobulin leader sequence, such as an IgE leader sequence. In some embodiments, the fragments do not contain a leader sequence. In some embodiments, the fragment does not contain a leader sequence, such as, for example, an IgE leader sequence.

As indicated herein with respect to linking 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 that is encoded by the start codon of the nucleic acid sequence encoding the protein without the signal peptide coding sequence.

(4) Melanoma-associated antigen 4 (MAGEA4)

The vaccine of the present invention may comprise the cancer antigen melanoma-associated antigen 4 (MAGEA4), a fragment thereof, or a variant thereof. MAGE-A4, encoded by the MAGE-A4 gene, is a 317-amino acid protein expressed in male germ cells and in tumor cells of various histological types, such as gastrointestinal, esophageal, and lung cancers. MAGE-A4 binds to the oncoprotein Gankyrin. This specific binding of MAGE-A4 is mediated by its C-terminus. Studies have shown that exogenous MAGE-A4 can partially inhibit the anchorage-independent growth of cells overexpressing Gankyrin in vitro and inhibit the formation of migratory tumors from these cells in nude mice. This inhibition is dependent on the binding between MAGE-A4 and Gankyrin, suggesting that the interaction between Gankyrin and MAGE-A4 inhibits Gankyrin-mediated carcinogenesis. MAGE expression in tumor tissue may not be a cause, but rather a consequence of tumorigenesis, and that MAGE genes participate in immune processes by targeting early-stage tumor cells for destruction.

Melanoma-associated antigen 4 (MAGEA4) protein may be involved in embryonic development as well as 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 a variety of tumors.

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

MAGEA4 antigens may comprise protein epitopes that make them particularly effective as immunogens against which an anti-MAGEA4 immune response can be induced. MAGEA4 antigens may comprise full-length translation products, variants thereof, fragments thereof, or combinations thereof. MAGEA4 antigens may comprise a consensus protein.

The nucleic acid sequence encoding the consensus MAGEA4 antigen can be optimized for codon usage and corresponding RNA transcripts. The nucleic acid encoding the consensus MAGEA4 antigen can be codon and RNA optimized for expression. In some embodiments, the nucleic acid sequence encoding the consensus MAGEA4 antigen can include a Kozak sequence (e.g., GCC ACC) to enhance translation efficiency. The nucleic acid encoding the consensus MAGEA4 antigen can include multiple stop codons (e.g., TGA TGA) to enhance translation termination efficiency.

The nucleic acid encoding the consensus MAGEA4 antigen may also encode an immunoglobulin E (IgE) leader sequence. The nucleic acid encoding the consensus Tyr antigen may 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 via a peptide bond. The nucleic acid encoding the consensus MAGEA4 antigen may also include a nucleotide sequence encoding the IgE leader sequence. In some embodiments, the nucleic acid encoding the consensus MAGEA4 antigen does not contain or contains a nucleotide sequence encoding the IgE leader sequence.

The consensus MAGEA4 antigen can 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 can be linked to an IgE leader sequence and an HA tag. In other embodiments, the consensus MAGEA4 protein may not contain or be linked to an IgE leader sequence and/or an HA tag.

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 shown 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 shown in SEQ ID NO: 8. The consensus MAGEA4 antigen can be 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 shown in SEQ ID NO:8.

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 as described herein and immunogenic fragments of proteins homologous to the proteins described herein are also provided.

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

Some embodiments relate to nucleic acid sequences encoding proteins having a specified percentage identity to the full-length MAGEA4 consensus protein, immunogenic fragments of the MAGEA4 consensus protein, and immunogenic fragments of proteins having identity to the MAGEA4 consensus protein. Such nucleic acid molecules can be provided that encode immunogenic proteins having up to 80% identity to the full-length MAGEA4 consensus sequence, up to 85% identity to the full-length 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. Likewise, nucleic acid sequences encoding immunogenic fragments set forth herein and immunogenic fragments of proteins having similar percentage identities to the MAGEA4 proteins set forth herein as specified above are also provided.

In some embodiments, the nucleic acid sequence does not contain a coding sequence that encodes a leader sequence. In some embodiments, the nucleic acid sequence does not contain a coding sequence that encodes an IgE leader sequence.

Some embodiments relate to fragments of SEQ ID NO: 7. The fragments 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% of SEQ ID NO: 7. The fragments can have at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology to the fragments of SEQ ID NO: 7. The fragments can have 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% identity to the fragments of SEQ ID NO: 7. In some embodiments, the fragment includes a sequence encoding a leader sequence, such as, for example, an immunoglobulin leader sequence, such as an IgE leader sequence. In some embodiments, the fragment does not contain a coding sequence encoding a leader sequence. In some embodiments, the fragment does not contain a coding sequence encoding a leader sequence, such as, for example, an IgE leader sequence.

In addition, 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 the IgE leader sequence is SEQ ID NO: 8. The amino acid sequence of the consensus MAGEA4 protein linked to the IgE leader sequence can be linked to an HA tag.

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 shown in SEQ ID NO: 8. Some embodiments relate to immunogenic proteins having 96% homology to the consensus protein sequence shown in SEQ ID NO: 8. Some embodiments relate to immunogenic proteins having 97% homology to the consensus protein sequence shown in SEQ ID NO: 8. Some embodiments relate to immunogenic proteins having 98% homology to the consensus protein sequence shown in SEQ ID NO: 8. Some embodiments relate to immunogenic proteins having 99% homology to the consensus protein sequence shown in SEQ ID NO: 8.

Some embodiments relate to proteins identical to SEQ ID NO: 8. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 80% identical to the full-length consensus amino acid sequence as shown in SEQ ID NO: 8. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 85% identical to the full-length consensus amino acid sequence as shown in SEQ ID NO: 8. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 90% identical to the full-length consensus amino acid sequence as shown in SEQ ID NO: 8. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 91% identical to the full-length consensus amino acid sequence as shown in SEQ ID NO: 8. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 92% identical to the full-length consensus amino acid sequence as shown in SEQ ID NO: 8. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 93% identical to the full-length consensus amino acid sequence as shown in SEQ ID NO: 8. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 94% identical to the full-length consensus amino acid sequence as shown in SEQ ID NO: 8. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 95% identical to the full-length consensus amino acid sequence as shown 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 as shown 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 as shown 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 as shown 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 as shown in SEQ ID NO: 8.

In some embodiments, the protein does not contain a leader sequence. In some embodiments, the protein does not contain an IgE leader sequence. 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. The immunogenic fragment may 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, such as, for example, an immunoglobulin leader sequence, such as an IgE leader sequence. In some embodiments, the fragment does not contain a leader sequence. In some embodiments, the fragment does not contain a leader sequence, such as, for example, an IgE leader sequence.

Immunogenic fragments of proteins having amino acid sequences homologous to immunogenic fragments of SEQ ID NO: 8 can be provided. 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 having 95% or greater homology 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 immunogenic fragments of the consensus protein sequences herein. Some embodiments relate to immunogenic fragments having 99% homology to immunogenic fragments of the consensus protein sequences herein. In some embodiments, the fragments include a leader sequence, such as, for example, an immunoglobulin leader sequence, such as an IgE leader sequence. In some embodiments, the fragments do not contain a leader sequence. In some embodiments, the fragments do not contain a leader sequence, such as, for example, an IgE leader sequence.

Immunogenic fragments of proteins having an amino acid sequence identical to an immunogenic fragment of SEQ ID NO: 8 can be provided. 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 having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence shown in SEQ ID NO: 8. In some embodiments, the fragments include a leader sequence, such as, for example, an immunoglobulin leader sequence, such as an IgE leader sequence. In some embodiments, the fragments do not contain a leader sequence. In some embodiments, the fragment does not contain a leader sequence, such as, for example, an IgE leader sequence.

As indicated herein with respect to linking 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 that is encoded by the start codon of the nucleic acid sequence encoding the protein without the signal peptide coding sequence.

(5) Growth hormone-releasing hormone (GHRH)

The vaccines of the present invention may comprise the cancer antigen growth hormone-releasing hormone (GHRH; also known as growth-hormone-releasing factor (GRF or GHRF) or somatokinin), 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, a regulator of growth, metabolism, and body structure, from the pituitary gland. GHRH also stimulates the production of growth hormone. Antagonists of GHRH can inhibit the growth of a variety of cancers, such as osteosarcoma, glioblastoma, prostate cancer, kidney cancer, pancreatic cancer, colorectal cancer, and breast cancer. Therefore, GHRH may be an antigen associated with a variety of tumors.

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

GHRH antigens may comprise protein epitopes that make them particularly effective as immunogens against which an anti-GHRH immune response can be induced. GHRH antigens may comprise full-length translation products, variants thereof, fragments thereof, or combinations thereof. GHRH antigens may comprise a consensus protein.

The nucleic acid sequence encoding the consensus GHRH antigen can be optimized for codon usage and corresponding RNA transcripts. The nucleic acid encoding the consensus GHRH antigen can be codons 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 enhance the efficiency of translation. The nucleic acid encoding the consensus GHRH antigen can include multiple stop codons (e.g., TGA TGA) to enhance the efficiency of translation termination.

The nucleic acid encoding the consensus GHRH antigen may also encode an immunoglobulin E (IgE) leader sequence. The nucleic acid encoding the consensus GHRH antigen may 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 via a peptide bond. The nucleic acid encoding the consensus GHRH antigen may also include a nucleotide sequence encoding the IgE leader sequence. In some embodiments, the nucleic acid encoding the consensus GHRH antigen does not contain or does not contain a nucleotide sequence encoding the IgE leader sequence.

The consensus GHRH antigen can be the nucleic acid sequence of SEQ ID NO:9, which encodes the amino acid sequence of SEQ ID NO:10. SEQ ID NO:9 encodes the consensus GHRH protein linked to an IgE leader sequence. The consensus GHRH protein can be linked to an IgE leader sequence and an HA tag. In other embodiments, the consensus GHRH protein may not contain or be linked to an IgE leader sequence and/or an HA tag.

In some embodiments, the consensus GHRH 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 shown in SEQ ID NO: 9. In other embodiments, the consensus GHRH 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 shown in SEQ ID NO: 10. The consensus GHRH antigen can be 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 shown in SEQ ID NO: 10.

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 encoding 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 can be provided. 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.

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

Some embodiments relate to nucleic acid sequences encoding proteins having a specified percentage identity to a full-length GHRH consensus protein, immunogenic fragments of a GHRH consensus protein, and immunogenic fragments of a protein having identity to a GHRH consensus protein. Such nucleic acid molecules can be provided that encode immunogenic proteins having up to 80% identity to the full-length GHRH consensus sequence, up to 85% identity to the full-length 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 set forth herein and immunogenic fragments of proteins having similar percentage identities to the GHRH proteins set forth herein as specified above are also provided.

In some embodiments, the nucleic acid sequence does not contain a coding sequence that encodes a leader sequence. In some embodiments, the nucleic acid sequence does not contain a coding sequence that encodes an IgE leader sequence.

Some embodiments relate to fragments of SEQ ID NO: 9. The fragments 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% of SEQ ID NO: 9. The fragments can have at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology to the fragments of SEQ ID NO: 9. The fragments can have 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% identity to the fragments of SEQ ID NO: 9. In some embodiments, the fragment includes a sequence encoding a leader sequence, such as, for example, an immunoglobulin leader sequence, such as an IgE leader sequence. In some embodiments, the fragment does not contain a coding sequence encoding a leader sequence. In some embodiments, the fragment does not contain a coding sequence encoding a leader sequence, such as, for example, an IgE leader sequence.

In addition, 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 the IgE leader sequence is SEQ ID NO: 10. The amino acid sequence of the consensus GHRH protein linked to the IgE leader sequence can be linked to an HA tag.

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 shown in SEQ ID NO: 10. Some embodiments relate to immunogenic proteins having 96% homology to the consensus protein sequence shown in SEQ ID NO: 10. Some embodiments relate to immunogenic proteins having 97% homology to the consensus protein sequence shown in SEQ ID NO: 10. Some embodiments relate to immunogenic proteins having 98% homology to the consensus protein sequence shown in SEQ ID NO: 10. Some embodiments relate to immunogenic proteins having 99% homology to the consensus protein sequence shown in SEQ ID NO: 10.

Some embodiments relate to proteins identical to SEQ ID NO: 10. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 80% identical to the full-length consensus amino acid sequence as shown in SEQ ID NO: 10. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 85% identical to the full-length consensus amino acid sequence as shown in SEQ ID NO: 10. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 90% identical to the full-length consensus amino acid sequence as shown in SEQ ID NO: 10. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 91% identical to the full-length consensus amino acid sequence as shown in SEQ ID NO: 10. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 92% identical to the full-length consensus amino acid sequence as shown in SEQ ID NO: 10. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 93% identical to the full-length consensus amino acid sequence as shown in SEQ ID NO: 10. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 94% identical to the full-length consensus amino acid sequence as shown 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 as shown 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 as shown 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 as shown 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 as shown 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 as shown in SEQ ID NO: 10.

In some embodiments, the protein does not contain a leader sequence. In some embodiments, the protein does not contain an IgE leader sequence. 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. The immunogenic fragment may 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, such as, for example, an immunoglobulin leader sequence, such as an IgE leader sequence. In some embodiments, the fragment does not contain a leader sequence. In some embodiments, the fragment does not contain a leader sequence, such as, for example, an IgE leader sequence.

Immunogenic fragments of proteins having amino acid sequences homologous to immunogenic fragments of SEQ ID NO: 10 can be provided. 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 having 95% or greater homology 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 an immunogenic fragment of a consensus protein sequence herein. Some embodiments relate to immunogenic fragments having 99% homology to an immunogenic fragment of a consensus protein sequence herein. In some embodiments, the fragment includes a leader sequence, such as, for example, an immunoglobulin leader sequence, such as an IgE leader sequence. In some embodiments, the fragment does not contain a leader sequence. In some embodiments, the fragment does not contain a leader sequence, such as, for example, an IgE leader sequence.

Immunogenic fragments of proteins having an amino acid sequence identical to an immunogenic fragment of SEQ ID NO: 10 can be provided. 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 having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence shown in SEQ ID NO: 10. In some embodiments, the fragments include a leader sequence, such as, for example, an immunoglobulin leader sequence, such as an IgE leader sequence. In some embodiments, the fragments do not contain a leader sequence. In some embodiments, the fragment does not contain a leader sequence, such as, for example, an IgE leader sequence.

As indicated herein with respect to linking 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 that is encoded by the start codon of the nucleic acid sequence encoding the protein without the signal peptide coding sequence.

(6)MART-1/Melan-A

The vaccine of the present invention may comprise the cancer antigen MART-1 (also referred to as Melan-A), a fragment thereof or a variant thereof. MART-1, encoded by the MLANA gene, is a 118-amino acid protein containing a single transmembrane domain and is expressed in most melanoma cells. MART-1 forms a complex with structural proteins and affects the transport and processing required for its expression, stability, melanosome structure and maturation. Therefore, MART-1 is necessary for regulating pigmentation in mammals. Defects in melanosome maturation are associated with susceptibility to cancer. MART-1 can be expressed in many types of cancer (including but not limited to melanoma).

Melan-A, also known as melanoma antigen recognized by T cells (MART-1), is a melanocyte differentiation antigen and is found in normal skin, retina and melanocytes. Melan-a can be associated with the endoplasmic reticulum and melanosomes. Melan-A can be recognized by cytotoxic T cells as an antigen on melanoma cells, but can also be associated with other tumors such as clear cell sarcoma and melanin-type neurofibroma with melanocyte origin or differentiation (i.e., cells have melanosomes). Therefore, Melan-A can be an antigen associated with a variety of tumors derived from cells with melanosomes.

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

Melan-A antigens may comprise protein epitopes that make them particularly effective as immunogens against which an anti-Melan-A immune response can be induced. Melan-A antigens may comprise full-length translation products, variants thereof, fragments thereof, or combinations thereof. Melan-A antigens may comprise a consensus protein.

The nucleic acid sequence encoding the consensus Melan-A antigen can be optimized for codon usage and corresponding RNA transcripts. The nucleic acid encoding the consensus Melan-A antigen can be codons and RNA optimized for expression. In some embodiments, the nucleic acid sequence encoding the consensus Melan-A antigen can include a Kozak sequence (e.g., GCC ACC) to enhance the efficiency of translation. The nucleic acid encoding the consensus Melan-A antigen can include multiple stop codons (e.g., TGA TGA) to enhance the efficiency of translation termination.

The nucleic acid encoding the consensus Melan-A antigen may also encode an immunoglobulin E (IgE) leader sequence. The nucleic acid encoding the consensus Melan-A antigen may 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 via a peptide bond. The nucleic acid encoding the consensus Melan-A antigen may also include a nucleotide sequence encoding the IgE leader sequence. In some embodiments, the nucleic acid encoding the consensus Melan-A antigen does not contain or does not contain a nucleotide sequence encoding the IgE leader sequence.

The consensus Melan-A antigen can 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 can be linked to an IgE leader sequence and an HA tag. In other embodiments, the consensus Melan-A protein may not contain or be linked to an IgE leader sequence and/or an HA tag.

In some embodiments, the consensus Melan-A 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 shown in SEQ ID NO: 11. In other embodiments, the consensus Melan-A 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 shown in SEQ ID NO: 12. The consensus Melan-A antigen can be 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 shown in SEQ ID NO:12.

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 encoding 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 can be provided. 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.

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

Some embodiments relate to nucleic acid sequences encoding proteins having a specified percentage identity to a full-length Melan-A consensus protein, immunogenic fragments of a Melan-A consensus protein, and immunogenic fragments of proteins having identity to a Melan-A consensus protein. Such nucleic acid molecules can be provided that 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. Likewise, nucleic acid sequences encoding immunogenic fragments set forth herein and immunogenic fragments of proteins having similar percentage identities to the Melan-A proteins set forth herein as specified above are also provided.

In some embodiments, the nucleic acid sequence does not contain a coding sequence that encodes a leader sequence. In some embodiments, the nucleic acid sequence does not contain a coding sequence that encodes an IgE leader sequence.

Some embodiments relate to fragments of SEQ ID NO: 11. The fragments 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% of SEQ ID NO: 11. The fragments can be at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous to a fragment of SEQ ID NO: 11. The fragment may 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, such as, for example, an immunoglobulin leader sequence, such as an IgE leader sequence. In some embodiments, the fragment does not contain a coding sequence encoding a leader sequence. In some embodiments, the fragment does not contain a coding sequence encoding a leader sequence, such as, for example, an IgE leader sequence.

In addition, 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 the IgE leader sequence is SEQ ID NO: 12. The amino acid sequence of the consensus Melan-A protein linked to the IgE leader sequence can be linked to an HA tag.

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 shown in SEQ ID NO: 12. Some embodiments relate to immunogenic proteins having 96% homology to the consensus protein sequence shown in SEQ ID NO: 12. Some embodiments relate to immunogenic proteins having 97% homology to the consensus protein sequence shown in SEQ ID NO: 12. Some embodiments relate to immunogenic proteins having 98% homology to the consensus protein sequence shown in SEQ ID NO: 12. Some embodiments relate to immunogenic proteins having 99% homology to the consensus protein sequence shown in SEQ ID NO: 12.

Some embodiments relate to a protein 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 as shown 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 as shown 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 as shown 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 as shown 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 as shown 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 as shown in SEQ ID NO: 12. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 94% identical to the full-length consensus amino acid sequence as shown 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 as shown 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 as shown 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 as shown 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 as shown 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 as shown in SEQ ID NO: 12.

In some embodiments, the protein does not contain a leader sequence. In some embodiments, the protein does not contain an IgE leader sequence. 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. The immunogenic fragment may 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, such as, for example, an immunoglobulin leader sequence, such as an IgE leader sequence. In some embodiments, the fragment does not contain a leader sequence. In some embodiments, the fragment does not contain a leader sequence, such as, for example, an IgE leader sequence.

