EA045958B1 - ANTI-CANCER VACCINES AND METHODS OF TREATMENT USING THEIR USE - Google Patents

Description

Cross reference to related applications

This application claims priority to US Provisional Patent Application No. 61/799952, filed March 15, 2013, the disclosure of which is incorporated herein by reference.

Technical field

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

State of the art

Cancer is a leading cause of death worldwide, and in the United States, it is the second most common cause of death, accounting for about 1 in every 4 deaths. Cancer arises from a single cell that has transformed from a normal cell to a tumor cell. This transformation is often a multistage process, proceeding from a pretumor state to malignant tumors. Several factors contribute to this development, including aging, genetic factors, and exposure to environmental factors such as physical carcinogens (eg, ultraviolet and ionizing radiation), chemical carcinogens (eg, asbestos, components of tobacco smoke, etc.), and biological carcinogens (for example, some viruses, bacteria and parasites).

Prevention, diagnosis and treatment of cancer can take many forms. Prevention may include screening for predisposing factors (eg, specific genetic variants (alleles)), behavioral changes (eg, smoking, diet, and amount of exercise), and vaccination against viruses (eg, human papillomavirus, hepatitis B virus). Treatment may include chemotherapy, radiation therapy, and surgical removal of the tumor or cancerous tissue. Despite the availability of various methods of prevention and treatment, in reality such methods often do not provide much success in the field of effective prevention and/or treatment of cancer.

Accordingly, there is a need to find and develop compositions and methods for preventing and/or treating cancer in order to facilitate clinical management of disease protection and progression. In addition, more effective treatments are needed to delay disease progression and/or reduce mortality in subjects suffering from cancer.

The essence of the invention

The present invention relates to a vaccine containing one or more nucleotide or amino acid sequences of cancer antigens that are no longer self-antigens and stimulate an immune response to a specific cancer or tumor caused by a specific cancer. This vaccine may further contain an immune checkpoint inhibitor, such as antibodies to PD-1 and PDL-1, which prevents suppression of any component in the immune system, such as MHC presentation, T cell presentation and/or differentiation, T cell presentation and/or differentiation B cells, any cytokine, chemokine or signaling for immune cell proliferation and/or differentiation. The one or more cancer antigens of a given vaccine may be a nucleic acid encoding one or more amino acid sequences or an amino acid sequence selected from the group consisting of: an amino acid sequence identical to 95% or more of the amino acid sequence of tyrosinase (Tyr); amino acid sequence of tyrosinase-dependent protein 1 (TYRP1); an amino acid sequence identical to 95% or more with the amino acid sequence of tyrosinase-dependent protein 2 (TYRP2); an amino acid sequence identical to 95% or more with the amino acid sequence of melanoma associated antigen 4 (MAGEA4); an amino acid sequence identical to 95% or more with the amino acid sequence of growth hormone releasing hormone (GHRH); an amino acid sequence identical to 95% or more with the amino acid sequence of the MART-1/melan-A antigen (MART-1/Melan-A); an amino acid sequence identical to 95% or more with the amino acid sequence of cancer-testis antigen (NY-ESO-1); an amino acid sequence identical to 95% or more with the amino acid sequence of cancer-testicular antigen II (NY-ESO-2); an amino acid sequence identical to 95% or more of the amino acid sequence of PRAME; an amino acid sequence that is 95% or more identical to the amino acid sequence of WT1; an amino acid sequence identical to 95% or more with the amino acid sequence of hTERT; or combinations thereof. The vaccine may further comprise a nucleic acid encoding one or more antigens selected from the group consisting of: PSA, PSMA, STEAP, PSCA, MAGE A1, gp100, viral antigen, and combinations thereof.