Immunogenic fragments of proteins having amino acid sequences homologous to immunogenic fragments of SEQ ID NO: 12 can be provided. 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 having 95% or greater homology 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 an immunogenic fragment of a consensus protein sequence herein. Some embodiments relate to immunogenic fragments having 99% homology to an immunogenic fragment of a consensus protein sequence herein. In some embodiments, the fragment includes a leader sequence, such as, for example, an immunoglobulin leader sequence, such as an IgE leader sequence. In some embodiments, the fragment does not contain a leader sequence. In some embodiments, the fragment does not contain a leader sequence, such as, for example, an IgE leader sequence.

Immunogenic fragments of proteins having an amino acid sequence identical to an immunogenic fragment of SEQ ID NO: 12 can be provided. 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 having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence shown in SEQ ID NO: 12. In some embodiments, the fragments include a leader sequence, such as, for example, an immunoglobulin leader sequence, such as an IgE leader sequence. In some embodiments, the fragments do not contain a leader sequence. In some embodiments, the fragment does not contain a leader sequence, such as, for example, an IgE leader sequence.

As indicated herein with respect to linking 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 that is encoded by the start codon of the nucleic acid sequence encoding the protein without the signal peptide coding sequence.

(7)NY-ESO-1

Vaccines of the present invention may comprise the cancer antigen New York Esophageal Cancer-1 (NY-ESO-1; also known as CTAG1), fragments thereof, or variants thereof. NY-ESO-1, encoded by the CTAG1B gene, is a 180-amino acid protein with a glycine-rich N-terminal region and an extremely hydrophobic C-terminal region. NY-ESO-1 has limited expression in normal tissues but is frequently found in cancer. NY-ESO-1 is expressed in a variety of cancers, including, but not limited to, bladder, colorectal, esophageal, gastric, liver, head and neck cancers, melanoma, non-small cell lung cancer, ovarian cancer, pancreatic cancer, synovial cancer, and prostate cancer.

Cancer-testis antigen (NY-ESO-1) is expressed in the testes and ovaries. NY-ESO-1 is associated with various cancers and can induce humoral immune responses. Subjects with cancer or tumors can develop immunogenicity against NY-ESO-1. Therefore, NY-ESO-1 may be an antigen associated with various tumors.

The NY-ESO-1 antigen can induce antigen-specific T cell and/or high-titer antibody responses, thereby inducing or eliciting an immune response against or reactive with 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-γ (IFN-γ) and/or tumor necrosis factor α (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 expressing the antigen, such as, but not limited to, factors that downregulate MHC presentation, factors that upregulate antigen-specific regulatory T cells (Tregs), PD-L1, FasL, cytokines such as IL-10 and TFG-β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by immunosuppressive cells, CTLA-4, PD-1, MDSC, MCP-1, and immune checkpoint molecules, which are described in more detail below.

The NY-ESO-1 antigen can enhance the cellular immune response of a subject administered with the NY-ESO-1 antigen by about 50-fold to about 6,000-fold, about 50-fold to about 5,500-fold, about 50-fold to about 5,000-fold, about 50-fold to about 4,500-fold, about 100-fold to about 6,000-fold, about 150-fold to about 6,000-fold, about 200-fold to about 6,000-fold, about 250-fold to about 6,000-fold, or about 300-fold to about 6,000-fold, compared to the cellular immune response of a subject not administered with the NY-ESO-1 antigen. In some embodiments, the NY-ESO-1 antigen can enhance the cellular immune response of a subject administered NY-ESO-1 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 more. 0 times, 2200 times, 2300 times, 2400 times, 2500 times, 2600 times, 2700 times, 2800 times, 2900 times, 3000 times, 3100 times, 3200 times, 3300 times, 3400 times, 3500 times, 3600 times, 3700 times, 3800 times, 3900 times, 4000 times, 4100 times, 4200 times, 4300 times, 4400 times, 4500 times, 4600 times, 4700 times, 4800 times, 4900 times, 5000 times, 5100 times, 5200 times, 5300 times, 5400 times, 5500 times, 5600 times, 5700 times, 5800 times, 5900 times or 6000 times.

The NY-ESO-1 antigen can increase interferon gamma (IFN-γ) levels in a subject administered with the NY-ESO-1 antigen by about 50-fold to about 6,000-fold, about 50-fold to about 5,500-fold, about 50-fold to about 5,000-fold, about 50-fold to about 4,500-fold, about 100-fold to about 6,000-fold, about 150-fold to about 6,000-fold, about 200-fold to about 6,000-fold, about 250-fold to about 6,000-fold, or about 300-fold to about 6,000-fold, compared to IFN-γ levels in a subject not administered with the NY-ESO-1 antigen. In some embodiments, the NY-ESO-1 antigen can increase IFN-γ levels in a subject administered with 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, 2000-fold, 2100-fold, 2200-fold, 2300-fold, 2400-fold, 2500-fold, 2600-fold, 2700-fold, 2800-fold, 2900-fold, 3000-fold, 3100-fold, 3200-fold, 3300-fold, 3400-fold, 3500-fold, 3600-fold, 3700-fold, 3800-fold, 3900-fold, 4000-fold, 4100-fold, 4200-fold, 4300-fold, 4400-fold 100 times, 2200 times, 2300 times, 2400 times, 2500 times, 2600 times, 2700 times, 2800 times, 2900 times, 3000 times, 3100 times, 3200 times, 3300 times, 3400 times, 3500 times, 3600 times, 3700 times, 3800 times, 3900 times, 4000 times, 4100 times, 4200 times, 4300 times, 4400 times, 4500 times, 4600 times, 4700 times, 4800 times, 4900 times, 5000 times, 5100 times, 5200 times, 5300 times, 5400 times, 5500 times, 5600 times, 5700 times, 5800 times, 5900 times or 6000 times.

NY-ESO-1 antigens may comprise protein epitopes that make them particularly effective as immunogens against which an anti-NY-ESO-1 immune response can be induced. NY-ESO-1 antigens may comprise full-length translation products, variants thereof, fragments thereof, or combinations thereof. NY-ESO-1 antigens may comprise consensus proteins.

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

The nucleic acid encoding the consensus NY-ESO-1 antigen may also encode an immunoglobulin E (IgE) leader sequence. The nucleic acid encoding the consensus NY-ESO-1 antigen may 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 via a peptide bond. The nucleic acid encoding the consensus NY-ESO-1 antigen may 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 contain or is not enriched with a nucleotide sequence encoding an IgE leader sequence.

The consensus NY-ESO-1 antigen can be the nucleic acid sequence of SEQ ID NO: 13, which encodes the amino acid sequence of 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 contain or be linked to an IgE leader sequence and/or an HA tag.

In some embodiments, the consensus NY-ESO-1 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 shown in SEQ ID NO: 13. In other embodiments, the consensus NY-ESO-1 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 shown in SEQ ID NO: 14. The consensus NY-ESO-1 antigen can be 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 shown in SEQ ID NO: 14.

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 as described herein and immunogenic fragments of proteins homologous to the proteins described herein are also provided.

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

Some embodiments relate to nucleic acid sequences encoding proteins having a specified percentage identity to a full-length NY-ESO-1 consensus protein, immunogenic fragments of a NY-ESO-1 consensus protein, and immunogenic fragments of proteins having identity to a NY-ESO-1 consensus protein. Such nucleic acid molecules can be provided that encode immunogenic proteins having up to 80% identity to the full-length NY-ESO-1 consensus sequence, up to 85% identity to the full-length 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, and up to 99% identity to the full-length NY-ESO-1 consensus sequence. Likewise, nucleic acid sequences encoding immunogenic fragments set forth herein and immunogenic fragments of proteins having similar percentage identities to the NY-ESO-1 proteins set forth herein as specified above are also provided.

In some embodiments, the nucleic acid sequence does not contain a coding sequence that encodes a leader sequence. In some embodiments, the nucleic acid sequence does not contain a coding sequence that encodes an IgE leader sequence.

Some embodiments relate to fragments of SEQ ID NO: 13. The fragments 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% of SEQ ID NO: 13. The fragments can be at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous to a fragment of SEQ ID NO: 13. The fragment may 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, such as, for example, an immunoglobulin leader sequence, such as an IgE leader sequence. In some embodiments, the fragment does not contain a coding sequence encoding a leader sequence. In some embodiments, the fragment does not contain a coding sequence encoding a leader sequence, such as, for example, an IgE leader sequence.

In addition, 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 the IgE leader sequence is SEQ ID NO: 14. The amino acid sequence of the consensus NY-ESO-1 protein linked to the IgE leader sequence can be linked to an HA tag.

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 shown in SEQ ID NO: 14. Some embodiments relate to immunogenic proteins having 96% homology to the consensus protein sequence shown in SEQ ID NO: 14. Some embodiments relate to immunogenic proteins having 97% homology to the consensus protein sequence shown in SEQ ID NO: 14. Some embodiments relate to immunogenic proteins having 98% homology to the consensus protein sequence shown in SEQ ID NO: 14. Some embodiments relate to immunogenic proteins having 99% homology to the consensus protein sequence shown in SEQ ID NO: 14.

Some embodiments relate to a protein 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 as shown 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 as shown 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 as shown 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 as shown 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 as shown 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 as shown in SEQ ID NO: 14. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 94% identical to the full-length consensus amino acid sequence as shown 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 as shown 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 as shown 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 as shown 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 as shown 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 as shown in SEQ ID NO: 14.

In some embodiments, the protein does not contain a leader sequence. In some embodiments, the protein does not contain an IgE leader sequence. 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. The immunogenic fragment may 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, such as, for example, an immunoglobulin leader sequence, such as an IgE leader sequence. In some embodiments, the fragment does not contain a leader sequence. In some embodiments, the fragment does not contain a leader sequence, such as, for example, an IgE leader sequence.

Immunogenic fragments of proteins having amino acid sequences homologous to immunogenic fragments of SEQ ID NO: 14 can be provided. 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 having 95% or greater homology 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 an immunogenic fragment of a consensus protein sequence herein. Some embodiments relate to immunogenic fragments having 99% homology to an immunogenic fragment of a consensus protein sequence herein. In some embodiments, the fragment includes a leader sequence, such as, for example, an immunoglobulin leader sequence, such as an IgE leader sequence. In some embodiments, the fragment does not contain a leader sequence. In some embodiments, the fragment does not contain a leader sequence, such as, for example, an IgE leader sequence.

Immunogenic fragments of proteins having an amino acid sequence identical to an immunogenic fragment of SEQ ID NO: 14 can be provided. 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 having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence shown in SEQ ID NO: 14. In some embodiments, the fragments include a leader sequence, such as, for example, an immunoglobulin leader sequence, such as an IgE leader sequence. In some embodiments, the fragments do not contain a leader sequence. In some embodiments, the fragment does not contain a leader sequence, such as, for example, an IgE leader sequence.

As indicated herein with respect to linking 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 that is encoded by the start codon of the nucleic acid sequence encoding the protein without the signal peptide coding sequence.

(8)NY-ESO-2

The vaccine of the present invention may comprise the cancer antigen New York Esophageal Cancer-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 is expressed in a variety of cancers, including melanoma, breast cancer, bladder cancer, and prostate cancer, and is typically expressed in testicular tissue. In addition, NY-ESO-2 can be observed in 25-50% of tumor samples from melanoma, non-small cell lung cancer, bladder, prostate, and head and neck cancers. The gene encoding NY-ESO-2 also contains an alternative open reading frame for a protein called CAMEL, a tumor antigen recognized by melanoma-specific cytotoxic T-lymphocytes.

Similar to NY-ESO-1, NY-ESO-2 is expressed in the testes and ovaries. NY-ESO-2 has also been associated with immunogenicity in various cancers and subjects with cancer or tumors. Therefore, NY-ESO-2 may be an antigen associated with many tumors.

The NY-ESO-2 antigen can induce antigen-specific T cell and/or high-titer antibody responses, thereby inducing or eliciting an immune response against or reactive with 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-γ (IFN-γ) and/or tumor necrosis factor α (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 expressing the antigen, such as, but not limited to, factors that downregulate MHC presentation, factors that upregulate antigen-specific regulatory T cells (Tregs), PD-L1, FasL, cytokines such as IL-10 and TFG-β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by immunosuppressive cells, CTLA-4, PD-1, MDSC, MCP-1, and immune checkpoint molecules, which are described in more detail below.

The NY-ESO-2 antigen can enhance the cellular immune response of a subject administered with the NY-ESO-2 antigen by about 50-fold to about 6,000-fold, about 50-fold to about 5,500-fold, about 50-fold to about 5,000-fold, about 50-fold to about 4,500-fold, about 100-fold to about 6,000-fold, about 150-fold to about 6,000-fold, about 200-fold to about 6,000-fold, about 250-fold to about 6,000-fold, or about 300-fold to about 6,000-fold, compared to the cellular immune response of a subject not administered with the NY-ESO-2 antigen. In some embodiments, the NY-ESO-2 antigen can enhance the cellular immune response of 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, 2100-fold, 2200-fold, 2300-fold, 2400-fold, 2500-fold, 2600-fold, 2700-fold, 2800-fold, 2900-fold, 3000-fold, 3100-fold, 3200-fold, 3300-fold, 3400-fold, 3500-fold, 3600-fold, 3700-fold, 3800-fold, 3900-fold, 4000-fold, 4100-fold, 4200-fold, 4300-fold, 4400-fold 00 times, 2200 times, 2300 times, 2400 times, 2500 times, 2600 times, 2700 times, 2800 times, 2900 times, 3000 times, 3100 times, 3200 times, 3300 times, 3400 times, 3500 times, 3600 times, 3700 times, 3800 times, 3900 times, 4000 times, 4100 times, 4200 times, 4300 times, 4400 times, 4500 times, 4600 times, 4700 times, 4800 times, 4900 times, 5000 times, 5100 times, 5200 times, 5300 times, 5400 times, 5500 times, 5600 times, 5700 times, 5800 times, 5900 times or 6000 times.

The NY-ESO-2 antigen can increase interferon gamma (IFN-γ) levels in a subject administered with the NY-ESO-2 antigen by about 50-fold to about 6,000-fold, about 50-fold to about 5,500-fold, about 50-fold to about 5,000-fold, about 50-fold to about 4,500-fold, about 100-fold to about 6,000-fold, about 150-fold to about 6,000-fold, about 200-fold to about 6,000-fold, about 250-fold to about 6,000-fold, or about 300-fold to about 6,000-fold, compared to IFN-γ levels in a subject not administered with the NY-ESO-2 antigen. In some embodiments, the NY-ESO-2 antigen can increase IFN-γ levels in a subject administered with 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, 2100-fold, 2200-fold, 2300-fold, 2400-fold, 2500-fold, 2600-fold, 2700-fold, 2800-fold, 2900-fold, 3000-fold, 3100-fold, 3200-fold, 3300-fold, 3400-fold, 3500-fold, 3600-fold, 3700-fold, 3800-fold, 3900-fold, 4000-fold, 4100-fold, 4200-fold, 4300-fold, 4400-fold 100 times, 2200 times, 2300 times, 2400 times, 2500 times, 2600 times, 2700 times, 2800 times, 2900 times, 3000 times, 3100 times, 3200 times, 3300 times, 3400 times, 3500 times, 3600 times, 3700 times, 3800 times, 3900 times, 4000 times, 4100 times, 4200 times, 4300 times, 4400 times, 4500 times, 4600 times, 4700 times, 4800 times, 4900 times, 5000 times, 5100 times, 5200 times, 5300 times, 5400 times, 5500 times, 5600 times, 5700 times, 5800 times, 5900 times or 6000 times.

NY-ESO-2 antigens may comprise protein epitopes that make them particularly effective as immunogens against which an anti-NY-ESO-2 immune response can be induced. NY-ESO-2 antigens may comprise full-length translation products, variants thereof, fragments thereof, or combinations thereof. NY-ESO-2 antigens may comprise consensus proteins.

The nucleic acid sequence encoding the consensus NY-ESO-2 antigen can be optimized for codon usage and corresponding RNA transcripts. The nucleic acid encoding the consensus NY-ESO-2 antigen can be codon and RNA optimized for expression. In some embodiments, the nucleic acid sequence encoding the consensus NY-ESO-2 antigen can include a Kozak sequence (e.g., GCC ACC) to enhance translation efficiency. The nucleic acid encoding the consensus NY-ESO-2 antigen can include multiple stop codons (e.g., TGA TGA) to enhance translation termination efficiency.

The nucleic acid encoding the consensus NY-ESO-2 antigen may also encode an immunoglobulin E (IgE) leader sequence. The nucleic acid encoding the consensus NY-ESO-2 antigen may 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 via a peptide bond. The nucleic acid encoding the consensus NY-ESO-2 antigen may 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 contain or is not enriched with a nucleotide sequence encoding an IgE leader sequence.

The consensus NY-ESO-2 antigen can be the nucleic acid sequence of SEQ ID NO: 15, which encodes the amino acid sequence of 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 contain or be linked to an IgE leader sequence and/or an HA tag.

In some embodiments, the consensus NY-ESO-2 antigen may 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 shown in SEQ ID NO: 15. In other embodiments, the consensus NY-ESO-2 antigen may 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 shown in SEQ ID NO: 16. The consensus NY-ESO-2 antigen can be 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 shown in SEQ ID NO: 16.

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 as set forth herein and immunogenic fragments of proteins homologous to the proteins set forth herein are also provided.

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

Some embodiments relate to nucleic acid sequences encoding proteins having a specified percentage identity to a full-length NY-ESO-2 consensus protein, immunogenic fragments of a NY-ESO-2 consensus protein, and immunogenic fragments of proteins having identity to a NY-ESO-2 consensus protein. Such nucleic acid molecules can be provided that encode immunogenic proteins having up to 80% identity to the full-length NY-ESO-2 consensus sequence, up to 85% identity to the full-length 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, and up to 99% identity to the full-length NY-ESO-2 consensus sequence. Likewise, nucleic acid sequences encoding immunogenic fragments set forth herein and immunogenic fragments of proteins having similar percentage identities to the NY-ESO-2 proteins set forth herein as specified above are also provided.

In some embodiments, the nucleic acid sequence does not contain a coding sequence that encodes a leader sequence. In some embodiments, the nucleic acid sequence does not contain a coding sequence that encodes an IgE leader sequence.

Some embodiments relate to fragments of SEQ ID NO: 15. The fragments 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% of SEQ ID NO: 15. The fragments can be at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous to a fragment of SEQ ID NO: 15. The fragment may 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, such as, for example, an immunoglobulin leader sequence, such as an IgE leader sequence. In some embodiments, the fragment does not contain a coding sequence encoding a leader sequence. In some embodiments, the fragment does not contain a coding sequence encoding a leader sequence, such as, for example, an IgE leader sequence.

In addition, 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 the IgE leader sequence is SEQ ID NO: 16. The amino acid sequence of the consensus NY-ESO-2 protein linked to the IgE leader sequence can be linked to an HA tag.

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 shown in SEQ ID NO: 16. Some embodiments relate to immunogenic proteins having 96% homology to the consensus protein sequence shown in SEQ ID NO: 16. Some embodiments relate to immunogenic proteins having 97% homology to the consensus protein sequence shown in SEQ ID NO: 16. Some embodiments relate to immunogenic proteins having 98% homology to the consensus protein sequence shown in SEQ ID NO: 16. Some embodiments relate to immunogenic proteins having 99% homology to the consensus protein sequence shown in SEQ ID NO: 16.

Some embodiments relate to proteins identical to SEQ ID NO: 16. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 80% identical to the full-length consensus amino acid sequence as shown in SEQ ID NO: 16. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 85% identical to the full-length consensus amino acid sequence as shown in SEQ ID NO: 16. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 90% identical to the full-length consensus amino acid sequence as shown in SEQ ID NO: 16. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 91% identical to the full-length consensus amino acid sequence as shown in SEQ ID NO: 16. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 92% identical to the full-length consensus amino acid sequence as shown in SEQ ID NO: 16. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 93% identical to the full-length consensus amino acid sequence as shown in SEQ ID NO: 16. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 94% identical to the full-length consensus amino acid sequence as shown 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 as shown 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 as shown 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 as shown 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 as shown 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 as shown in SEQ ID NO: 16.

In some embodiments, the protein does not contain a leader sequence. In some embodiments, the protein does not contain an IgE leader sequence. 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. The immunogenic fragment may 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 includes a leader sequence, such as, for example, an immunoglobulin leader sequence, such as an IgE leader sequence. In some embodiments, the fragment does not contain a leader sequence. In some embodiments, the fragment does not contain a leader sequence, such as, for example, an IgE leader sequence.

Immunogenic fragments of proteins having amino acid sequences homologous to immunogenic fragments of SEQ ID NO: 16 can be provided. 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 having 95% or greater homology to SEQ ID NO: 16. 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 an immunogenic fragment of a consensus protein sequence herein. Some embodiments relate to immunogenic fragments having 99% homology to an immunogenic fragment of a consensus protein sequence herein. In some embodiments, the fragment includes a leader sequence, such as, for example, an immunoglobulin leader sequence, such as an IgE leader sequence. In some embodiments, the fragment does not contain a leader sequence. In some embodiments, the fragment does not contain a leader sequence, such as, for example, an IgE leader sequence.

Immunogenic fragments of proteins having an amino acid sequence identical to an immunogenic fragment of SEQ ID NO: 16 can be provided. 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 having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence shown in SEQ ID NO: 16. In some embodiments, the fragments include a leader sequence, such as, for example, an immunoglobulin leader sequence, such as an IgE leader sequence. In some embodiments, the fragments do not contain a leader sequence. In some embodiments, the fragment does not contain a leader sequence, such as, for example, an IgE leader sequence.

As indicated herein with respect to linking 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 that is encoded by the start codon of the nucleic acid sequence encoding the protein without the signal peptide coding sequence.

(9)PRAME

The vaccine of the present invention may comprise the cancer antigen PRAME, its fragment or variant thereof. PRAME, encoded by the PRAME gene, is a protein consisting of 509 amino acids and is expressed in the testis, placenta, endometrium, ovary, adrenal gland, and in tissues derived from melanoma, lung cancer, renal cancer, and head and neck cancer. PRAME is also expressed in adult and pediatric acute leukemia and multiple myeloma. PRAME contains an immunogenic nine-peptide that can elicit a cytotoxic response when presented by HLA-A24. Studies have shown that overexpression of PRAME in cultured cells induces cell death that is independent of caspase, resulting in a slower proliferation rate. Other studies have shown that overexpression of PRAME also confers growth or survival advantages by antagonizing retinoic acid receptor (RAR) signal transduction, and is causally involved in the tumorigenesis process. Interference with RAR signal transduction leads to the loss of regulated cell proliferation, development, and differentiation.

PRAME can have an expression pattern similar to 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 cytotoxic T lymphocytes. Therefore, PRAME can be associated with melanoma and leukemia.

PRAME antigen can induce antigen-specific T cells and/or high-titer antibody responses, thereby inducing or initiating an immune response to a cancer or tumor expressing the antigen or to the cancer or tumor reaction. In some embodiments, the immune response induced or initiated can be a cellular immune response, a humoral immune response, or a cellular and humoral immune response. In some embodiments, the cellular immune response induced or initiated can include the induction or secretion of interferon-γ (IFN-γ) and/or tumor necrosis factor α (TNF-α). In other embodiments, the immune response induced or initiated can reduce or inhibit one or more immunosuppressive factors that promote the growth of a tumor or cancer expressing the antigen, for example, but not limited to factors that lower MHC presentation, factors that raise antigen-specific regulatory T cells (Treg), PD-L1, FasL, cytokines such as IL-10 and TFG-β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by immunosuppressive cells, CTLA-4, PD-1, MDSC, MCP-1, and immune checkpoint molecules, described in more detail below.

The PRAME antigen can enhance the cellular immune response of a subject administered with the PRAME antigen by about 50-fold to about 6000-fold, about 50-fold to about 5500-fold, about 50-fold to about 5000-fold, about 50-fold to about 4500-fold, about 100-fold to about 6000-fold, about 150-fold to about 6000-fold, about 200-fold to about 6000-fold, about 250-fold to about 6000-fold, or about 300-fold to about 6000-fold, compared to the cellular immune response of a subject not administered with the PRAME antigen. In some embodiments, the PRAME antigen can enhance the cellular immune response of 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, 2200-fold, 2300-fold, 2400-fold, 2500-fold, 2600-fold, 2700-fold, 2800-fold, 2900-fold, 3000-fold, 3100-fold, 3200-fold, 3300-fold, 3400-fold, 3500-fold, 3600-fold, 3700-fold, 3800-fold, 3900-fold, 4000-fold, 4100-fold, 4200-fold, 4300-fold, 4400-fold, 4500-fold, 4600-fold, 4700-fold, 4800-fold, 4900-fold, 5000-fold, 5100-fold, 5200-fold, 5300-fold, 2200 times, 2300 times, 2400 times, 2500 times, 2600 times, 2700 times, 2800 times, 2900 times, 3000 times, 3100 times, 3200 times, 3300 times, 3400 times, 3500 times, 3600 times, 3700 times, 3800 times, 3900 times, 4000 times, 4100 times, 4200 times, 4300 times, 4400 times, 4500 times, 4600 times, 4700 times, 4800 times, 4900 times, 5000 times, 5100 times, 5200 times, 5300 times, 5400 times, 5500 times, 5600 times, 5700 times, 5800 times, 5900 times or 6000 times.

The PRAME antigen can increase interferon gamma (IFN-γ) levels in a subject administered with the PRAME antigen by about 50-fold to about 6000-fold, about 50-fold to about 5500-fold, about 50-fold to about 5000-fold, about 50-fold to about 4500-fold, about 100-fold to about 6000-fold, about 150-fold to about 6000-fold, about 200-fold to about 6000-fold, about 250-fold to about 6000-fold, or about 300-fold to about 6000-fold, compared to IFN-γ levels in a subject not administered with the PRAME antigen. In some embodiments, the PRAME antigen can increase the level of IFN-γ in a subject administered with 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. , 2200 times, 2300 times, 2400 times, 2500 times, 2600 times, 2700 times, 2800 times, 2900 times, 3000 times, 3100 times, 3200 times, 3300 times, 3400 times, 3500 times, 3600 times, 3700 times, 3800 times, 3900 times, 4000 times, 4100 times, 4200 times, 4300 times, 4400 times, 4500 times, 4600 times, 4700 times, 4800 times, 4900 times, 5000 times, 5100 times, 5200 times, 5300 times, 5400 times, 5500 times, 5600 times, 5700 times, 5800 times, 5900 times or 6000 times.

PRAME antigens may comprise protein epitopes that make them particularly effective as immunogens against which an anti-PRAME immune response can be induced. PRAME antigens may comprise full-length translation products, variants thereof, fragments thereof, or combinations thereof. PRAME antigens may comprise a consensus protein.

Can be used for codon and corresponding RNA transcript optimization coding total PRAME antigen nucleic acid sequence.Coding total PRAME antigen nucleic acid can be for expression and optimized codon and RNA.In some embodiments, coding total PRAME antigen nucleic acid sequence can comprise Kozak sequence (for example, GCC ACC) to enhance the efficiency of translation.Coding total PRAME antigen nucleic acid can comprise multiple termination codons (for example, TGA TGA) to enhance the efficiency of translation termination.

The nucleic acid encoding the consensus PRAME antigen may also encode an immunoglobulin E (IgE) leader sequence. The nucleic acid encoding the consensus PRAME antigen may further encode an IgE leader sequence so 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 may also include a nucleotide sequence encoding the IgE leader sequence. In some embodiments, the nucleic acid encoding the consensus PRAME antigen does not contain or does not contain a nucleotide sequence encoding the IgE leader sequence.

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 contain or be linked to an IgE leader sequence and/or an HA tag.

In some embodiments, the consensus PRAME antigen may 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 shown in SEQ ID NO: 17. In other embodiments, the consensus PRAME antigen may 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 shown in SEQ ID NO: 18. The consensus PRAME antigen may be 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 shown in SEQ ID NO: 18.

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 encoding 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 can be provided. 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.

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

Some embodiments relate to nucleic acid sequences encoding proteins having a specified percentage identity to the full-length PRAME consensus protein, immunogenic fragments of the PRAME consensus protein, and immunogenic fragments of proteins having identity to the PRAME consensus protein. Such nucleic acid molecules encoding immunogenic proteins having up to 80% identity to the full-length PRAME consensus sequence, up to 85% identity to the full-length 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 are provided. Similarly, nucleic acid sequences encoding immunogenic fragments set forth herein and immunogenic fragments of proteins having similar percentage identities as specified above to the PRAME proteins set forth herein are also provided.

In some embodiments, the nucleic acid sequence does not contain a coding sequence that encodes a leader sequence. In some embodiments, the nucleic acid sequence does not contain a coding sequence that encodes an IgE leader sequence.

Some embodiments relate to fragments of SEQ ID NO: 17. The fragments 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% of SEQ ID NO: 17. The fragments can be at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous to a fragment of SEQ ID NO: 17. The fragment may 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, such as, for example, an immunoglobulin leader sequence, such as an IgE leader sequence. In some embodiments, the fragment does not contain a coding sequence encoding a leader sequence. In some embodiments, the fragment does not contain a coding sequence encoding a leader sequence, such as, for example, an IgE leader sequence.

In addition, 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 the IgE leader sequence is SEQ ID NO: 18. The amino acid sequence of the consensus PRAME protein linked to the IgE leader sequence can be linked to an HA tag.

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 shown in SEQ ID NO: 18. Some embodiments relate to immunogenic proteins having 96% homology to the consensus protein sequence shown in SEQ ID NO: 18. Some embodiments relate to immunogenic proteins having 97% homology to the consensus protein sequence shown in SEQ ID NO: 18. Some embodiments relate to immunogenic proteins having 98% homology to the consensus protein sequence shown in SEQ ID NO: 18. Some embodiments relate to immunogenic proteins having 99% homology to the consensus protein sequence shown in SEQ ID NO: 18.

Some embodiments relate to proteins identical to SEQ ID NO: 18. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 80% identical to the full-length consensus amino acid sequence as shown in SEQ ID NO: 18. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 85% identical to the full-length consensus amino acid sequence as shown in SEQ ID NO: 18. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 90% identical to the full-length consensus amino acid sequence as shown in SEQ ID NO: 18. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 91% identical to the full-length consensus amino acid sequence as shown in SEQ ID NO: 18. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 92% identical to the full-length consensus amino acid sequence as shown in SEQ ID NO: 18. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 93% identical to the full-length consensus amino acid sequence as shown in SEQ ID NO: 18. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 94% identical to the full-length consensus amino acid sequence as shown 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 as shown 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 as shown 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 as shown 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 as shown 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 as shown in SEQ ID NO: 18.

In some embodiments, the protein does not contain a leader sequence. In some embodiments, the protein does not contain an IgE leader sequence. 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. The immunogenic fragment may 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 includes a leader sequence, such as, for example, an immunoglobulin leader sequence, such as an IgE leader sequence. In some embodiments, the fragment does not contain a leader sequence. In some embodiments, the fragment does not contain a leader sequence, such as, for example, an IgE leader sequence.

Immunogenic fragments of proteins having amino acid sequences homologous to immunogenic fragments of SEQ ID NO: 18 can be provided. 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 having 95% or greater homology 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 an immunogenic fragment of a consensus protein sequence herein. Some embodiments relate to immunogenic fragments having 99% homology to an immunogenic fragment of a consensus protein sequence herein. In some embodiments, the fragment includes a leader sequence, such as, for example, an immunoglobulin leader sequence, such as an IgE leader sequence. In some embodiments, the fragment does not contain a leader sequence. In some embodiments, the fragment does not contain a leader sequence, such as, for example, an IgE leader sequence.

Immunogenic fragments of proteins having an amino acid sequence identical to an immunogenic fragment of SEQ ID NO: 18 can be provided. 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 having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence shown in SEQ ID NO: 18. In some embodiments, the fragments include a leader sequence, such as, for example, an immunoglobulin leader sequence, such as an IgE leader sequence. In some embodiments, the fragments do not contain a leader sequence. In some embodiments, the fragment does not contain a leader sequence, such as, for example, an IgE leader sequence.

As indicated herein with respect to linking 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 that is encoded by the start codon of the nucleic acid sequence encoding the protein without the signal peptide coding sequence.

(10)PSA

The vaccine of the present invention may comprise the cancer antigen prostate-specific antigen (PSA; also known as gamma-sperm 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 cells and encoded by the KLK3 gene. PSA is commonly used as a serum marker for prostate cancer. PSA is a member of the tissue kallikrein family and cleaves seminal coagulants in semen coagulates after the cleavage zymogen is cleaved to release the active enzyme, thereby liquefying the semen to enable sperm to swim freely. In addition, PSA enzymatic activity is regulated by zinc concentration, i.e., high zinc concentrations inhibit the enzymatic activity of PSA.

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

PSA antigens may comprise protein epitopes that make them particularly effective as immunogens against which an anti-PSA immune response can be induced. PSA antigens may comprise full-length translation products, variants thereof, fragments thereof, or combinations thereof. PSA antigens may comprise consensus proteins.

The nucleic acid sequence encoding the consensus PSA antigen can be optimized for codon usage and corresponding RNA transcripts. The nucleic acid encoding the consensus PSA antigen can be codon and RNA optimized for expression. In some embodiments, the nucleic acid sequence encoding the consensus PSA antigen can include a Kozak sequence (e.g., GCC ACC) to enhance the efficiency of translation. The nucleic acid encoding the consensus PSA antigen can include multiple stop codons (e.g., TGA TGA) to enhance the efficiency of translation termination.

The nucleic acid encoding the consensus PSA antigen may also encode an immunoglobulin E (IgE) leader sequence. The nucleic acid encoding the consensus PSA antigen may 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 P antigen via a peptide bond. The nucleic acid encoding the consensus PSA antigen may also include a nucleotide sequence encoding the IgE leader sequence. In some embodiments, the nucleic acid encoding the consensus PSA antigen does not contain or contains a nucleotide sequence encoding the IgE leader sequence.

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

(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 (NAALAD enzyme 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 found in membranes and the extracellular space. PSMA is highly expressed in the human prostate and is upregulated in prostate cancer. PSMA has also been found to be overexpressed in other cancers such as solid tumors of the kidney, breast, and colon.

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

PSMA antigens may comprise protein epitopes that make them particularly effective as immunogens against which an anti-PSMA immune response can be induced. PSMA antigens may comprise full-length translation products, variants thereof, fragments thereof, or combinations thereof. PSMA antigens may comprise consensus proteins.

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

The nucleic acid encoding the consensus PSMA antigen may also encode an immunoglobulin E (IgE) leader sequence. The nucleic acid encoding the consensus PSMA antigen may 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 P antigen via a peptide bond. The nucleic acid encoding the consensus PSMA antigen may also include a nucleotide sequence encoding the IgE leader sequence. In some embodiments, the nucleic acid encoding the consensus PSMA antigen does not contain or contains a nucleotide sequence encoding an IgE leader sequence.

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

(12)STEAP

The vaccine of the present invention may comprise the cancer antigen six transmembrane epithelial antigen of the prostate (STEAP), a fragment thereof, or a variant thereof. STEAP is a metalloreductase encoded by the STEAP1 gene. STEAP is primarily expressed in prostate tissue and is upregulated in cancer cells. STEAP is predicted to be a six-transmembrane protein and a cell surface antigen found in intercellular junctions.

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

STEAP antigens may comprise protein epitopes that make them particularly effective as immunogens against which an anti-STEAP immune response can be induced. STEAP antigens may comprise full-length translation products, variants thereof, fragments thereof, or combinations thereof. STEAP antigens may comprise a consensus protein.

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

The nucleic acid encoding the consensus STEAP antigen may also encode an immunoglobulin E (IgE) leader sequence. The nucleic acid encoding the consensus STEAP antigen may 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 via a peptide bond. The nucleic acid encoding the consensus STEAP antigen may also include a nucleotide sequence encoding the IgE leader sequence. In some embodiments, the nucleic acid encoding the consensus STEAP antigen does not contain or contains a nucleotide sequence encoding an IgE leader sequence.

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

(13)PSCA

The vaccine of the present invention may comprise 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 and is 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.

The PSCA antigen can induce antigen-specific T cell and/or high-titer antibody responses, thereby inducing or eliciting an immune response against or reactive with 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. In some embodiments, the induced or elicited cellular immune response can include the induction or secretion of interferon-γ (IFN-γ) and/or tumor necrosis factor α (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 expressing the antigen, such as, but not limited to, factors that downregulate MHC presentation, factors that upregulate antigen-specific regulatory T cells (Tregs), PD-L1, FasL, cytokines such as IL-10 and TFG-β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by immunosuppressive cells, CTLA-4, PD-1, MDSCs, MCP-1, and immune checkpoint molecules, which are described in more detail below.

PSCA antigens may comprise protein epitopes that make them particularly effective as immunogens against which an anti-PSCA immune response can be induced. PSCA antigens may comprise full-length translation products, variants thereof, fragments thereof, or combinations thereof. PSCA antigens may comprise consensus proteins.

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

The nucleic acid encoding the consensus PSCA antigen may also encode an immunoglobulin E (IgE) leader sequence. The nucleic acid encoding the consensus PSCA antigen may 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 via a peptide bond. The nucleic acid encoding the consensus PSCA antigen may also include a nucleotide sequence encoding the IgE leader sequence. In some embodiments, the nucleic acid encoding the consensus PSCA antigen does not contain or does not include a nucleotide sequence encoding an IgE leader sequence.

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

(14)hTERT

The vaccine of the present invention may comprise the cancer antigen hTERT, a fragment thereof or a variant thereof. hTERT is a human telomerase reverse transcriptase that synthesizes a TTAGGG tag at the end of telomerase to prevent cell death caused by chromosome shortening. Hyperproliferative cells may have abnormally high hTERT expression. Abnormal expression of hTERT may also occur in hyperproliferative cells infected with HCV and HPV. Therefore, immunotherapy for HPV and HCV can be enhanced by targeting cells that express hTERT at abnormal levels. HPV and HCV antigens are discussed in more detail below. The hTERT cancer antigen can be further defined by U.S. patent application No. 14/139,660, filed December 23, 2013, which is incorporated by reference in its entirety.

In addition, hTERT in dendritic cells transfected with the hTERT gene can induce CD8 ⁺ cytotoxic T cells and trigger CD4 ⁺ T cells in an antigen-specific manner. Therefore, using hTERT expression in antigen-presenting cells (APCs) to delay senescence and support their ability to present selected antigens can be used in immunotherapy methods such as those described herein.

The hTERT antigen can be associated with or expressed in a variety 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 blood cancers. Thus, when the hTERT antigen described herein is included, the vaccine can be used to treat subjects with a variety 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 blood cancers.

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

hTERT antigens may comprise protein epitopes that make them particularly effective as immunogens against which an anti-hTERT immune response can be induced. hTERT antigens may comprise full-length translation products, variants thereof, fragments thereof, or combinations thereof. hTERT antigens may comprise a consensus protein.

The nucleic acid sequence encoding hTERT antigen or a total hTERT antigen can be optimized for codon usage and corresponding RNA transcripts. The nucleic acid encoding hTERT antigen or a total hTERT antigen can be a codon and RNA optimized for expression. In some embodiments, the nucleic acid sequence encoding hTERT antigen or a total hTERT antigen can include a Kozak sequence (e.g., GCC ACC) to enhance the efficiency of translation. The nucleic acid encoding hTERT antigen or a total hTERT antigen can include multiple stop codons (e.g., TGA TGA) to enhance the efficiency of translation termination.

The nucleic acid encoding hTERT antigen or the shared hTERT antigen may also encode an immunoglobulin E (IgE) leader sequence. The nucleic acid encoding hTERT antigen or the shared hTERT antigen may further encode an IgE leader sequence so that the amino acid sequence of the IgE leader sequence is linked to the amino acid sequence of the hTERT antigen or the shared hTERT antigen by a peptide bond, respectively. The nucleic acid encoding hTERT antigen or the shared hTERT antigen may also include a nucleotide sequence encoding an IgE leader sequence. In some embodiments, the nucleic acid encoding hTERT antigen or the shared hTERT antigen does not contain or does not contain a nucleotide sequence encoding an IgE leader sequence.