The present invention further provides a method for preventing or treating cancer in a subject in need thereof, the method comprising administering to the subject in need thereof a vaccine containing a predetermined amount of cancer antigens to treat or prevent a particular type of cancer. The method may include administering to a subject in need thereof a vaccine containing a CMV cancer antigen for the treatment or prevention of glioblastoma, or administering to a subject in need thereof a vaccine containing a CMV cancer antigen in combination with any one or more hTERT, NY- cancer antigens. ESO-1, MAGE-A1 or WT1 for treatment or

- 1 045958 prevention of glioblastoma; administering to a subject in need thereof a vaccine containing one or more cancer antigens PSA, PSMA or STEAP for the treatment or prevention of prostate cancer, or administering to a subject in need thereof a vaccine containing PSA, PSMA or STEAP in combination with any one or more cancer antigens hTERT, NY-ESO-1, MAGE-A1 or WT1 for the treatment or prevention of prostate cancer; administering to a subject in need thereof a vaccine containing one or more cancer antigens tyrosinase, PRAME or GP-100 for the treatment or prevention of melanoma, or administering to a subject in need thereof a vaccine containing tyrosinase, PRAME or GP-100 in combination with any one or more hTERT, NY-ESO-1, MAGE-A1 or WT1 cancer antigens for the treatment or prevention of melanoma; administering to a subject in need thereof a vaccine containing one or more HPV 16 E6 or HPV 16 E7 cancer antigens for the treatment or prevention of head and neck cancer, or administering to a subject in need thereof a vaccine containing HPV 16 E6 or HPV 16 E7 in combinations with any one or more hTERT, NY-ESO-1, MAGE-A1 or WT1 cancer antigens for the treatment or prevention of head and neck cancer; administering to a subject in need thereof a vaccine containing one or more cancer antigens tyrosinase, PRAME or GP-100 for the treatment or prevention of melanoma, or administering to a subject in need thereof a vaccine containing tyrosinase, PRAME or GP-100 in combination with any one or more hTERT, NY-ESO-1, MAGE-A1 or WT1 cancer antigens for the treatment or prevention of melanoma; administering to a subject in need thereof a vaccine containing one or more HPV 6, HPV 11 or HPV 16 cancer antigens for the treatment or prevention of anal cancer, or administering to a subject in need thereof a vaccine containing HPV 6, HPV 11 or HPV 16 in combination with any one or more hTERT, NY-ESO-1, MAGE-A1 or WT1 cancer antigens for the treatment or prevention of anal cancer; administering to a subject in need thereof a vaccine containing 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, or administration to a subject in need thereof, a vaccine containing HBV core antigen, HBV surface antigen, HCV NS34A, HCV NS5A, HCV NS5B or HCV NS4B in combination with any one or more hTERT, NY-ESO-1, MAGE-A1 or WT1 cancer antigens for the treatment or prevention of liver cancer ; administering to a subject in need thereof a vaccine containing one or more HPV 16 E6/E7 or HPV 18 E6/E7 cancer antigens for the treatment or prevention of cervical cancer, or administering to a subject in need thereof a vaccine containing HPV 16 E6/E7 or HPV 18 E6/E7 in combination with any one or more hTERT, NY-ESO-1, MAGE-A1 or WT1 cancer antigens for the treatment or prevention of cervical cancer; or administering to a subject in need thereof a vaccine containing one or more PRAME, WT-1 or hTERT cancer antigens for the treatment or prevention of blood cancer, or administering to a subject in need thereof a vaccine containing PRAME, WT-1 or hTERT in combination with any one or more cancer antigens NY-ESO-1 or MAGE-A1 for the treatment or prevention of blood cancer, wherein the method may further comprise combining administration steps (a)-(i), wherein the immune checkpoint inhibitor is selected from the group , consisting of: anti-PD-1 antibodies, anti-PD-L1 antibodies and combinations thereof.

Brief description of graphic materials

Figures 1A-E illustrate the construction of pTyr.

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

Fig. 3 illustrates fluorescence activated cell sorting (FACS) of control and immunized mice.

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

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

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

Fig. 7 illustrates MDSC staining in mice immunized with pVax1 and pTyr.

Figures 8A and 8B illustrate the secretion of MCP-1 by MDSC cells.

Fig. 9 illustrates the phylogenetic relationship of Tyr nucleotide sequences among these organisms.

Fig. 10 illustrates (A) a schematic map of plasmid pPRAME (also referred to herein as pGX1411); (B) staining of RD and 293T cells for nuclei with DAPI and for the PRAME consensus antigen; and (C) Western blotting for the PRAME consensus antigen in lysates from untransfected cells (control), pVAX-transfected cells (pVAX), and pPRAME-transfected cells (PRAME-pVAX).