In some embodiments, the nucleic acid encoding the hTERT antigen or the shared hTERT antigen can be a heterologous nucleic acid sequence and/or contain one or more heterologous nucleic acid sequences. The nucleic acid encoding the hTERT antigen or the shared hTERT antigen can be mutated relative to the wild-type hTERT antigen so that one or more amino acids or residues in the amino acid sequence of the hTERT antigen or the shared hTERT antigen are replaced or substituted with another amino acid or residue, respectively. The nucleic acid encoding the hTERT antigen or the shared hTERT antigen can be mutated relative to the wild-type hTERT antigen so that one or more residues in the amino acid sequence of the hTERT antigen or the shared hTERT antigen are replaced or substituted with another residue, respectively, so that the immune system of a mammal administered with the nucleic acid encoding the hTERT antigen or the shared hTERT antigen, the hTERT antigen or the shared hTERT antigen, or a combination thereof is no longer tolerant to hTERT. The nucleic acid encoding the hTERT antigen or the consensus hTERT antigen is mutated relative to the wild-type hTERT antigen so that arginine 589, aspartate 1005, or arginine 589 and aspartate 1005 in the amino acid sequence of the hTERT antigen or the consensus hTERT antigen are replaced or substituted with a tyrosine residue.

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 the 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 contain or be linked to an IgE leader sequence and/or an HA tag.

In some embodiments, the hTERT 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 to the nucleic acid sequence set forth in SEQ ID NO: 23. In other embodiments, the hTERT 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 to the amino acid sequence set forth in SEQ ID NO: 24. The hTERT antigen can be 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 shown in SEQ ID NO:24.

Some embodiments relate to nucleic acid sequences encoding proteins homologous to hTERT proteins, immunogenic fragments of hTERT proteins, and immunogenic fragments of homologous proteins. Such nucleic acid molecules encoding 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 can be provided. Similarly, nucleic acid sequences encoding immunogenic fragments as described herein and immunogenic fragments of proteins homologous to the proteins described herein are also provided.

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

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

In some embodiments, the nucleic acid sequence does not contain a coding sequence that encodes a leader sequence. In some embodiments, the nucleic acid sequence does not contain a coding sequence that encodes an IgE leader sequence.

Some embodiments relate to fragments of SEQ ID NO: 23. The fragments 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% of SEQ ID NO: 23. The fragments can be at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous to a fragment of SEQ ID NO: 23. The fragment may 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, such as, for example, an immunoglobulin leader sequence, such as an IgE leader sequence. In some embodiments, the fragment does not contain a coding sequence encoding a leader sequence. In some embodiments, the fragment does not contain a coding sequence encoding a leader sequence, such as, for example, an IgE leader sequence.

In addition, the amino acid sequence of the hTERT protein is SEQ ID NO: 24. The amino acid sequence of the hTERT protein linked to the IgE leader sequence is SEQ ID NO: 24. The amino acid sequence of the hTERT protein linked to the IgE leader sequence can be linked to an HA tag.

Some embodiments relate to proteins homologous to SEQ ID NO: 24. Some embodiments relate to immunogenic proteins having 95% homology to the consensus protein sequence shown in SEQ ID NO: 24. Some embodiments relate to immunogenic proteins having 96% homology to the consensus protein sequence shown in SEQ ID NO: 24. Some embodiments relate to immunogenic proteins having 97% homology to the consensus protein sequence shown in SEQ ID NO: 24. Some embodiments relate to immunogenic proteins having 98% homology to the consensus protein sequence shown in SEQ ID NO: 24. Some embodiments relate to immunogenic proteins having 99% homology to the consensus protein sequence shown in SEQ ID NO: 24.

Some embodiments relate to proteins identical to SEQ ID NO: 24. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 80% identical to the full-length amino acid sequence as shown in SEQ ID NO: 24. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 85% identical to the full-length amino acid sequence as shown in SEQ ID NO: 24. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 90% identical to the full-length amino acid sequence as shown in SEQ ID NO: 24. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 91% identical to the full-length amino acid sequence as shown in SEQ ID NO: 24. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 92% identical to the full-length amino acid sequence as shown in SEQ ID NO: 24. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 93% identical to the full-length amino acid sequence as shown in SEQ ID NO: 24. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 94% identical to the full-length amino acid sequence as shown in SEQ ID NO: 24. Some embodiments relate to immunogenic proteins having an amino acid sequence that is 95% identical to the full-length amino acid sequence as shown 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 as shown 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 as shown 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 as shown 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 as shown in SEQ ID NO: 24.

In some embodiments, the protein does not contain a leader sequence. In some embodiments, the protein does not contain an IgE leader sequence. A fragment of the 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. The immunogenic fragment may 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 includes a leader sequence, such as, for example, an immunoglobulin leader sequence, such as an IgE leader sequence. In some embodiments, the fragment does not contain a leader sequence. In some embodiments, the fragment does not contain a leader sequence, such as, for example, an IgE leader sequence.

Immunogenic fragments of proteins having amino acid sequences homologous to immunogenic fragments of SEQ ID NO: 24 can be provided. 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 having 95% or greater homology 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 immunogenic fragments of the protein sequences herein. In some embodiments, the fragments include a leader sequence, such as, for example, an immunoglobulin leader sequence, such as an IgE leader sequence. In some embodiments, the fragments do not contain a leader sequence. In some embodiments, the fragments do not contain a leader sequence, such as, for example, an IgE leader sequence.

Immunogenic fragments of proteins having an amino acid sequence identical to an immunogenic fragment of SEQ ID NO: 24 can be provided. 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 having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence shown in SEQ ID NO: 24. In some embodiments, the fragments include a leader sequence, such as, for example, an immunoglobulin leader sequence, such as an IgE leader sequence. In some embodiments, the fragments do not contain a leader sequence. In some embodiments, the fragment does not contain a leader sequence, such as, for example, an IgE leader sequence.

As indicated herein with respect to linking 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 that is encoded by the start codon of the nucleic acid sequence encoding the protein without the signal peptide coding sequence.

Fragments of SEQ ID NO: 23 may comprise 30 or more nucleotides, including preferred sequences encoding immunodominant epitopes. In some embodiments, fragments of SEQ ID NO: 23 may comprise 45 or more nucleotides, including preferred sequences encoding immunodominant epitopes. In some embodiments, fragments of SEQ ID NO: 23 may comprise 60 or more nucleotides, including preferred sequences encoding immunodominant epitopes. In some embodiments, fragments of SEQ ID NO: 23 may comprise 75 or more nucleotides, including preferred sequences encoding immunodominant epitopes. In some embodiments, fragments of SEQ ID NO: 23 may comprise 90 or more nucleotides, including preferred sequences encoding immunodominant epitopes. In some embodiments, fragments of SEQ ID NO: 23 may comprise 120 or more nucleotides, including preferred sequences encoding immunodominant epitopes. In some embodiments, fragments of SEQ ID NO: 23 may comprise 150 or more nucleotides, including preferred sequences encoding immunodominant epitopes. In some embodiments, a fragment of SEQ ID NO: 23 may comprise 180 or more nucleotides, including a preferred sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO: 23 may comprise 210 or more nucleotides, including a preferred sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO: 23 may comprise 240 or more nucleotides, including a preferred sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO: 23 may comprise 270 or more nucleotides, including a preferred sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO: 23 may comprise 300 or more nucleotides, including a preferred sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO: 23 may comprise 360 or more nucleotides, including a preferred sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO: 23 may comprise 420 or more nucleotides, including a preferred sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO: 23 may comprise 480 or more nucleotides, including a preferred sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO: 23 may comprise 540 or more nucleotides, including a preferred sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO: 23 may comprise 600 or more nucleotides, including a preferred sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO: 23 may comprise 300 or more nucleotides, including a preferred sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO: 23 may comprise 660 or more nucleotides, including a preferred sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO: 23 may comprise 720 or more nucleotides, including a preferred sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO: 23 may comprise 780 or more nucleotides, including a preferred sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO: 23 may comprise 840 or more nucleotides, including a preferred sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO: 23 may comprise 900 or more nucleotides, including a preferred sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO: 23 may comprise 960 or more nucleotides, including a preferred sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO: 23 may comprise 1020 or more nucleotides, including a preferred sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO: 23 may comprise 1080 or more nucleotides, including a preferred sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO: 23 may comprise 1140 or more nucleotides, including a preferred sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO: 23 may comprise 1200 or more nucleotides, including a preferred sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO: 23 may comprise 1260 or more nucleotides, including a preferred sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO: 23 may comprise 1320 or more nucleotides, including a preferred sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO: 23 may comprise 1380 or more nucleotides, including a preferred sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO: 23 may comprise 1440 or more nucleotides, including a preferred sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO: 23 may comprise 1500 or more nucleotides, including a preferred sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO: 23 may comprise 1560 or more nucleotides, including a preferred sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO: 23 may comprise 1620 or more nucleotides, including a preferred sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO: 23 may comprise 1680 or more nucleotides, including a preferred sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO: 23 may comprise 1740 or more nucleotides, including a preferred sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO: 23 may comprise 1800 or more nucleotides, including a preferred sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO: 23 may comprise 1860 or more nucleotides, including a preferred sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO: 23 may comprise 1920 or more nucleotides, including a preferred sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO: 34 may comprise 1980 or more nucleotides, including a preferred sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO: 23 may comprise 2040 or more nucleotides, including a preferred sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO: 23 may comprise 2100 or more nucleotides, including a preferred sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO: 23 may comprise 2160 or more nucleotides, including a preferred sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO: 23 may comprise 2220 or more nucleotides, including a preferred sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO: 23 may comprise 2280 or more nucleotides, including a preferred sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO: 23 may comprise 2340 or more nucleotides, including a preferred sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO: 23 may comprise 2400 or more nucleotides, including a preferred sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO: 23 may comprise 2460 or more nucleotides, including a preferred sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO: 23 may comprise 2520 or more nucleotides, including a preferred sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO: 23 may comprise 2580 or more nucleotides, including a preferred sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO: 23 may comprise 2640 or more nucleotides, including a preferred sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO: 23 may comprise 2700 or more nucleotides, including a preferred sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO: 23 may comprise 2760 or more nucleotides, including a preferred sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO: 23 may comprise 2820 or more nucleotides, including a preferred sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO: 23 may comprise 2880 or more nucleotides, including a preferred sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO: 23 may comprise 2940 or more nucleotides, including a preferred sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO: 23 may comprise 3000 or more nucleotides, including a preferred sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO: 23 may comprise 3060 or more nucleotides, including a preferred sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO: 23 may comprise 3120 or more nucleotides, including a preferred sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO: 23 may comprise 3180 or more nucleotides, including a preferred sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO: 23 may comprise 3240 or more nucleotides, including a preferred sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO: 23 may comprise 3300 or more nucleotides, including a preferred sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO: 23 may comprise 3360 or more nucleotides, including a preferred sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO: 23 may comprise 3420 or more nucleotides, including a preferred sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO: 23 may comprise 3480 or more nucleotides, including a preferred sequence encoding an immunodominant epitope. In some embodiments, a fragment of SEQ ID NO: 23 may comprise a coding sequence for an IgE leader sequence. In some embodiments, a fragment of SEQ ID NO: 23 does not comprise a coding sequence for an IgE leader sequence.

A fragment may comprise 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, the number of nucleotides is less than 780, in some embodiments, less than 840, in some embodiments, less than 900, in some embodiments, less than 960, in some embodiments, less than 1020, in some embodiments, less than 1080, in some embodiments, less than 1140, in some embodiments, less than 1200, in some embodiments, less than 1260, in some embodiments, less than 1320, in some embodiments, less than 1380, in some embodiments, less than 1440, in some embodiments, less than 1500, in some embodiments, less than 1560, in some embodiments, less than 1620, 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 less than 2040 nucleotides, in some embodiments less than 2100 nucleotides, in some embodiments less than 2160 nucleotides, in some embodiments less than 2220 nucleotides, in some embodiments less than 2280 nucleotides, in some embodiments less than 2340 nucleotides, in some embodiments less than 2400 nucleotides, in some embodiments less than 2460 nucleotides, in some embodiments less than 2520 nucleotides, in some embodiments less than 2580 nucleotides, in some embodiments In some embodiments, the present invention comprises less than 2640 nucleotides, in some embodiments less than 2700 nucleotides, in some embodiments less than 2760 nucleotides, in some embodiments less than 2820 nucleotides, in some embodiments less than 2860 nucleotides, in some embodiments less than 2940 nucleotides, in some embodiments less than 3000 nucleotides, in some embodiments less than 3060 nucleotides, in some embodiments less than 3120 nucleotides, in some embodiments less than 3180 nucleotides, in some embodiments less than 3240 nucleotides, in some embodiments less than 3300 nucleotides, in some embodiments less than 3360 nucleotides, in some embodiments less than 3420 nucleotides, in some embodiments less than 3480 nucleotides, and in some embodiments less than 3510 nucleotides.

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

Fragments may comprise less than 24 amino acids, in some embodiments less than 30 amino acids, in some embodiments less than 36 amino acids, in some embodiments less than 42 amino acids, in some embodiments less than 48 amino acids, in some embodiments less than 54 amino acids, in some embodiments less than 60 amino acids, in some embodiments less than 72 amino acids, in some embodiments less than 90 amino acids, in some embodiments less than 120 amino acids, in some embodiments less than 150 amino acids, in some embodiments less than 180 amino acids, in some embodiments less than 210 amino acids, in some embodiments less than 240 amino acids, in some embodiments less than 260 amino acids, in some embodiments less than 290 amino acids, in some embodiments less than 320 amino acids, in some embodiments less than 350 amino acids, in some embodiments less than 380 amino acids, in some embodiments less than 410 amino acids, in some embodiments less than 440 amino acids, in some embodiments 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 700 amino acids. In some embodiments, the protein comprises 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 In some embodiments, the present invention comprises less than 1040 amino acids, in some embodiments less than 1070 amino acids, in some embodiments less than 1200 amino acids, in some embodiments less than 1230 amino acids, in some embodiments less than 1260 amino acids, in some embodiments less than 1290 amino acids, in some embodiments less than 1320 amino acids, in some embodiments less than 1350 amino acids, in some embodiments less than 1380 amino acids, in some embodiments less than 1410 amino acids, in some embodiments less than 1440 amino acids, in some embodiments less than 1470 amino acids, and in some embodiments less than 1500 amino acids.

(15)MAGE A1

The vaccine of the present invention may comprise the cancer antigen melanoma-associated antigen 1 (MAGE A1), a fragment thereof, or a variant thereof. MAGE A1, encoded by the MAGE A1 gene, is a 280-amino acid protein and has been found to be expressed only by tumor cells and spermatocytes. The expression of MAGE A1 in normal somatic tissues is dependent on DNA methylation. These genes are activated in many types of tumors during the whole-genome demethylation process that typically accompanies tumorigenesis. Specifically, during tumorigenic transformation, these genes are activated, expressed, and can become antigenic targets recognized and attacked by the immune system. Thus, MAGE genes participate in the immune process by targeting some early-stage tumor cells for immune destruction. MAGE A1 is expressed in many types of cancer, including, but not limited to, melanoma, lung cancer, and esophageal squamous cell carcinoma.

The MAGE A1 antigen can induce antigen-specific T cells and/or high-titer antibody responses, thereby inducing or triggering an immune response to a cancer or tumor expressing the antigen or reacting with the cancer or tumor. In some embodiments, the induced or triggered immune response can be a cellular immune response, a humoral immune response, or a cellular and humoral immune response. In some embodiments, the induced or triggered cellular immune response can include the induction or secretion of interferon-γ (IFN-γ) and/or tumor necrosis factor α (TNF-α). In other embodiments, the induced or triggered immune response can reduce or inhibit one or more immunosuppressive factors that promote the growth of tumors or cancers expressing the antigen, such as, but not limited to, factors that downregulate MHC presentation, factors that upregulate antigen-specific regulatory T cells (Treg), PD-L1, FasL, cytokines such as IL-10 and TFG-β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by immunosuppressive cells, CTLA-4, PD-1, MDSC, MCP-1, and immune checkpoint molecules, which are described in more detail below.

(16)WT1

The vaccine of the present invention may comprise the cancer antigen Wilms' tumor 1 (WT1), a fragment thereof, or a variant thereof. WT1 is a transcription factor containing a proline/glutamine-rich DNA binding domain at its N-terminus and four zinc finger motifs at its C-terminus. WT1 plays a role in the normal development of the urogenital system and interacts with numerous factors, such as p53 (a known tumor suppressor) and the serine protease HtrA2 (which cleaves WT1 at multiple sites after treatment with cytotoxic drugs).

Mutations in WT1 can lead to the formation of tumors or cancers such as Wilms' tumors or tumors expressing WT1. Wilms' tumors typically form in one or both kidneys before metastasizing to other tissues such as, but not limited to, liver tissue, urinary system tissue, lymphoid tissue, and lung tissue. Therefore, Wilms' tumors can be considered to be metastatic tumors. Wilms' tumors typically occur in young children (e.g., less than 5 years old) and occur in sporadic and hereditary forms. The WT1 cancer antigen can be further defined by PCT/US13/75141, filed December 23, 2013, which is incorporated herein by reference in its entirety.

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

The vaccine can be used to treat subjects with Wilms' tumor. The vaccine can be used to treat subjects with many types of cancer (including but not limited to melanoma, prostate cancer, liver cancer, cervical cancer, recurrent respiratory papillomatosis (RRP), anal cancer, head and neck cancer and blood cancer). The vaccine can also be used to treat subjects with cancer or tumors that express WT1 to prevent such tumors from developing in subjects. The WT1 antigen can be different from the natural "normal" WT1 gene, thereby providing treatment or prevention for tumors that express the WT1 antigen. Therefore, provided herein are WT1 antigen sequences (i.e., mutated WT1 genes or sequences) that are different from the natural WT1 gene.

The transcript of the native WT1 gene can be processed into multiple mRNAs, and the resulting proteins are not completely equivalent in inducing an immune response. The mutated WT1 gene described herein avoids selective processing, thereby producing a full-length transcript and resulting in a stronger induction of effector T cell and B cell responses. The first mutated WT1 sequence is referred to as CON WT1 with modified zinc fingers or ConWT1-L. SEQ ID NO: 19 is a nucleic acid sequence encoding the WT1 antigen CON WT1 with modified zinc fingers. SEQ ID NO: 20 is an amino acid sequence of the WT1 antigen CON WT1 with modified zinc fingers. The second mutated WT1 sequence is referred to as CON WT1 without zinc fingers or ConWT1-S. SEQ ID NO: 21 is a nucleic acid sequence encoding the WTI antigen CON WT1 without zinc fingers. SEQ ID NO: 22 is an amino acid sequence of the WT1 antigen CON WT1 without modified zinc fingers.

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

The WT1 consensus antigen may comprise one or more mutations in one or more zinc fingers, thereby eliciting a stronger and broader cellular and/or humoral immune response than the corresponding codon-optimized WT1 antigen. The one or more mutations may be substitutions of one or more amino acids that coordinate zinc ions in one or more zinc fingers. The one or more amino acids that coordinate zinc ions may be a CCHH motif. Thus, in some embodiments, the one or more mutations may replace 1, 2, 3, or all 4 amino acids of the CCHH motif.

In other embodiments, the one or more mutations are mutations in which residues 312, 317, 342, and 347 of SEQ ID NO: 20 are any residue except cysteine (C) and residues 330, 334, 360, and 364 of SEQ ID NO: 20 are any residue except histidine (H). Specifically, the one or more mutations are mutations in which residues 312, 317, 330, 334, 342, 347, 360, and 364 of SEQ ID NO: 20 are glycine (G).

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.

The WT1 consensus antigen may be the nucleic acid SEQ ID NO: 19 encoding SEQ ID NO: 20. In some embodiments, the WT1 consensus antigen may 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%, or 99% identical over the entire length of the nucleic acid sequence shown in SEQ ID NO: 19. In other embodiments, the WT1 consensus antigen can be a nucleic acid sequence encoding 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% or 99% identical over the entire length of the amino acid sequence shown in SEQ ID NO:20.

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 amino acid sequence shown in SEQ ID NO: 20, so long as 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 other embodiments, the WT1 consensus antigen can be a nucleic acid sequence encoding 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% or 99% identical over the entire length of the amino acid sequence shown in SEQ ID NO:20, so long as residues 312, 317, 330, 334, 342, 347, 360 and 364 of SEQ ID NO:20 are glycine (G).

The WT1 consensus antigen may be the amino acid sequence of SEQ ID NO: 20. In some embodiments, the WT1 consensus antigen may be 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%, or 99% identical over the entire length of the amino acid sequence shown in SEQ ID NO: 20. 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 amino acid sequence shown in SEQ ID NO: 20, so long as 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 sequence can be 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%, or 99% identical over the entire length of the amino acid sequence shown in SEQ ID NO:20, so long as residues 312, 317, 330, 334, 342, 347, 360, and 364 of SEQ ID NO:20 are glycine (G).

The WT1 consensus antigen can be the nucleic acid SEQ ID NO: 21, which encodes SEQ ID NO: 22. In some embodiments, the WT1 consensus 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%, or 99% identical over the entire length of the nucleic acid sequence shown in SEQ ID NO: 21. In other embodiments, the WT1 consensus antigen can be a nucleic acid sequence encoding 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% or 99% identical over the entire length of the amino acid sequence shown in SEQ ID NO:22.

The WT1 consensus antigen may be the amino acid sequence of SEQ ID NO: 22. In some embodiments, the WT1 consensus antigen may be 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%, or 99% identical over the entire length of the amino acid sequence shown in SEQ ID NO: 22.

Immunogenic fragments can be provided of SEQ ID NO: 20 and SEQ ID NO: 22. The 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: 20 and/or SEQ ID NO: 22. In some 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, provided that residues 312, 317, 342, and 347 of SEQ ID NO:20, if present in the immunogenic fragment, are any residues other than cysteine (C), and provided that residues 330, 334, 360, and 364 of SEQ ID NO:20, if present in the immunogenic fragment, are any residues 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, so that if residues 312, 317, 330, 334, 342, 347, 360, and 364 of SEQ ID NO:20 are present in the immunogenic fragment, then these residues are glycine (G).

In some embodiments, the immunogenic fragment includes a leader sequence, eg, an immunoglobulin leader sequence, such as an immunoglobulin E (IgE) leader sequence. In some embodiments, the immunogenic fragment does not contain a leader sequence.

Immunogenic fragments of proteins containing amino acid sequences identical to immunogenic fragments of SEQ ID NOs: 20 and 22 can be provided. 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 NOs: 20 and/or SEQ ID NOs: 22. Some embodiments relate to immunogenic fragments having 96% or greater identity to immunogenic fragments of the WT1 protein sequences herein. Some embodiments relate to immunogenic fragments having 97% or greater identity to immunogenic fragments of the WT1 protein sequences herein. Some embodiments relate to immunogenic fragments having 98% or greater identity to immunogenic fragments of the WT1 protein sequences herein. Some embodiments relate to immunogenic fragments having 99% or greater identity to immunogenic fragments of the WT1 protein sequences 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 contain a leader sequence.

Some embodiments relate to immunogenic fragments of SEQ ID NO: 19 and SEQ ID NO: 21. The 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: 19 and/or SEQ ID NO: 21. In some embodiments, the immunogenic fragments comprise a sequence encoding a leader sequence, e.g., an immunoglobulin leader sequence such as an IgE leader sequence. In some embodiments, the immunogenic fragments do not contain a coding sequence encoding a leader sequence.

Immunogenic fragments of nucleic acids having nucleotide sequences identical to immunogenic fragments of SEQ ID NO: 19 and SEQ ID NO: 21 can be provided. 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 the WT1 nucleic acid sequences herein. Some embodiments relate to immunogenic fragments having 97% or greater identity to immunogenic fragments of the WT1 nucleic acid sequences herein. Some embodiments relate to immunogenic fragments having 98% or greater identity to immunogenic fragments of the WT1 nucleic acid sequences herein. Some embodiments relate to immunogenic fragments having 99% or greater identity to immunogenic fragments of the WT1 nucleic acid sequences herein. In some embodiments, the immunogenic fragment comprises a sequence encoding a leader sequence, e.g., an immunoglobulin leader sequence such as an IgE leader sequence. In some embodiments, the immunogenic fragment does not contain a coding sequence encoding a leader sequence.

(17)gp100

The vaccine of the present invention may comprise 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. It is a 70kDa type 1 transmembrane glycoprotein composed of 661 amino acids, which plays an important role in the biogenesis of melanosomes because it is involved in the maturation of melanosomes from stage I to stage II. gp100 drives the formation of stripes from multivesicular bodies and is directly involved in the biogenesis of premelanosomes. Relative to mature melanosomes, gp100 is enriched in premelanosomes but is overexpressed by proliferating neomelanosomes during tumor growth. The gp100 protein includes multiple immunogenic epitopes that are recognized by cytotoxic T lymphocytes from the peripheral blood of melanoma patients and from tumor-infiltrating lymphocytes.

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

(18) Viral antigens

Cancer antigens can be viral antigens, fragments thereof, or variants thereof. Viral antigens can be antigens from hepatitis B virus, hepatitis C virus, or human papillomavirus (HPV). HPV can be HPV 6, HPV 11, HPV 16, or HPV 18 as discussed below.

Viral antigens can induce antigen-specific T cells and/or high-titer antibody responses, thereby inducing or triggering an immune response to a cancer or tumor expressing the antigen or to the cancer or tumor reaction. In some embodiments, the immune response induced or triggered can be a cellular immune response, a humoral immune response, or a cellular and humoral immune response. In some embodiments, the cellular immune response induced or triggered may include the induction or secretion of interferon-γ (IFN-γ) and/or tumor necrosis factor α (TNF-α). In other embodiments, the immune response induced or triggered can reduce or inhibit one or more immunosuppressive factors that promote the growth of tumors or cancers expressing the antigen, for example, but not limited to factors that downregulate MHC presentation, factors that upregulate antigen-specific regulatory T cells (Treg), PD-L1, FasL, cytokines such as IL-10 and TFG-β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by immunosuppressive cells, CTLA-4, PD-1, MDSC, MCP-1, and immune checkpoint molecules, described in more detail below.

(a) Hepatitis B virus antigen

The viral antigen can be an antigen 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, such as a plasmid, encoding one or more antigens from HBV. The HBV antigen can be a full-length or immunogenic fragment of a full-length protein.

HBV antigens may comprise a consensus sequence and/or one or more modifications to improve expression. Genetic modifications (including codon optimization, RNA optimization, and the addition of efficient immunoglobulin leader sequences (to enhance the immunogenicity of the construct)) may be included in the modified consensus sequence. The consensus HBV antigens may comprise a signal peptide, such as an immunoglobulin signal peptide, such as an IgE or IgG signal peptide, and in some embodiments, an HA tag may be included. Immunogens may be designed to elicit a stronger and broader cellular immune response than corresponding codon-optimized immunogens.

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 common HBV core protein or a common HBV surface protein.

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 of the HBV genotype A core protein, or a HBV genotype A consensus core protein sequence.

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 of the HBV genotype B core protein, or a HBV genotype B consensus core protein sequence.

In 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 of the HBV genotype C core protein, or a HBV genotype C consensus core protein sequence.

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 of the HBV genotype D core protein, or a HBV genotype D consensus core protein sequence.

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 of the HBV genotype E core protein, or a HBV genotype E consensus core protein sequence.

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 of the HBV genotype F core protein, or a HBV genotype F consensus core protein sequence.

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 of the HBV genotype G core protein, or a HBV genotype G consensus core protein sequence.

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 of the HBV genotype H core protein, or a HBV genotype H consensus core protein sequence.

In 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 of an HBV genotype A surface protein, or an HBV genotype A consensus surface protein sequence.

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 of an HBV genotype B surface protein, or an HBV genotype B consensus surface protein sequence.

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 of an HBV genotype C surface protein, or an HBV genotype C consensus surface protein sequence.

In other embodiments, the HBV antigen can be an HBV genotype D consensus surface DNA sequence construct, an IgE leader sequence linked to a consensus sequence of an HBV genotype D surface protein, or an HBV genotype D consensus surface protein sequence.

In some embodiments, the HBV antigen can be an HBV genotype E consensus surface DNA sequence construct, an IgE leader sequence linked to a consensus sequence of an HBV genotype E surface protein, or an HBV genotype E consensus surface protein sequence.

In other embodiments, the HBV antigen can be an HBV genotype F consensus surface DNA sequence construct, an IgE leader sequence linked to a consensus sequence of an HBV genotype F surface protein, or an HBV genotype F consensus surface protein sequence.

In other embodiments, the HBV antigen can be an HBV genotype G consensus surface DNA sequence construct, an IgE leader sequence linked to a consensus sequence of an HBV genotype G surface protein, or an HBV genotype G consensus surface protein sequence.

In other embodiments, the HBV antigen can be an HBV genotype H consensus surface DNA sequence construct, an IgE leader sequence linked to a consensus sequence of an HBV genotype H surface protein, or an HBV genotype H consensus surface protein sequence.

(b) Hepatitis C virus antigen

The viral antigen can be an antigen from hepatitis C virus (HCV), a fragment thereof, or a variant thereof. HCV antigens can be associated with or cause liver cancer. In some embodiments, HCV antigens can be heterologous nucleic acid molecules encoding one or more antigens from HCV, such as plasmids. HCV antigens can be full-length or immunogenic fragments of a full-length protein.

In some embodiments, the HCV antigen can comprise a consensus sequence and/or one or more modifications to improve expression.Genetic modification (comprising the interpolation (to enhance the immunogenicity of construct) of codon optimization, RNA optimization and efficient immunoglobulin leader sequence) can be included in the modified consensus sequence.Total HCV antigen can comprise a signal peptide, such as an immunoglobulin signal peptide, such as IgE or IgG signal peptide, and in some embodiments, can comprise the HA tag.Immunogen can be designed to cause a stronger and wider cellular immune response than corresponding codon optimized immunogen.

The HCV antigen can be an HCV nucleocapsid protein (i.e., core protein), an HCV envelope protein (e.g., E1 and E2), an HCV nonstructural protein (e.g., NS1, NS2, NS3, NS4a, NS4b, NS5a, and NS5b), a fragment thereof, a variant thereof, or a combination thereof.

(c) Human papillomavirus

The viral antigen can be an antigen from HPV, a fragment thereof, or a variant thereof. HPV antigens can be from HPV types 16, 18, 31, 33, 35, 45, 52, and 58, which can cause cervical cancer, colorectal cancer, and/or other cancers. HPV antigens can be from HPV types 6 and/or 11, which cause genital warts and are known to be a cause of head and neck cancer. HPV antigens can be from HPV types 16 and/or 18, which cause cervical cancer. HPV antigens can be from HPV types 6, 11, and/or 16, which cause RRP and anal cancer. HPV cancer antigens may be further defined by U.S. Patent No. 8,168,769, filed on July 30, 2007, U.S. Patent No. 8,389,706, filed on January 21, 2010, U.S. Patent Application No. 13/271,576, filed on October 21, 2011, and U.S. Patent Application No. 61/777,198, filed on March 12, 2013 (each of which is incorporated by reference in its entirety).

The HPV antigens can be HPV E6 or E7 domains from each HPV type. For example, for HPV type 16 (HPV16), the HPV16 antigens can include HPV16 E6 antigen, HPV16 E7 antigen, fragments, variants, or combinations thereof. Similarly, the HPV antigens can be HPV 6 E6 and/or E7, HPV 11 E6 and/or E7, HPV 16 E6 and/or E7, HPV 18 E6 and/or E7, HPV 31 E6 and/or E7, HPV 33 E6 and/or E7, HPV 52 E6 and/or E7, or HPV 58 E6 and/or E7, fragments, variants, or combinations thereof.

(d) Herpes virus

The viral antigen may be a herpes virus antigen. The herpes virus antigen may be an antigen selected from the group consisting of: CMV, HSV1, HSV2, VZV, CeHV1, EBV, Roseolavirus, Kaposi's sarcoma-associated herpesvirus or MuHV, and preferably CMV, HSV1, HSV2, CeHV1 and VZV.

Common protein HCMV-gB (SEQ ID NO: 26), common protein HCMV-gM (SEQ ID NO: 28), common protein HCMV-gN (SEQ ID NO: 30), common protein HCMV-gH (SEQ ID NO: 32), common protein HCMV-gL (SEQ ID NO: 34), common protein HCMV-gO (SEQ ID NO: 36), common protein HCMV-UL128 (SEQ ID NO: 38), common protein HCMV-UL130 (SEQ ID NO: 40), common protein HCMV-UL-131A (SEQ ID NO: 42), common protein HCMV-UL-83 (pp65) (SEQ ID NO: 44).

The nucleic acid sequences include sequences encoding SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, or SEQ ID NO: 44. Nucleic acid molecules encoding consensus amino acid sequences are generated. The vaccine may comprise one or more nucleic acid sequences encoding one or more consensus forms of an immunogenic protein selected from the group of sequences generated to optimize stability and expression in humans. Nucleic acid sequence encoding the consensus protein HCMV-gB (SEQ ID NO: 25), nucleic acid sequence encoding the consensus protein HCMV-gM (SEQ ID NO: 27), nucleic acid sequence encoding the consensus protein HCMV-gN (SEQ ID NO: 29), nucleic acid sequence encoding the consensus protein HCMV-gH (SEQ ID NO: 31), nucleic acid sequence encoding the consensus protein HCMV-gL (SEQ ID NO: 33), nucleic acid sequence encoding the consensus protein HCMV-gO (SEQ ID NO: 35), nucleic acid sequence encoding the consensus protein HCMV-UL128 (SEQ ID NO: 37), nucleic acid sequence encoding the consensus protein HCMV-UL130 (SEQ ID NO: 39), nucleic acid sequence encoding the consensus protein HCMV-UL-131A (SEQ ID NO: 41), nucleic acid sequence encoding the consensus protein HCMV-UL-83 (pp65) (SEQ ID NO: 43). The nucleic acid sequence may additionally have an IgE leader sequence attached to the 5' end.

Given the evolutionary divergence from clinical isolates and the extensive genetic differences between circulating human strains, consensus amino acid sequences have been generated for each immunogenic protein. The consensus amino acid sequences for gB, gM, gH, gL, gE, gI, gK, gC, gD, UL128, UL130, UL-131A, and UL-83 (pp65) are based on sequences from human clinical isolates. Due to the vast evolutionary divergence of the gN protein, a consensus sequence was generated from only one (gN-4c) of the seven serotypes representing the most seroprevalent (gN-4). Similarly, in the case of gO, a consensus amino acid sequence was generated from one (gO-5) of the eight serotypes (because this particular serotype is reported to be related to the gN-4c serotype).

As mentioned above, herpesvirus antigen can be total herpesvirus.Total herpesvirus antigen can be provided with signal peptide.In some embodiments, the IgE leader sequence is connected to the N-terminal.As described herein, when referring to the signal peptide that is connected to the N-terminal of consensus sequence, it is intended to clearly include the embodiment that the N-terminal Xaa residue of wherein consensus sequence is replaced by signal peptide.That is, as used herein, Xaa is intended to refer to any amino acid or no amino acid.The protein that comprises the consensus sequence shown in SEQ ID NO:26,28,30,32,34,36,38,40,42,44 herein can comprise those sequences that do not contain N-terminal Xaa.

In some embodiments, the present invention provides the nucleic acid construct of the fusion protein that is expressed as two or more herpesvirus antigens and is connected to each other by the proteolytic cleavage site.The nucleic acid construct of the fusion protein that is expressed as two or more herpesvirus antigens and is connected to each other by the proteolytic cleavage site is provided.The furin proteolytic cleavage site is the example of the proteolytic cleavage site that can connect herpesvirus antigen in the fusion protein expressed by construct.The viral cancer antigen of herpes family can also be any antigen disclosed in U.S. Patent Application No. 13/982,457 (its content is incorporated to by reference in its entirety).

3. Vaccines combined with immune checkpoint inhibitors

Vaccines may also include one or more inhibitors (i.e., immune checkpoint inhibitors) of one or more immune checkpoint molecules. Immune checkpoint molecules are described in more detail below. Immune checkpoint inhibitors are any nucleic acid or protein that prevents any component in the immune system from presenting, presenting and/or differentiating, presenting and/or differentiating in MHC class, T cells, B cells, any cytokine, chemokine or signal transduction for immunocyte proliferation and/or differentiation.

Such inhibitors can be nucleic acid sequences, amino acid sequences, small molecules or combinations thereof. The nucleic acid sequence can be DNA, RNA, cDNA, variants thereof, fragments thereof or combinations thereof. The nucleic acid can also include another sequence encoding a linker or tag sequence connected to an immune checkpoint inhibitor by a peptide bond. Small molecules can be low molecular weight, such as organic or inorganic compounds with less than 800 daltons, which can be used as enzyme substrates, ligands (analogs thereof) bound by proteins or nucleic acids, or regulators of biological processes. The amino acid sequence can be a protein, a peptide, variants thereof, fragments thereof or combinations thereof.

In some embodiments, the immune checkpoint inhibitor can be one or more nucleic acid sequences encoding an antibody, a variant thereof, a fragment thereof, or a combination thereof. In other embodiments, the immune checkpoint inhibitor can be an antibody, a variant thereof, a fragment thereof, or a combination thereof.

a. Immune checkpoint molecules

The immune checkpoint molecule can be a nucleic acid sequence, an amino acid sequence, a small molecule 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 may also include another sequence encoding a linker or tag sequence connected to an immune checkpoint inhibitor by a peptide bond. Small molecules can be low molecular weight, such as organic or inorganic compounds with less than 800 daltons, which can be used as enzyme substrates, ligands (analogs thereof) bound by proteins or nucleic acids, or regulators of biological processes. The amino acid sequence can be a protein, a peptide, a variant thereof, a fragment thereof or a combination thereof.

(1) PD-1 and PD-L1

Immune checkpoint molecules can be programmed cell death protein 1 (PD-1), programmed cell death ligand 1 (PD-L1), fragments thereof, variants thereof, or combinations thereof. PD-1 is a cell surface protein encoded by the PDCD1 gene. PD-1 is a member of the immunoglobulin superfamily and is expressed on T cells and proto-B cells, and therefore contributes to the fate and/or differentiation of these cells. In particular, PD-1 is a type 1 membrane protein of the CD28/CTLA-4 family of T cell regulators and negatively regulates T cell receptor (TCR) signals, thereby negatively regulating the immune response. PD-1 can negatively regulate CD8+ T cell responses, and thereby inhibit CD8-mediated cytotoxicity and enhance tumor growth.

PD-1 has two ligands, PD-L1 and PD-L2, which are members of the B7 family. PD-L1 is upregulated on macrophages and dendritic cells (DCs) in response to LPS and GM-CSF treatment, as well as on T cells and B cells during TCR and B cell receptor signaling. PD-L1 is expressed by many tumor cell lines, including myeloma, mastocytoma, and melanoma.

b. Anti-immune checkpoint molecule antibodies

As described above, the immune checkpoint inhibitor can be an antibody. The antibody can bind to or react with an antigen (i.e., the immune checkpoint molecule described above). Therefore, the antibody can be considered an anti-immune checkpoint molecule antibody or an immune checkpoint molecule antibody. The antibody can be encoded by a nucleic acid sequence contained in

An antibody may include a heavy chain polypeptide and a light chain polypeptide. The heavy chain polypeptide may include a variable heavy chain (VH) region and/or at least one constant heavy chain (CH) region. The at least one constant heavy chain region may include a constant heavy chain region 1 (CH1), a constant heavy chain region 2 (CH2), a constant heavy chain region 3 (CH3), and/or a hinge region.

In some embodiments, the heavy chain polypeptide may include a VH region and a CH1 region. In other embodiments, the heavy chain polypeptide may include a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region.

A heavy chain polypeptide may include a set of complementary determining regions ("CDRs"). The CDR set may contain three hypervariable regions of the VH region. Starting from the N-terminus of the heavy chain polypeptide, these CDRs are designated "CDR1," "CDR2," and "CDR3," respectively. The CDR1, CDR2, and CDR3 of the heavy chain polypeptide may contribute to the binding or recognition of an antigen.