Fig. 11 illustrates in graphs (A) and (B) a mouse group as a function of spot forming units (SFU)/10 ⁶ splenocytes for gamma interferon (IFN-γ).

Fig. 12 illustrates (A) a schematic map of plasmid pNY-ESO-1 (also referred to herein as

- 2 045958 mente as pGX1409); (B) cell staining for nuclei with DAPI and for the NY-ESO-1 consensus antigen; and (C) Western blotting for the NY-ESO-1 consensus antigen in RD and 293T lysates from untransfected cells (control), cells transfected with pVAX (pVAX), and cells transfected with pNY-ESO-1 (pNY-ESO- 1).

Fig. 13 illustrates mouse group according to spot forming units (SFU)/10 ⁶ splenocytes for gamma interferon (IFN-γ).

Fig. 14 illustrates the mouse group according to spot forming units (SFU)/10 ⁶ splenocytes for gamma interferon (IFN-γ).

Fig. 15 illustrates a diagram of various cancers with some of their associated cancer antigen(s).

Detailed Description of the Invention

The present invention relates to a vaccine that can be adapted to certain types of cancer and tumors. Consensus antigen sequences have been constructed for certain cancer-associated antigens such as tyrosinase (Tyr), predominantly melanoma expressed antigen (PRAME), tyrosinase-dependent protein 1 (Tyrp1), cancer-testis antigen (NY-ESO-1), viral antigen hepatitis B and Wilms tumor antigen 1 (WT-1), which will be used in the vaccine to enable the vaccine to be adapted for the prevention and treatment of certain types of cancer. For example, tyrosinase antigen can be used in a vaccine to prevent or treat melanomas. The vaccine 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 method for constructing a nucleic acid and its encoded recombinant cancer antigen amino acid sequence is to introduce mutations that alter specific amino acids throughout the amino acid sequence of the native cancer antigen. Introduction of mutations does not change the cancer antigen so much that it cannot be applied universally to mammals, preferably humans or dogs, but changes it enough that the resulting amino acid sequence breaks tolerance or is considered a foreign antigen to generate an immune response. Another method may be the creation of a consensus recombinant cancer antigen that has at least 85% to 99% amino acid sequence identity to its corresponding native cancer antigen; preferably at least 90% to 98% sequence identity; more preferably, at least 93% to 98% sequence identity; or even more preferably, at least 95% to 98% sequence identity. In some cases, the recombinant cancer antigen has 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to its corresponding native cancer antigen. This native cancer antigen is an antigen that is typically associated with a specific type of cancer or cancerous tumor. Depending on the cancer antigen, the cancer antigen consensus sequence may be present in mammals or within subtypes of a species, or in viral strains or serotypes. Some cancer antigens are not very different from the amino acid sequence of the wild type cancer antigen. Some cancer antigens have nucleotide/amino acid sequences that are so divergent among species that a consensus sequence cannot be obtained. In these cases, a recombinant cancer antigen is produced that will break tolerance and generate an immune response, an antigen that has at least 85% to 99% amino acid sequence identity to its corresponding native cancer antigen; preferably at least 90% to 98% sequence identity; more preferably, at least 93% to 98% sequence identity; or even more preferably, at least 95% to 98% sequence identity. In some cases, the recombinant cancer antigen has 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to its corresponding native cancer antigen. The above approaches can be combined such that the final recombinant cancer antigen has the same percentage similarity to the amino acid sequence of the native cancer antigen as described above.

The recombinant cancer antigen is capable of inducing antigen-specific T cell and/or potent humoral immune responses, thereby inducing or eliciting an immune response that targets or responds to the cancer or tumor by expression of the antigen. In some embodiments, the induced or evoked immune response may be cellular, humoral, or both cellular and humoral immune responses. In some embodiments, the induced or evoked cellular immune response may include the induction or secretion of interferon gamma (IFN-γ) and/or tumor necrosis factor alpha (TNF-α). In other embodiments, the induced or elicited immune response may reduce or inhibit one or more immune response suppressive factors that

- 3 045958 They stimulate the development of a tumor or cancer by expressing an antigen, for example, but not limited to, factors that suppress MHC presentation, factors that activate 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 immune suppressor cells, CTLA-4, PD-1, MDSC, MCP-1 and immune checkpoint molecules.