A light chain polypeptide may include a variable light chain (VL) region and/or a constant light chain (CL) region. A light chain polypeptide may include a complementarity determining region ("CDR") set. The CDR set may contain three hypervariable regions of the VL region. Starting from the N-terminus of the light chain polypeptide, these CDRs are designated as "CDR1," "CDR2," and "CDR3," respectively. The CDR1, CDR2, and CDR3 of a light chain polypeptide may contribute to the binding or recognition of an antigen.

The antibody may comprise a set of heavy and light chain complementary determining regions ("CDRs") interposed between heavy and light chain framework ("FR") sets, respectively, which provide support for the CDRs and define the spatial relationship between the CDRs. The CDR set may contain three hypervariable regions from the heavy or light chain V region. Starting from the N-terminus of the heavy or light chain, these regions are designated "CDR1," "CDR2," and "CDR3," respectively. The antigen-binding site may thus comprise six CDRs, comprising a CDR set from each of the heavy and light chain V regions.

The antibody can be an immunoglobulin (Ig). Ig can be, for example, IgA, IgM, IgD, IgE, and IgG. An immunoglobulin can include a heavy chain polypeptide and a light chain polypeptide. The heavy chain polypeptide of an immunoglobulin can include a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region. The light chain polypeptide of an immunoglobulin can include a VL region and a CL region.

In addition, the proteolytic enzyme papain preferentially cleaves IgG molecules to produce several fragments, two of which (F(ab) fragments) each comprise a covalent heterodimer comprising a complete antigen-binding site. Pepsin can cleave IgG molecules to provide several fragments, including F(ab') ₂ fragments, which contain two antigen-binding sites. Thus, the antibody can be a Fab or F(ab') _2. Fab can include a heavy chain polypeptide and a light chain polypeptide. The heavy chain polypeptide of Fab can include a VH region and a CH1 region. The light chain of Fab can include a VL region and a CL region.

The antibody can be a polyclonal or monoclonal antibody. The antibody can be a chimeric antibody, a single-chain antibody, an affinity matured antibody, a human antibody, a humanized antibody or a fully human antibody. A humanized antibody can be an antibody from a non-human species that combines with one or more complementary determining regions (CDRs) from a non-human species and with the desired antigen from the framework region of a human immunoglobulin molecule.

(1) PD-1 antibodies

The anti-immune checkpoint molecule antibody can be an anti-PD-1 antibody (also referred to herein as a "PD-1 antibody"), a variant thereof, a fragment thereof, or a combination thereof. The PD-1 antibody can be nivolumab. The anti-PD-1 antibody can inhibit PD-1 activity, thereby inducing, triggering, or enhancing an immune response against a tumor or cancer and reducing tumor growth.

(2) PD-L1 antibodies

The anti-immune checkpoint molecule antibody can be an anti-PD-L1 antibody (also referred to herein as a "PD-L1 antibody"), a variant thereof, a fragment thereof, or a combination thereof. The anti-PD-L1 antibody can inhibit PD-L1 activity, thereby inducing, triggering, or enhancing an immune response against a tumor or cancer or reducing tumor growth.

4. Vaccine Constructs and Plasmids

The vaccine may comprise a nucleic acid construct or plasmid encoding the above-mentioned antigens and/or antibodies. The nucleic acid construct or plasmid may include or contain one or more heterologous nucleic acid sequences. Provided herein are genetic constructs that may include nucleic acid sequences encoding the above-mentioned antigens and/or antibodies. The genetic construct may be present in the cell as a functional extrachromosomal molecule. The genetic construct may be a linear minichromosome or plasmid or cosmid comprising a centromere and telomeres. The genetic construct may include or contain one or more heterologous nucleic acid sequences.

The genetic construct may be in the form of a plasmid that expresses the above-mentioned antigens and/or antibodies in any order.

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

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

The nucleic acid sequence may be formed into a genetic construct that may be a vector. The vector may be capable of expressing the antigen and/or antibody in mammalian cells in an amount effective to elicit an immune response in the mammal. The vector may be a recombinant. The vector may contain heterologous nucleic acid encoding the antigen and/or antibody. The vector may be a plasmid. The vector may be used to transfect cells with the nucleic acid encoding the antigen and/or antibody, and the transformed cells may be cultured and maintained under conditions where expression of the antigen and/or antibody can occur.

The coding sequence can be optimized for stability and high levels of expression.In some cases, codons are selected to reduce the formation of secondary structure of the RNA, such as that formed by intramolecular bonding.

The vector may comprise a heterologous nucleic acid encoding the above-mentioned antigens and/or antibodies and may further comprise a start codon (which may be upstream of one or more cancer antigen coding sequences) and a stop codon (which may be downstream of the coding sequences of the above-mentioned antigens and/or antibodies). The start and stop codons may be in frame with the coding sequences of the above-mentioned antigens and/or antibodies. The vector may further comprise a promoter operatively linked to the coding sequences of the above-mentioned antigens and/or antibodies. The promoter operatively linked to the above-mentioned antigens and/or antibodies may be a promoter from Simian Virus 40 (SV40), a mouse mammary tumor virus (MMTV) promoter, a human immunodeficiency virus (HIV) promoter such as a bovine immunodeficiency virus (BIV) long terminal repeat (LTR) promoter, a Moloney virus promoter, an avian leukocytogenes virus (ALV) promoter, a cytomegalovirus (CMV) promoter such as a CMV immediate early promoter, an Epstein-Barr virus (EBV) promoter, or a Rous sarcoma virus (RSV) promoter. The promoter can also be a promoter from a human gene such as human actin, human myosin, human hemoglobin, human muscle creatine, or human metallothionein. The promoter can also be a tissue-specific promoter, such as a muscle or skin-specific promoter (natural or synthetic). Examples of such promoters are described in U.S. Patent Application Publication No. US20040175727 (the contents of which are incorporated herein in their entirety).

The vector may further comprise a polyadenylation signal, which may be downstream of the coding sequence for the above-mentioned antigen and/or antibody. The polyadenylation signal may be an SV40 polyadenylation signal, an LTR polyadenylation signal, a bovine growth hormone (bGH) polyadenylation signal, a human growth hormone (hGH) polyadenylation signal, or a human β-globin polyadenylation signal. The SV40 polyadenylation signal may be a polyadenylation signal from a pCEP4 vector (Invitrogen, San Diego, CA).

The vector may also contain an enhancer upstream of the above-mentioned antigen and/or antibody. The enhancer may be necessary for DNA expression. The enhancer may be human actin, human myosin, human hemoglobin, human muscle creatine, or a viral enhancer, such as an enhancer from CMV, HA, RSV, or EBV. Polynucleotide functional enhancers are described in U.S. Patent Nos. 5,593,972, 5,962,428, and WO94/016737, the contents of each of which are fully incorporated by reference.

The vector may also contain a mammalian origin of replication to maintain the vector extrachromosomally and to produce multiple copies of the vector in the cell. The vector may be pVAX1, pCEP4, or pREP4 from Invitrogen (San Diego, CA), which may contain the Epstein-Barr virus origin of replication and the EBNA-1 coding region, which can produce high-copy episomal replication without the need for integration. The vector may be pVAX1 or a modified pVax1 variant such as the variant plasmid described herein. The variant pVax1 plasmid is a 2998 base pair variant of the backbone vector plasmid pVAX1 (Invitrogen, Carlsbad, CA). The CMV promoter is located at bases 137-724. The T7 promoter/priming site is located at bases 664-683. The multiple cloning site is located at bases 696-811. The bovine GH polyadenylation signal is located at bases 829-1053. The kanamycin resistance gene is located at bases 1226-2020. The pUC origin of replication is located at bases 2320-2993.

Based on the sequence of pVAX1 available from Invitrogen, the following mutations were found in the sequence of pVAX1 used as the backbone for plasmids 1-6 presented herein:

Base pairs 2, 3, and 4 in the backbone upstream of the CMV promoter were changed from ACT to CTG.

The backbone of the vector may be pAV0242. The vector may be a replication-defective adenovirus type 5 (Ad5) vector.

The vector may also contain regulatory sequences that may be well suited for gene expression in mammalian or human cells to which the vector is administered.One or more cancer antigen sequences disclosed herein may contain codons that may allow for more efficient transcription of the coding sequence in the host cell.

The vector can be pSE420 (Invitrogen, San Diego, Calif.), which can be used to produce proteins in Escherichia coli (E. coli). The vector can also be pYES2 (Invitrogen, San Diego, Calif.), which can be used to produce proteins in yeast strains of Saccharomyces cerevisiae. The vector can also have a MAXBAC ^™ complete baculovirus expression system (Invitrogen, San Diego, Calif.), which can be used to produce proteins in insect cells. The vector can also be pcDNA 1 or pcDNA3 (Invitrogen, San Diego, Calif.), which can be used to produce proteins in mammalian cells such as Chinese hamster ovary (CHO) cells. The vector can be an expression vector or system for producing proteins by conventional techniques and readily available starting materials (including Sambrook et al., Molecular Cloning and Laboratory Manual, 2nd edition, Cold Spring Harbor (1989), which is incorporated by reference in its entirety).

In some embodiments, the vector may comprise one or more of the nucleic acid sequences of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15 and/or 17.

5. Pharmaceutical compositions of vaccines

The vaccine may be in the form of a pharmaceutical composition. The pharmaceutical composition may comprise the vaccine. The pharmaceutical composition may comprise from about 5 nanograms to about 10 mg of the vaccine's DNA. In some embodiments, a pharmaceutical composition according to the present invention comprises from about 25 nanograms to about 5 mg of the vaccine's DNA. In some embodiments, the pharmaceutical composition contains from about 50 nanograms to about 1 mg of the vaccine's DNA. In some embodiments, the pharmaceutical composition contains from about 0.1 to about 500 micrograms of the vaccine's DNA. In some embodiments, the pharmaceutical composition contains from about 1 to about 350 micrograms of the vaccine's DNA. In some embodiments, the pharmaceutical composition contains from about 5 to about 250 micrograms of the vaccine's DNA. In some embodiments, the pharmaceutical composition contains from about 10 to about 200 micrograms of the vaccine's DNA. In some embodiments, the pharmaceutical composition contains from about 15 to about 150 micrograms of the vaccine's DNA. In some embodiments, the pharmaceutical composition contains from about 20 to about 100 micrograms of the vaccine's DNA. In some embodiments, the pharmaceutical composition contains from about 25 to about 75 micrograms of the vaccine's DNA. In some embodiments, the pharmaceutical composition contains from about 30 to about 50 micrograms of the vaccine's DNA. In some embodiments, the pharmaceutical composition contains about 35 to about 40 micrograms of vaccine DNA. In some embodiments, the pharmaceutical composition contains about 100 to about 200 micrograms of vaccine DNA. In some embodiments, the pharmaceutical composition contains about 10 micrograms to about 100 micrograms of vaccine DNA. In some embodiments, the pharmaceutical composition contains about 20 micrograms to about 80 micrograms of vaccine DNA. In some embodiments, the pharmaceutical composition contains about 25 micrograms to about 60 micrograms of vaccine DNA. In some embodiments, the pharmaceutical composition contains about 30 nanograms to about 50 micrograms of vaccine DNA. In some embodiments, the pharmaceutical composition contains about 35 nanograms to about 45 micrograms of vaccine DNA. In some preferred embodiments, the pharmaceutical composition contains about 0.1 to about 500 micrograms of vaccine DNA. In some preferred embodiments, the pharmaceutical composition contains about 1 to about 350 micrograms of vaccine DNA. In some preferred embodiments, the pharmaceutical composition contains about 25 to about 250 micrograms of vaccine DNA. In some preferred embodiments, the pharmaceutical composition contains about 100 to about 200 micrograms of vaccine DNA.

In some embodiments, the pharmaceutical composition according to the present invention comprises at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nanograms of the DNA of the vaccine. In some embodiments, the pharmaceutical composition may comprise at least 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230 30, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 605, 610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695, 700, 705, 710, 715, 720, 725, 730, 735, 740, 745, 750, 755, 760, 765, 770, 775, 780, 785 , 790, 795, 800, 805, 810, 815, 820, 825, 830, 835, 840, 845, 850, 855, 860, 865, 870, 875, 880, 885, 890, 895. 900, 905, 910, 915, 920, 925, 930, 935, 940, 945, 950, 955, 960, 965, 970, 975, 980, 985, 990, 995 or 1000 micrograms of vaccine DNA. In some embodiments, the pharmaceutical composition may comprise at least 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 mg or more of the DNA of the vaccine.

In other embodiments, the pharmaceutical composition may comprise up to and including 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nanograms of vaccine DNA. In some embodiments, the pharmaceutical composition may comprise up to and including 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350 25, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 460 55, 460, 465, 470, 475, 480, 485, 490, 495, 500, 605, 610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695, 700, 705, 710, 715, 720, 725, 730, 735, 740, 745, 750, 755, 760, 765, 770, 775, 780, 85, 790, 795, 800, 805, 810, 815, 820, 825, 830, 835, 840, 845, 850, 855, 860, 865, 870, 875, 880, 885, 890, 895, 900, 905, 910, 915, 920, 925, 930, 935, 940, 945, 950, 955, 960, 965, 970, 975, 980, 985, 990, 995, or 1000 micrograms of vaccine DNA. In some embodiments, the pharmaceutical composition may contain up to and including 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 mg of the vaccine's DNA.

The pharmaceutical composition may also contain other agents for the purpose of formulation of the mode of administration to be used. In the case where the pharmaceutical composition is an injectable pharmaceutical composition, it is sterile, pyrogen-free, and particle-free. Isotonic formulations are preferably used. Generally, additives for isotonicity may include sodium chloride, glucose, mannitol, sorbitol, and lactose. In some cases, isotonic solutions such as phosphate-buffered saline are preferred. Stabilizers include gelatin and albumin. In some embodiments, a vasoconstrictor is added to the formulation.

The vaccine can also include a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient can be a functional molecule as a vehicle, adjuvant, carrier or diluent. The pharmaceutically acceptable excipient can be a transfection facilitator, which can include a surfactant, such as immune-stimulating complexes (ISCOMS), Freund's incomplete adjuvant, LPS analogs, monophosphoryl lipid A, muramyl peptides, quinone analogs, vesicles such as squalene and squalene, hyaluronic acid, lipids, liposomes, calcium ions, viral proteins, polyanions, polycations or nanoparticles or other known transfection facilitators.

The transfection facilitator is a polyanion, a polycation, including poly-L-glutamic acid (LGS) or a lipid. The transfection facilitator is poly-L-glutamic acid, and more preferably, poly-L-glutamic acid is present in the vaccine at a concentration lower than 6 mg/ml. The transfection facilitator may also include surfactants such as immune-stimulating complexes (ISCOMS), Freund's incomplete adjuvant, LPS analogs, including monophosphoryl lipid A, muramyl peptides, quinone analogs, and vesicles such as squalene and squalene, and hyaluronic acid may also be administered in conjunction with a genetic construct. In some embodiments, the DNA vector vaccine may also include a transfection facilitator (such as a lipid, a liposome, including lecithin liposomes or other liposomes known in the art), as a DNA-liposome mixture (see, for example, WO9324640), calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other known transfection facilitators. Preferably, the transfection facilitator is a polyanion, a polycation, including poly-L-glutamate (LGS) or a lipid. The concentration of the transfection agent in the vaccine is less than 4 mg/ml, less than 2 mg/ml, less than 1 mg/ml, less than 0.750 mg/ml, less than 0.500 mg/ml, less than 0.250 mg/ml, less than 0.100 mg/ml, less than 0.050 mg/ml or less than 0.010 mg/ml.

The pharmaceutically acceptable excipient can be an adjuvant. The adjuvant can be other genes expressed in a selectable plasmid or be delivered as a protein combined with the above-mentioned plasmid in a vaccine. The adjuvant can be selected from the material of the following group consisting of: alpha-interferon (IFN-α), beta-interferon (IFN-β), gamma-interferon, platelet-derived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermal growth factor (EGF), skin T cell capture chemokine (CTACK), thymic epithelial cell expression chemokine (TECK), mucosa-associated epithelial chemokine (MEC), IL-12, IL-15, MHC, CD80, CD86, including IL-15 (deletion signal sequence and optionally comprising a signal peptide from IgE). The adjuvant can be IL-12, IL-15, IL-28, CTACK, TECK, platelet-derived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermal growth factor (EGF), IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-18 or a combination thereof. In an exemplary embodiment, the adjuvant is IL-12.

Other genes that may be useful adjuvants include those encoding MCP-1, MIP-1a, MIP-1p, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, p150.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, mutant forms of IL-18, CD40, CD40L, vascular endothelial growth factor receptor agonists, and leukocyte antigen 1 (VEGF). Growth factors, fibroblast growth factor, IL-7, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Flt, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, inactive NIK, SAP K, SAP-1, JNK, interferon response genes, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, Ox40, Ox40LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1, TAP2 and their functional fragments.

6. Combination vaccines for treating specific cancers

Vaccine can be in the form of various combinations of cancer antigens as described above to treat cancer or tumor.Depending on the combination of one or more cancer antigens, various cancers or other tumor types can be targeted by vaccine.These cancers can include melanoma, blood cancer (for example, leukemia, lymphoma, myeloma), lung cancer, esophageal squamous cell carcinoma, bladder cancer, colorectal cancer, esophageal cancer, gastric cancer, liver cancer, head and neck cancer, brain cancer, anal cancer, non-small cell lung cancer, pancreatic cancer, synovial cancer, prostate cancer, testicular cancer, liver cancer, cervical cancer, recurrent respiratory papillomatosis, skin cancer and gastric cancer.Figure 15 provides the example of the specific combination of antigens that can be used for treating specific cancers.

a. Melanoma

Vaccines can combine one or more cancer antigens such as tyrosinase, PRAME, or GP100-Trp2 to treat or prevent melanoma (see Figure 15). Vaccines can further combine one or more cancer antigens tyrosinase, PRAME, or GP100-Trp2 with any one or more cancer antigens hTERT, NY-ESO-1, MAGE-A1, or WTI to treat or prevent melanoma. Other combinations of cancer antigens can also be used to treat or prevent melanoma.

b. Head and neck cancer

Vaccines can include the cancer antigens HPV 16 E6/E7 to treat or prevent head and neck cancer (see Figure 15). Vaccines can further combine the cancer antigens HPV 16 E6/E7 with any one or more of the cancer antigens hTERT, NY-ESO-1, MAGE-A1, or WTI for the treatment or prevention of head and neck cancer. Other combinations of cancer antigens can also be used to treat or prevent head and neck cancer.

c. Recurrent respiratory papillomatosis/anal cancer

The vaccine can combine one or more cancer antigens such as HPV 6, HPV11 or HPV 16 to treat or prevent recurrent respiratory papillomatosis or anal cancer (see Figure 15). The vaccine can further combine one or more cancer antigens HPV 6, HPV11 or HPV16 with one or more cancer antigens hTERT, NY-ESO-1, MAGE-A1 or WTI to treat or prevent recurrent respiratory papillomatosis or anal cancer. Other combinations of cancer antigens can also be used to treat or prevent recurrent respiratory papillomatosis or anal cancer.

d. Cervical cancer

Vaccines can combine one or more cancer antigens such as HPV 16 E6/E7 or HPV 18 E6/E7 to treat or prevent cervical cancer (see Figure 15). Vaccines can further combine one or more cancer antigens HPV 16 E6/E7 or HPV 18 E6/E7 with one or more cancer antigens hTERT, NY-ESO-1, MAGE-A1, or WTI to treat or prevent cervical cancer. Other combinations of cancer antigens can also be used to treat or prevent cervical cancer.

e. Liver cancer

Vaccines can combine one or more cancer antigens such as HBV core antigen, HBV surface antigen, HCV NS34A, HCV NS5A, HCV NS5B, or HCV NS4B to treat or prevent liver cancer (see Figure 15). Vaccines can further combine one or more cancer antigens HBV core antigen, HBV surface antigen, HCV NS34A, HCV NS5A, HCV NS5B, or HCV NS4B with one or more cancer antigens hTERT, NY-ESO-1, MAGE-A1, or WTI to treat or prevent liver cancer. Other combinations of cancer antigens can also be used to treat or prevent liver cancer.

f. Glioblastoma

The vaccine may comprise CMV to treat or prevent glioblastoma (see Figure 15). The vaccine may further combine CMV with one or more of the cancer antigens hTERT, NY-ESO-1, MAGE-A1, or WTI for the treatment or prevention of glioblastoma. Other combinations of cancer antigens may also be used to treat or prevent glioblastoma.

g. Prostate

Vaccines can combine one or more cancer antigens such as PSA, PSMA, or STEAP to treat or prevent prostate cancer (see Figure 15). Vaccines can further combine one or more cancer antigens PSA, PSMA, or STEAP with one or more cancer antigens hTERT, NY-ESO-1, MAGE-A1, or WTI to treat or prevent prostate cancer. Other combinations of cancer antigens can also be used to treat or prevent prostate cancer.

h. Blood cancer (e.g., leukemia, lymphoma, myeloma)

Vaccines can combine one or more cancer antigens such as PRAME, WT-1, hTERT to treat or prevent blood cancers such as leukemia, lymphoma, and myeloma (see Figure 51). Vaccines can further combine one or more cancer antigens PRAME, WT-1, hTERT with one or more of the cancer antigens NY-ESO-1 or MAGE-A1 to treat or prevent blood cancers such as leukemia, lymphoma, and myeloma. Other combinations of cancer antigens can also be used to treat or prevent blood cancers such as leukemia, lymphoma, and myeloma.

7. Inoculation Method

Provided herein are methods for treating or preventing cancer using pharmaceutical preparations (to provide genetic constructs and proteins of one or more cancer antigens as described above), wherein the antigens contain epitopes that make them particularly effective immunogens for which an immune response to one or more cancer antigens can be induced. Methods for administering vaccines or vaccinations can be provided to induce therapeutic and/or prophylactic immune responses. Vaccination methods can produce an immune response to one or more cancer antigens as described above in mammals. The vaccine can be administered to an individual to modulate the activity of the mammalian immune system and enhance the immune response. Administration of the vaccine can be a transfection in which one or more cancer antigens as disclosed herein are expressed in a cell and thereby delivered to a nucleic acid molecule on the cell surface, where the immune system recognizes the antigens on the cell surface and induces a cellular response, a humoral response, or a cellular and humoral response. Administration of the vaccine can be used to induce or elicit an immune response to one or more cancer antigens of a mammal by administering a vaccine as described herein to the mammal.

When a vaccine is administered to a mammal and the vector is administered to the cells of the mammal, the transfected cells will express and secrete one or more cancer antigens as disclosed herein. These secreted proteins or synthetic antigens will be recognized as foreign by the immune system, which will stimulate an immune response that may include the following responses: antibodies produced against the one or more cancer antigens, and T cell responses specific for the one or more cancer antigens. In some instances, a mammal utilizing the vaccination discussed herein will have an activated immune system, and when attacked with one or more cancer antigens as disclosed herein, the activated immune system will allow for rapid clearance of subsequent cancer antigens as disclosed herein, whether by humoral immune response, cellular immune response, or both cellular and humoral immune response. Vaccines can be administered to an individual to modulate the activity of the individual's immune system, thereby enhancing the immune response.

Methods of administering vaccine DNA are described in US Patent Nos. 4,945,050 and 5,036,006, both of which are incorporated herein by reference in their entirety.

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

The vaccine dosage may be 1 μg to 10 mg of active ingredient/kg body weight/time, and may be 20 μg to 10 mg of component/kg body weight/time. The vaccine may be administered once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days. The number of doses of the vaccine for effective treatment may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more doses.

a. Methods of generating immune responses using vaccines

Vaccines can be used to generate an immune response in a mammal, including a therapeutic or prophylactic immune response. The immune response can generate antibodies and/or killer T cells against one or more cancer antigens as disclosed herein. Such antibodies and T cells can be isolated.

Some embodiments provide methods for generating an immune response against one or more of the cancer antigens disclosed herein, comprising administering a vaccine to an individual. Some embodiments provide methods for prophylactically vaccinating an individual against one or more cancers or tumors expressing the cancer antigens described above, comprising administering a vaccine. Some embodiments provide methods for therapeutically vaccinating an individual suffering from a cancer or tumor expressing one or more of the cancer antigens, comprising administering a vaccine. Prior to administering the vaccine, diagnosis of a cancer or tumor expressing one or more of the cancer antigens disclosed herein may be routinely performed.

b. Cancer treatment methods using vaccines

Vaccines can be used to generate or elicit an immune response in a mammal that is reactive with or directed against a cancer or tumor (e.g., melanoma, head and neck cancer, cervical cancer, liver cancer, prostate cancer, blood cancer, esophageal squamous cell carcinoma, gastric cancer) in a mammal or subject in need thereof. The elicited immune response can prevent the growth of the cancer or tumor.

The immune response elicited can prevent and/or reduce the metastasis of cancer cells or tumor cells. Therefore, vaccines can be used to treat and/or prevent cancer or tumors in mammals or subjects to which the vaccine is administered. Depending on the antigen used in the vaccine, the growth of the cancer or tumor being treated can be any type of cancer such as, but not limited to, melanoma, blood cancer (e.g., leukemia, lymphoma, myeloma), lung cancer, esophageal squamous cell carcinoma, bladder cancer, colorectal cancer, esophageal cancer, gastric cancer, liver cancer, head and neck cancer, brain cancer, anal cancer, non-small cell lung cancer, pancreatic cancer, synovial cancer, prostate cancer, testicular cancer, liver cancer, cervical cancer, recurrent respiratory papillomatosis, skin cancer and gastric cancer.

In some embodiments, administration of the vaccine can mediate clearance of tumor cells or prevent their growth by inducing the following responses: (1) humoral immunity via a B cell response (to produce antibodies that block the production of monocyte chemoattractant protein-1 (MCP-1), thereby delaying myeloid-derived suppressor cells (MDSCs) and inhibiting tumor growth); (2) increasing cytotoxic T lymphocytes such as CD8 ⁺ (CTLs) to attack and kill tumor cells; (3) enhancing T helper cell responses; (4) and enhancing inflammatory responses via IFN-γ and TFN-α, or preferably all of the foregoing responses.

In some embodiments, the immune response can produce a humoral immune response and/or an antigen-specific cytotoxic T lymphocyte (CTL) response that does not cause damage to or inflammation of various tissues or systems (e.g., the brain or nervous system, etc.) of the subject to which the vaccine is administered.

In some embodiments, administration of the vaccine can increase tumor-free survival in a subject, reduce tumor mass, increase tumor survival, or a combination thereof. Administration of the vaccine can increase tumor-free survival in a subject by 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 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%. Administration of the vaccine can reduce tumor mass in immunized subjects by 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 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%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69 and 70%. Administration of the vaccine can prevent and block the increase of monocyte chemoattractant protein 1 (MCP-1), a cytokine secreted by myeloid-derived suppressor cells, in the subject. In some embodiments, administration of the vaccine can prevent and block the increase of MCP-1 in the cancer tissue or tumor tissue of the subject, thereby reducing the angiogenesis of the cancer tissue or tumor tissue of the subject.

Administration of the vaccine increased tumor survival in subjects by 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 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%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% and 70%. In some embodiments, vaccines can be administered peripherally (as described in more detail below) to establish an antigen-specific immune response that targets cancerous or tumor cells or tissues to clear or eliminate cancers or tumors that express one or more cancer antigens without injuring or causing disease or death in the subject to whom the vaccine is administered.

Administration of the vaccine can enhance the subject's cellular immune response by about 50-fold to about 6000-fold, about 50-fold to about 5500-fold, about 50-fold to about 5000-fold, about 50-fold to about 4500-fold, about 100-fold to about 6000-fold, about 150-fold to about 6000-fold, about 200-fold to about 6000-fold, about 250-fold to about 6000-fold, or about 300-fold to about 6000-fold. In some embodiments, administration of the vaccine may enhance the subject's cellular immune response 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, 2200-fold, 2300-fold, 2400-fold, 2500-fold, 00 times, 2600 times, 2700 times, 2800 times, 2900 times, 3000 times, 3100 times, 3200 times, 3300 times, 3400 times, 3500 times, 3600 times, 3700 times, 3800 times, 3900 times, 4000 times, 4100 times, 4200 times, 4300 times, 4400 times, 4500 times, 4600 times, 4700 times, 4800 times, 4900 times, 5000 times, 5100 times, 5200 times, 5300 times, 5400 times, 5500 times, 5600 times, 5700 times, 5800 times, 5900 times or 6000 times.

Administration of the vaccine can increase the subject's interferon gamma (IFN-γ) levels by about 50-fold to about 6000-fold, about 50-fold to about 5500-fold, about 50-fold to about 5000-fold, about 50-fold to about 4500-fold, about 100-fold to about 6000-fold, about 150-fold to about 6000-fold, about 200-fold to about 6000-fold, about 250-fold to about 6000-fold, or about 300-fold to about 6000-fold. In some embodiments, administration of the vaccine may increase IFN-γ levels in a subject 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, 2200-fold, 2300-fold, 2400-fold, 2500-fold, 2600-fold, 2700-fold, 2800-fold, 2900-fold, 3000-fold, 3100-fold, 3200-fold, 3300-fold, 3400-fold, 3500-fold, 3600-fold, 3700-fold, 3800-fold, 3900-fold, 4000-fold, 4100-fold, 4200-fold, 4300-fold, 4400-fold, 4500-fold, 4600-fold, 4700-fold, 4800-fold, 4900-fold, 5000-fold, 5100-fold, 5100-fold, 5100-fold 500 times, 2600 times, 2700 times, 2800 times, 2900 times, 3000 times, 3100 times, 3200 times, 3300 times, 3400 times, 3500 times, 3600 times, 3700 times, 3800 times, 3900 times, 4000 times, 4100 times, 4200 times, 4300 times, 4400 times, 4500 times, 4600 times, 4700 times, 4800 times, 4900 times, 5000 times, 5100 times, 5200 times, 5300 times, 5400 times, 5500 times, 5600 times, 5700 times, 5800 times, 5900 times or 6000 times.

The vaccine dosage may be 1 μg to 10 mg of active ingredient/kg body weight/time and may be 20 μg to 10 mg of ingredient/kg body weight/time. The vaccine may be administered once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days. The number of doses of the vaccine for effective treatment may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses.

(1) Combination therapy using PD-1 and/or PD-L1 antibodies

The present invention also relates to a method for enhancing the immune response of a mammal using a vaccine as described above. The vaccine as described above may comprise a cancer antigen and a PD1 antibody and/or a PDL1 antibody as described above. The combination may be present in a single formulation or may be separated and administered sequentially (first cancer antigen, then PD1 antibody or PDL1 antibody, or first PD1 antibody or PDL1 antibody, then cancer antigen). In some embodiments, about 30 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 0.25 hours, 0.5 hours, 0.75 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours before the PD-1 antibody or PD-L1 antibody is administered to the subject. The cancer antigen can be administered to the subject at 1 hour, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, 96 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks. In other embodiments, the present invention is administered to a subject approximately 30 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 0.25 hours, 0.5 hours, 0.75 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, The subject may be administered a PD-1 antibody or a PD-L1 antibody for 21 hours, 22 hours, 23 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, 96 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks.

The combination of cancer antigen and PD1 antibody or PDL1 antibody induces the immune system more efficiently than a vaccine comprising a separate cancer antigen. This more efficient immune response provides enhanced efficiency in the treatment and/or prevention of specific cancers. Depending on the antigen used in the vaccine in combination with PDL1 antibody or PD1 antibody, the growth of the cancer or tumor to be treated can be any type of cancer such as, but not limited to, melanoma, blood cancer (e.g., leukemia, lymphoma, myeloma), lung cancer, esophageal squamous cell carcinoma, bladder cancer, colorectal cancer, esophageal cancer, gastric cancer, liver cancer, head and neck cancer, brain cancer, anal cancer, non-small cell lung cancer, pancreatic cancer, synovial cancer, prostate cancer, testicular cancer, liver cancer, cervical cancer, recurrent respiratory papillomatosis, skin cancer and gastric cancer.

In some embodiments, the immune response can be enhanced from about 0.5-fold to about 15-fold, from about 0.5-fold to about 10-fold, or from about 0.5-fold to about 8-fold. Alternatively, the immune response of a subject administered the vaccine can be enhanced by at least about 0.5-fold, at least about 1.0-fold, at least about 1.5-fold, at least about 2.0-fold, at least about 2.5-fold, at least about 3.0-fold, at least about 3.5-fold, at least about 4.0-fold, at least about 4.5-fold, at least about 5.0-fold, at least about 5.5-fold, at least about 6.0-fold, at least about 6.5-fold, at least about 7.0-fold, at least about 7.5-fold, at least about 8.0-fold, at least about 8.5-fold, at least about 9.0-fold, at least about 9.5-fold, at least about 10.0-fold, at least about 10.5-fold, at least about 11.0-fold, at least about 11.5-fold, at least about 12.0-fold, at least about 12.5-fold, at least about 13.0-fold, at least about 13.5-fold, at least about 14.0-fold, at least about 14.5-fold, or at least about 15.0-fold.

In other alternative embodiments, the immune response of a subject administered the vaccine may be enhanced by about 50% to about 1500%, about 50% to about 1000%, or about 50% to about 800%. In other embodiments, the immune response of a subject administered the vaccine may be enhanced by at least about 50%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 450%, at least about 500%, at least about 550%, at least about 600%, at least about 650%, at least about 700%, at least about 750%, at least about 800%, at least about 850%, at least about 900%, at least about 950%, at least about 1000%, at least about 1050%, at least about 1100%, at least about 1150%, at least about 1200%, at least about 1250%, at least about 1300%, at least about 1350%, at least about 1450%, or at least about 1500%.

The vaccine dosage may be 1 μg to 10 mg of active ingredient/kg body weight/time and may be 20 μg to 10 mg of ingredient/kg body weight/time. The vaccine may be administered once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days. The number of doses of the vaccine for effective treatment may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses.

(2) Melanoma

Vaccines can be used to generate or elicit an immune response in a mammal that is reactive with or directed against a melanoma in a mammal or subject in need thereof. The elicited immune response can prevent the growth of the melanoma. The elicited immune response can slow the growth of the melanoma. The elicited immune response can prevent and/or reduce the metastasis of cancer cells or tumor cells from the melanoma. Thus, vaccines can be used in methods of treating and/or preventing melanoma in a mammal or subject to which the vaccine is administered.

In some embodiments, administration of the vaccine can mediate clearance of melanoma cells or prevent their growth by inducing the following responses: (1) humoral immunity via a B cell response (to produce antibodies that block the production of monocyte chemoattractant protein-1 (MCP-1), thereby delaying myeloid-derived suppressor cells (MDSCs) and inhibiting tumor growth); (2) increasing cytotoxic T lymphocytes such as CD8 ⁺ (CTLs) to attack and kill melanoma cells; (3) enhancing T helper cell responses; (4) and enhancing inflammatory responses via IFN-γ and TFN-α, or preferably all of the foregoing responses.

In some embodiments, administration of the vaccine can increase the subject's melanoma-free survival rate, reduce melanoma mass, increase melanoma survival rate, or a combination thereof. Administration of the vaccine can increase the subject's melanoma-free survival rate by 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, and 45%. Administration of the vaccine can reduce the mass of melanoma in immunized subjects 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%. Administration of the vaccine can prevent and prevent the increase of the monocyte chemoattractant protein 1 (MCP-1) (a cytokine secreted by myeloid-derived suppressor cells) of the subject. In some embodiments, administration of the vaccine can prevent or prevent the MCP-1 in the subject's melanoma from increasing, thereby reducing the angiogenesis of the subject's melanoma tissue. Administration of the vaccine can increase the melanoma survival rate of the subject 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%.

8. Route of administration

Vaccines or pharmaceutical compositions can be administered by different routes (including oral, parenteral, sublingual, transdermal, rectal, mucosal, topical, via inhalation, buccal administration, intrapleural, intravenous, intraarterial, intraperitoneal, subcutaneous, intramuscular, intranasal intrathecal and intraarticular or a combination thereof). For veterinary purposes, compositions can be administered as appropriately acceptable formulations according to normal veterinary practice. Veterinarians can easily determine the dosage regimen and route of administration that are most suitable for specific animals. Vaccines can be administered by conventional syringes, needleless injection devices, "microparticle bombardment gene guns" or other physical methods such as electroporation ("EP"), "hydrodynamic methods" or ultrasound.

Several well-known techniques can be used to administer the vectors of the vaccine to mammals, including DNA injection with and without in vivo electroporation (also known as DNA vaccination), liposome-mediated, nanoparticle-promoted recombinant vectors such as recombinant adenovirus, recombinant adeno-associated virus, and recombinant vaccinia virus. One or more cancer antigens of the vaccine of the present invention can be administered via DNA injection in conjunction with in vivo electroporation.

a. Electroporation

Vaccine or pharmaceutical composition can be administered by electroporation. Electroporation device can be used to realize the administration of the vaccine via the electroporation of the plasmid of the present invention, and the electroporation device is constructed to deliver the pulse of the energy that effectively causes reversible pore formation in cell membrane to the tissue of mammalian expectation, and preferred energy pulse is a constant current similar to the current input preset by the user. Electroporation device may include electroporation assembly and electrode parts or operating parts. Electroporation assembly may include and comprise one or more of the various elements of electroporation device, including: controller, current waveform generator, impedance tester, waveform recorder, input element, status reporting element, communication port, memory component, power supply and power switch. For example, electroporation device such as EP system (Inovio Pharmaceuticals, Inc., BlueBell, PA) or Elgen electroporator (Inovio Pharmaceuticals, Inc.) in vivo can be used to promote the transfection of plasmid to cell and realize electroporation.

Examples of electroporation devices and methods that can facilitate administration of the DNA vaccines of the present invention include those described in U.S. Patent No. 7,245,963 to Draghia-Akli et al., U.S. Patent Publication 2005/0052630 to Smith et al. (the contents of which are hereby incorporated by reference in their entirety). Other electroporation devices and methods that can be used to facilitate administration of DNA vaccines include those provided in co-pending and co-owned U.S. Patent Application Serial No. 11/874,072, filed October 17, 2007, which claims the benefit of U.S. Provisional Application Serial Nos. 60/852,149, filed October 17, 2006, and 60/978,982, filed October 10, 2007, all of which are hereby incorporated by reference in their entirety.

U.S. Patent No. 7,245,963 to Draghia-Akli et al. describes standard electrode systems and their use for facilitating the introduction of biomolecules into cells of selected tissues of the body or plant. The standard electrode system may include a plurality of needle electrodes; a subcutaneous needle; an electrical connector that provides an electrically conductive connection from a programmable constant current pulse controller to the plurality of needle electrodes; and a power source. An operator may grasp the plurality of needle electrodes mounted on a support structure and securely insert them into the selected tissue of the body or plant. The biomolecules are then administered to the selected tissue via the subcutaneous needles. The programmable constant current pulse controller is activated, and a constant current electrode pulse is applied to the plurality of needle electrodes. The applied constant current electrical pulse facilitates the introduction of the biomolecules into cells between the plurality of electrodes. The entire contents of U.S. Patent No. 7,245,963 are hereby incorporated by reference in their entirety.

US Patent Publication 2005/0052630 filed by Smith et al. describes an electroporation device that can be used to effectively facilitate the introduction of biomolecules into cells of selected tissues of the body or plants. The electroporation device includes an electrokinetic device ("EKD device") whose operation is specified by software or firmware. The EKD device generates a series of programmable constant current pulse waveforms between electrodes in an array based on user control and input of pulse parameters, and allows storage and retrieval of current waveform data. The electroporation device also includes a replaceable electrode tray having an array of needle electrodes, a central injection channel for an injection needle, and a removable guide tray. The entire contents of US Patent Publication 2005/0052630 are hereby incorporated by reference in their entirety.

The electrode arrays and methods described in U.S. Patent No. 7,245,963 and U.S. Patent Publication 2005/0052630 are suitable for deep penetration not only into tissues such as muscle, but also into other tissues or organs. Due to the configuration of the electrode array, the injection needle (biomolecules selected with the deneurological system) can also be fully inserted into the target organ and the injection can be administered perpendicular to the target tissue in the area pre-delineated by the electrodes. The electrodes described in U.S. Patent No. 7,245,963 and U.S. Patent Publication 2005/005263 are preferably 20 mm long and 21 gauge.