This vaccine can be further combined with antibodies to checkpoint inhibitors such as PD-1 and PDL-1 to enhance the stimulation of both cellular and humoral immune responses. The use of antibodies to PD-1 or PDL-1 prevents PD-1 or PDL-1 from suppressing T cell and/or B cell responses. In general, designing cancer antigens so that they are recognized by the immune system helps overcome other forms of suppression of the immune response by tumor cells, and these vaccines can be used in combination with suppression or inhibition therapies (such as anti-PD-1 and PDL-1 antibody therapies ) to further increase T cell and/or B cell responses.

1. Definitions.

Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In the event of a conflict, this document, including definitions, is subject to review. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein may be used in the practice 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 described herein are illustrative only and are not intended to be limiting. The terminology used herein is intended to describe certain embodiments of the invention and is not intended to be limiting.

The terms constitute, include, have, has, may, contain, and variations thereof, when used throughout this document, are intended to be used as non-limiting transitional phrases, terms or words that do not exclude the possibility of additional actions or structures. The singular includes a reference to the plural unless the context clearly indicates otherwise. The present invention also provides for other embodiments comprising, consisting of, and consisting essentially of, the embodiments or elements presented herein, whether expressly set forth or not.

To list numeric ranges, this document explicitly assumes every number in between to the same degree of precision. For example, for the range 6-9, the numbers 7 and 8 are assumed 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 are explicitly assumed. 4, 6.5, 6, 6, 6, 7 6, 8, 6, 9 and 7.0.

Adjuvant, as used herein, means any molecule added to the plasmid DNA vaccines described herein for the purpose of enhancing the immunogenicity of the antigens encoded by the DNA plasmids and encoding the nucleotide sequences described below.

Antibody, as used herein, means an antibody of the classes IgG, IgM, IgA, IgD or IgE, or fragments or derivatives thereof, including Fab, F(ab') ₂ , Fd, as well as single-chain antibodies, diabodies, bispecific antibodies, bifunctional antibodies and their derivatives. The antibody may be an antibody isolated from a mammalian serum sample, a polyclonal antibody, an affinity purified antibody, or mixtures thereof that exhibit sufficient binding specificity for the desired epitope or sequence derived therefrom.

Coding sequence or coding nucleic acid, as used herein, means nucleic acids (RNA or DNA molecule) containing a nucleotide sequence that encodes a protein. The coding sequence may further comprise initiation and termination signals operably linked to regulatory elements, including a promoter and a polyadenylation signal, capable of regulating expression in the cells of the individual or mammal to which the nucleic acid is administered.

Complement or complementary, as used herein, means that a nucleic acid may refer to a Watson-Crick (eg, A-T/U and C-G) or Hoogsteen base pairing between nucleotides or nucleotide analogues of nucleic acid molecules.

Consensus or consensus sequence, as used throughout this document, means a polypeptide sequence based on analysis of the alignment of multiple sequences for the same gene from different organisms. Nucleic acid sequences that encode a consensus polypeptide sequence can be obtained. To induce broad immunity to an antigen, vaccines containing proteins that contain consensus sequences and/or nucleic acid molecules encoding such proteins can be used.

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Electroporation, electropermeabilization or electrokinetic enhancement (EP), used interchangeably throughout this document, refers to the use of a transmembrane electric field pulse to induce microscopic pathways (pores) in a bio-membrane; their presence allows biomolecules such as plasmids, oligonucleotides, siRNAs, drugs, ions and water to pass from one side of the cell membrane to the other.

Fragment, as used herein, in relation to nucleic acid sequences, means a nucleotide sequence or portion thereof encoding a polypeptide capable of eliciting an immune response in a mammal that cross-reacts with an antigen described herein. These fragments may be DNA fragments selected from at least one of various nucleotide sequences that encode protein fragments described below. The fragments may contain at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% , at least 90% or at least 95% of one or more nucleotide sequences described below. In some embodiments, the fragments 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 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 than, 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 nucleotide sequences described below.

A fragment or immunogenic fragment, with respect to polypeptide sequences, means a polypeptide capable of eliciting an immune response in a mammal that cross-reacts with an antigen described herein. These fragments may be polypeptide fragments selected from at least one of the various amino acid sequences below. The consensus protein fragments may 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, the consensus protein fragments may contain 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 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 of a protein sequence described in this document.