Furthermore, in some embodiments comprising electroporation devices and uses thereof, it is contemplated that there are electroporation devices such as those described in the following patents: U.S. Pat. No. 5,273,525, issued December 28, 1993; U.S. Pat. No. 6,110,161, issued August 29, 2000; U.S. Pat. No. 6,261,281, issued July 17, 2001; and U.S. Pat. No. 6,958,060, issued October 25, 2005; and U.S. Pat. No. 6,939,862, issued September 6, 2005. Furthermore, patents encompassing the subject matter provided in U.S. Pat. No. 6,697,669, issued February 24, 2004 (which relates to administering DNA using any of a variety of devices) and U.S. Pat. No. 7,328,064, issued February 5, 2008, are contemplated herein for methods of injecting DNA. The foregoing patents are incorporated by reference in their entirety.

9. Methods for preparing vaccines

Provided herein are methods for preparing DNA plasmids comprising the vaccines discussed herein. The DNA plasmids, after being cloned into mammalian expression plasmids through a final subcloning step, can be used to inoculate cell cultures in large-scale fermenters using methods known in the art.

The DNA plasmids for use with the EP devices of the present invention can be formulated or manufactured using a combination of known devices and techniques, but are preferably manufactured using the optimized plasmid manufacturing technology described in U.S. Published Application No. 20090004716, filed May 23, 2007. In some instances, the DNA plasmids used in these studies can be formulated at a concentration of greater than or equal to 10 mg/mL. The manufacturing technology includes or incorporates various devices and protocols known to those skilled in the art, in addition to those described in U.S. Serial No. 60/939,792 (including those described in U.S. Patent No. 7,238,522, issued July 3, 2007). The aforementioned applications and patents (U.S. Serial No. 60/939,792 and U.S. Patent No. 7,238,522, respectively) are incorporated herein in their entirety.

The present invention has several aspects, which are illustrated by the following non-limiting examples.

10. Examples

Example 1

Construction of pTyr

As shown in Figures 1A and 9, tyrosinase (Tyr) can be found in many different organisms. Therefore, a consensus Tyr was generated by aligning the sequences corresponding to Tyr from the organisms shown in Figure 1A and selecting the most common amino acids and/or nucleotides of the consensus Tyr. The corresponding Tyr sequence for each organism was obtained from GenBank (NCBI). Therefore, the consensus Tyr reflects the conserved elements of Tyr sequences across species.

The nucleic acid sequence encoding the consensus Tyr can be modified to include an IgE leader sequence. Specifically, the IgE leader sequence is fused in frame upstream of the consensus Tyr nucleic acid sequence ( FIG. 1B ). The resulting sequence is then inserted into the pVax1 expression vector to generate a tyrosinase construct or plasmid (pTyr), such that the Kozak sequence is present before the nucleotide sequence encoding the IgE leader sequence and the consensus Tyr.

Insertion of the consensus Tyr nucleic acid sequence into pVax1 was confirmed by restriction enzyme analysis. As shown in FIG1C , the consensus Tyr nucleic acid sequence was separated from the pVax1 plasmid on a DNA agarose gel (i.e., the lane labeled BamH1/Xho1), thereby confirming that the pVax1 vector contains the consensus Tyr nucleic acid sequence.

The expression of the shared Tyr protein was confirmed by transfecting HeLa cells with pTyr. Immunoblotting with human anti-Tyr antibodies confirmed the expression of the shared Tyr protein in HeLa cells (Figure 1D). GPF staining further demonstrated the expression of the shared Tyr protein in transfected HeLa cells (Figure 1E). In immunoblotting and staining experiments.

Example 2

Induction of cell-mediated immune responses using pTyr vaccination

The above-mentioned pTyr was used to vaccinate mice to evaluate whether a cellular immune response was induced by pTyr. C57/B6 mice were immunized using the immunization strategy shown in Figure 2A. Some mice were immunized with pVax1, while others were immunized with pTyr. The mice immunized with pTyr were further divided into the following groups: (1) 5 μg of pTyr; (2) 20 μg of pTyr; (3) 30 μg of pTyr; and (4) 60 μg of pTyr.

On day 35 of the immunization strategy, splenocytes were isolated from C57B/6 mice and evaluated for interferon-γ (IFN-γ) induction by IFN-γ ELISpot analysis. As shown in Figure 2B, the 20 μg dose of pTyr induced the highest level of IFN-γ.

Cellular immune responses to pTyr were further evaluated in immunized Balb/c and C57B/6 mice. Mice were immunized with either pVax1 or pTyr. Splenocytes were isolated 2 weeks after the third immunization and stimulated with the consensus Tyr peptide. After stimulation, IFN-γ-secreting splenocytes were counted as the average number of spots in triplicate stimulation wells. This assay demonstrated that C57/B6 mice are suitable for pTyr vaccination (data not shown).

Example 3

pTyr vaccination increases the cytokines IFN-γ and TNF-α

The cytokine production of mice immunized with pTyr and pVax1 was examined. Mice were immunized using the strategy shown in Figure 2A. On day 35 of the immunization strategy, cells isolated from immunized mice were stimulated with Tyr peptides overnight. After stimulation, analysis of multifunctional responses was measured by FACS. Specifically, CD8 ⁺ and CD4 ⁺ T cells were analyzed. FACS allowed the identification of T cells that were positive for cytokines IL-2, TNF-α, and IFN-γ. Among CD44hi cells, a significant percentage of CD8 ⁺ T cells produced IFN-γ in mice immunized with pTyr compared to mice immunized with pVax1 (Figure 3).

Example 4

Production of Tyr-specific antibodies in response to pTyr vaccination

The humoral immune response of mice vaccinated with pTyr was examined. Specifically, C57BI/6 mice (n=4) were immunized 3 times with pTyr or pVax1 at intervals of 2 weeks. Each immunization consisted of 20 μg/intramuscular injection and subsequent electroporation using MID-EP. After the third immunization (i.e., day 35), serum was collected from the mice and antibodies were measured by ELISA using a total IgG-specific HRP-labeled secondary antibody. The diluted serum was as indicated in Figure 4A. As shown in Figure 4A, specific antibodies against Tyr were produced by mice immunized with pTyr. Mice immunized with pTyr are represented by solid circles in Figure 4A, while mice immunized with pVax1 are represented by hollow triangles in Figure 4A.

In addition, serum from immunized mice was serially diluted at 1:20, 1:40, 1:80, 1:160, 1:320, and 1:640. Each serum dilution was added to a single well (50 μl/well) containing a Tyr peptide in triplicate. The peak increase in Tyr-specific titers of the mice in all immunized groups was checked on the 90th and 120th recheck days compared to the pre-immune serum. Representative results of 3 independent experiments at each serial dilution point are shown in Figure 4B. These data further indicate that immunization with pTyr induces the production of Tyr-specific antibodies in mice immunized with pTyr.

Example 5

Mice vaccinated with pTyr have increased survival to tumor challenge

pTry was further analyzed to determine whether pTyr vaccination could provide protection against tumors. Specifically, C57BI/6 mice (10/group) were immunized with pTyr or pVax1 at 2-week intervals. Each immunization consisted of a 20 μg intramuscular injection followed by electroporation using MID-EP. One week after the third immunization (i.e., day 35), immunized mice were challenged intradermally with B16 melanoma tumors until tumor diameter exceeded 200 mm ² .

Subsequently, tumor-free survival and tumor volume were evaluated in the immunized groups of mice. As shown in Figure 5A (Kaplan-Meier survival curves), mice immunized with pTyr had improved tumor-free survival compared to mice vaccinated with pVax1 (i.e., 40% at day 40 and after tumor challenge) (p=0.05), and all mice vaccinated with pVax1 died on day 20 after tumor challenge. Mice immunized with pTyr also had reduced tumor volume (i.e., approximately 50%) compared to mice immunized with pVax1 (Figure 5B). For Figures 5A and 5B, mice immunized with pVax1 are represented by solid squares, while mice immunized with pTyr are represented by solid circles. Therefore, these data show that pTyr vaccination provides protection against melanoma, i.e., increased tumor-free survival and reduced tumor volume.

Example 6

MDSC populations are reduced in tumors from mice vaccinated with pTyr

The MDSC populations of mice immunized with pTyr and non-immunized mice were examined to examine whether vaccination with pTyr altered the levels of MDSCs in tumors from each group of mice. Specifically, the percentages of Gr-1+ and CD11b+ cells were examined in immunized and non-immunized mice.

As shown in Figures 6 and 7, MDSC levels were significantly reduced in tumors from mice immunized with pTyr compared to non-immunized mice (p = 0.0004). The percentage of MDSC populations in non-immunized mice was 40.00 ± 4.826. The percentage of MDSC populations in mice immunized with pTyr was 5.103 ± 0.7718. Therefore, these data show that immunity with pTyr reduces the MDSC population in tumors of mice inoculated with pTyr.

Example 7

pTyr inoculation reduces MCP-1 levels

MDSCs can secrete the cytokine MCP-1, which promotes angiogenesis, or blood vessel formation, through the migration of endothelial cells. Given the above-mentioned effects of pTyr vaccination on MDSC levels in tumors, MCP-1 levels were examined after vaccination with pTyr.

As shown in Figure 8A, MDSCs within B16 melanoma can secrete MCP-1. Therefore, mice immunized with pTyr and mice immunized with pVax1 were challenged with B16 melanoma to examine whether pTyr immunity changes MCP-1 levels. Mice that were first exposed to the experiment were included as additional controls. After the attack, MDSCs were isolated directly from the tumor tissue and analyzed for MCP-1 cytokine levels by ELISA. The experiment was performed in triplicate and repeated 2 times.

As shown in Figure 8B, MDSCs in B16 melanoma or tumor tissue significantly secrete MCP-1 (see mice immunized with pVax1). However, mice immunized with pTyr do not have a significant increase in MCP-1 levels. On the contrary, the MCP-1 levels in mice immunized with pTyr are approximately 1/3 of those in mice immunized with pVax1. Therefore, these data show that pTyr inoculation reduces the level of MCP-1 secreted by MDSCs in tumors of mice immunized with pTyr.

Example 8

Construction of pPRAME

A consensus sequence for PRAME was generated, and the nucleotide sequence encoding the consensus PRAME antigen was inserted into the BamHI and XhoI restriction enzyme sites of the expression vector or plasmid pVAX (also referred to herein as pVAX1) to generate pGX1411 (also referred to herein as pPRAME) (see Figure 10A).

In order to confirm that pPRAME causes the expression of total PRAME antigen, pVAX and pPRAME are transfected into RD cells and 293T cells. DAPI is used for dyeing the nucleus, and total PRAME antigen is also fluorescently stained. This dyeing is shown in Figure 10 B together with the merging of DAPI and total PRAME antigen dyeing. These dyeings show that PRAME total antigen is expressed from pPRAME and does not detect total PRAME antigen in the cell transfected with pVAX (that is, negative control).

In addition, immunoblotting analysis of lysates from transfected cells was used to confirm the expression of the total PRAME antigen in transfected cells (Figure 10 C). Non-transfected cells and cells transfected with pVAX were used as negative controls (see lanes labeled "control" and "pVAX" in Figure 10 C). In Figure 10 C, β-actin detection was used as a loading control. In short, staining of transfected cells and immunoblotting of lysates from transfected cells showed that vector pPRAME provided expression of the total PRAME antigen in cells.

Example 9

Interferon gamma response to vaccination with pPRAME

The above-mentioned pPRAME was used to inoculate mice to evaluate whether the cellular immune response was induced by pPRAME. C57BL/6 mice were divided into groups. The first group was naive and did not receive pPRAME. The second, third, fourth, fifth, and sixth groups of mice received 5 μg, 10 μg, 15 μg, 25 μg, and 50 μg of pPRAME, respectively.

After immunization, splenocytes were separated from C57BL/6 mice and the induction of interferon gamma (IFN-γ) was evaluated by IFN-γ ELISpot analysis. As shown in Figures 11A and 11B, the pPRAME of each dosage induced the generation or secretion of IFN-γ different from the mice of the negative control first contact experiment. In particular, IFN-γ levels were increased by approximately 3000 times to approximately 4500 times in the mice of inoculation compared to uninoculated mice. Therefore, these data show that the inoculation of pPRAME utilizing the total PRAME antigen of encoding induces cellular immune response, as demonstrated by the IFN-γ levels compared to those not inoculated.

Example 10

Construction of pNY-ESO-1

A consensus sequence for NY-ESO-1 was generated, and the nucleotide sequence encoding the consensus NY-ESO-1 antigen was inserted into the BamHI and XhoI restriction enzyme sites of the expression vector or plasmid pVAX (also referred to herein as pVAX1) to generate pGX1409 (also referred to herein as pNY-ESO-1) (see Figure 12A).

To confirm that pNY-ESO-1 results in the expression of the consensus NY-ESO-1 antigen, pVAX and pNY-ESO-1 were transfected into cells. DAPI was used to stain the cell nuclei, and the consensus NY-ESO-1 antigen was also fluorescently stained. This staining, along with a merge of DAPI and consensus NY-ESO-1 antigen staining, is shown in FIG12B . These stainings demonstrate that the consensus NY-ESO-1 antigen is expressed from pNY-ESO-1 and that no consensus NY-ESO-1 antigen was detected in cells transfected with pVAX (i.e., a negative control).

In addition, immunoblot analysis of lysates from 293T and RD transfected cells was used to confirm the expression of the consensus NY-ESO-1 antigen in the transfected cells ( FIG12C ). Non-transfected cells and cells transfected with pVAX served as negative controls (see lanes labeled "control" and "pVAX," respectively, in FIG12C ). In FIG12C , α-actin detection was used as a loading control. In summary, staining of transfected cells and immunoblotting of lysates from transfected cells demonstrated that the vector pNY-ESO-1 provides for intracellular expression of the consensus NY-ESO-1 antigen.

Example 11

Interferon gamma response to vaccination with pNY-ESO-1

The pNY-ESO-1 described above was used to inoculate mice to evaluate whether pNY-ESO-1 induces a cellular immune response. C57BL/6 mice were divided into groups. The first group was naive and did not receive pNY-ESO-1. Groups 2 and 3 received 25 μg and 50 μg of pNY-ESO-1, respectively.

After immunization, splenocytes were isolated from C57BL/6 mice and evaluated for interferon gamma (IFN-γ) induction by IFN-γ ELISpot analysis. As shown in Figure 13, each dose of pPRAME induced IFN-γ production or secretion that was different from that of negative control naive mice. Specifically, IFN-γ levels were increased by approximately 700-fold to approximately 1100-fold in vaccinated mice compared to non-vaccinated mice. Therefore, these data indicate that vaccination with pNY-ESO-1 encoding the consensus NY-ESO-1 antigen induces a cellular immune response, as evidenced by elevated IFN-γ levels compared to non-vaccinated mice.

Example 12

Interferon gamma response to vaccination with pNY-ESO-2

A consensus sequence for NY-ESO-2 is generated, and the nucleotide sequence encoding the consensus NY-ESO-2 antigen is inserted into the multiple cloning site of the expression vector or plasmid pVAX (also referred to herein as pVAX1) to generate pNY-ESO-2.

This pNY-ESO-2 was used to inoculate mice to evaluate whether pNY-ESO-2 induces a cellular immune response. C57BL/6 mice were divided into groups. The first group was naive and did not receive pNY-ESO-2. Groups 2 and 3 received 25 μg and 50 μg of pNY-ESO-2, respectively.

After immunization, splenocytes were isolated from C57BL/6 mice and the induction of interferon gamma (IFN-γ) was assessed by IFN-γ ELISpot analysis. As shown in Figure 14, each dose of pNY-ESO-2 induced IFN-γ production or secretion that was different from that of negative control naive mice. Specifically, IFN-γ levels were increased by approximately 400-fold to approximately 500-fold in vaccinated mice compared to unvaccinated mice. Therefore, these data indicate that vaccination with pNY-ESO-2 encoding the consensus NY-ESO-2 antigen induces a cellular immune response, as evidenced by elevated IFN-γ levels compared to non-vaccinated mice.

It is to be understood that the foregoing detailed description and accompanying examples are exemplary only and are not intended to be taken as limiting the scope of the invention, which is to be determined solely by the appended claims and their equivalents.

Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications may be made without departing from the spirit and scope thereof, including but not limited to those related to the chemical structures, substituents, derivatives, intermediates, syntheses, compositions, formulations or methods of use of the present invention.

Claims (24)

1. A vaccine comprising a nucleic acid encoding an amino acid sequence of PRAME shown in SEQ ID NO:18. 2. The vaccine according to claim 1 further comprises a nucleic acid encoding one or more antigens selected from the group consisting of: PSA, PSMA, STEAP, PSCA, MAGE A1, gp100, viral antigens and combinations thereof. 3. The vaccine according to claim 2, wherein the viral antigen is an antigen derived from hepatitis B virus (HBV), hepatitis C virus (HCV), or human papillomavirus (HPV). 4. The vaccine according to claim 3, wherein the HBV antigen is the HBV core antigen or the HBV surface antigen or a combination thereof. 5. The vaccine according to claim 3, wherein the HCV antigen is HCV NS34A antigen, HCV NS5A antigen, HCV NS5B antigen, HCV NS4B antigen, or a combination thereof. 6. The vaccine according to claim 3, wherein the HPV antigen is HPV type 6 E6 antigen, HPV type 6 E7 antigen, HPV type 11 E6 antigen, HPV type 11 E7 antigen, HPV type 16 E6 antigen, HPV type 16 E7 antigen, HPV type 18 E6 antigen, HPV type 18 E7 antigen, or a combination thereof. 7. The vaccine according to claim 1 further comprises an immune checkpoint inhibitor selected from the group consisting of: anti-PD-1 antibody, anti-PD-L1 antibody, and combinations thereof. 8. The vaccine according to claim 1, wherein the nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO:17. 9. The vaccine according to claim 1, wherein the nucleic acid is a plasmid. 10. The vaccine of claim 1, wherein the nucleic acid is one or more plasmids. 11. The vaccine according to claim 1, further comprising an adjuvant. 12. The vaccine of claim 11, wherein the adjuvant is IL-12, IL-15, IL-28 or RANTES. 13. Use of the vaccine of claim 1 in the preparation of an formulation for treating cancer in a subject with such need. 14. The application according to claim 13, wherein the administration of the vaccine includes an electroporation step. 15. The application according to claim 13, wherein the treatment further comprises administering an immune checkpoint inhibitor to the subject. 16. The application according to claim 15, wherein the immune checkpoint inhibitor is selected from the group consisting of: anti-PD-1 antibody, anti-PD-L1 antibody, and combinations thereof. 17. The application according to claim 15, wherein the vaccine and the immune checkpoint inhibitor are administered to the subject in a single formulation. 18. The application according to claim 15, wherein the vaccine and the immune checkpoint inhibitor are administered to the subject, respectively. 19. The application according to 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 papillomavirus (RRP), anal cancer, leukemia, and combinations thereof. 20. A nucleic acid molecule comprising the nucleotide sequence shown in SEQ ID NO:17. 21. A plasmid comprising the nucleotide sequence shown in SEQ ID NO:17. 22. One or more plasmids comprising the nucleotide sequence shown in SEQ ID NO:17. 23. A polypeptide molecule comprising the amino acid sequence shown in SEQ ID NO:18. 24. Use of a vaccine in the preparation of an formulation for the prevention or treatment of cancer in a subject with such need, said vaccine being: (a) Vaccines containing PRAME; (b) A vaccine containing PRAME in combination with one or more cancer antigens selected from the group consisting of tyrosinase, GP 100, hTERT, NY-ESO-1, MAGE A1 and WT1; (c) Vaccines containing PRAME and immune checkpoint inhibitors; or (d) A vaccine comprising PRAME in combination with one or more cancer antigens selected from the group consisting of tyrosinase, GP 100, hTERT, NY-ESO-1, MAGE A1 and WT1, and further in combination with an immune checkpoint inhibitor; PRAME consists of the amino acid sequence shown in SEQ ID NO:18.