As used throughout this document, the term genetic construct refers to DNA or RNA molecules containing a nucleotide sequence that encodes a protein. The coding sequence contains initiation and termination signals operably linked to regulatory elements, including a promoter and a polyadenylation signal, capable of regulating expression in the cells of the individual to whom the nucleic acid molecule is administered. As used herein, the term expressible form refers to genetic constructs containing necessary regulatory elements operably linked to a coding sequence that encodes a protein such that, when present in a cell of an individual, expression of the coding sequence will occur.

The term homology, as used throughout this document, refers to the degree of complementarity. There may be partial homology or complete homology (i.e., identity). A partially complementary sequence that at least partially inhibits a fully complementary sequence from hybridizing with a target nucleic acid is referred to using the functional term substantially homologous. When used in relation to a double-stranded nucleotide sequence, such as a cDNA or genomic clone, the term substantially homologous, as used herein, refers to a probe that can hybridize to a strand of the double-stranded nucleotide sequence under conditions

- 5 045958 reduced rigidity. When used in relation to a single-stranded nucleotide sequence, the term essentially homologous, as used herein, refers to a probe that can hybridize (ie is complementary) to a single-stranded template nucleotide sequence under conditions of reduced stringency.

Identical or identity, as used herein, in the context of two or more nucleic acid or polypeptide sequences means that the sequences have a certain percentage of residues that are the same throughout the specified region. This percentage can be calculated by optimally aligning the two sequences, comparing the two sequences in the specified region, determining the number of positions at which identical residues are present in both 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 result to 100 to obtain the percentage of sequence identity. In cases where the two sequences are of different lengths and the alignment results in one or more stepped ends, and the specified comparison region contains only one sequence, the residues of one sequence are included in the denominator rather than in the numerator of the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered as equivalents. Identification can be done manually or using a computer sequencing algorithm such as BLAST or BLAST 2.0.

Immune response, as used herein, means the activation of the immune system of a host, for example, a mammal, in response to the introduction of an antigen. The immune response can be in the form of a cellular or humoral response, or both.

Nucleic acid or oligonucleotide or polynucleotide, as used herein, means at least two nucleotides covalently linked to each other. The description of a single strand also determines the sequence of the complementary strand. Thus, the nucleic acid also spans the complementary strand of the given single strand. Many nucleic acid variants can be used for the same purpose as a given nucleic acid. Thus, nucleic acid also covers essentially identical nucleic acids and their complements. The single strand provides a probe that can hybridize to the target sequence under stringent hybridization conditions. Thus, the nucleic acid also covers the probe, which hybridizes under stringent hybridization conditions.

Nucleic acids may be single-stranded or double-stranded, or may contain portions of both double-stranded and single-stranded sequences. The nucleic acid may be DNA, either genomic or cDNA, RNA or a hybrid, in which the nucleic acid may contain combinations of deoxyribo- and ribonucleotides, as well as combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine and isoguanine. Nucleic acids can be obtained using chemical synthesis methods or using recombinant methods.

Functionally linked, as used herein, means that the expression of a gene is controlled by a promoter to which it is spatially linked. A promoter can be located 5' (upstream of transcription) or 3' (downstream of transcription) from the gene it controls. The distance between a promoter and a gene may be approximately the same as the distance between said promoter and the gene it controls in the gene from which the promoter was derived. As is known in the art, a change in this distance can be coordinated without loss of promoter function.

A peptide, protein or polypeptide, as used herein, can mean a linked sequence of amino acids and can be natural, synthetic, or a modification or combination of natural and synthetic.

A promoter, as used herein, means a synthetic or naturally occurring molecule that is capable of promoting, activating, or enhancing the expression of a nucleic acid in a cell. A promoter may contain one or more specific transcriptional regulatory sequences that provide further enhancement of expression and/or regulation of expression in space and/or time. The promoter may also contain distal enhancer or repressor elements, which may be located several thousand base pairs away from the transcription start site. The promoter can be obtained from sources including viral, bacterial, fungal, plant, insect and animal. A promoter may regulate expression of a gene component constitutively or differentially with respect to the cell, tissue or organ where expression occurs, or with respect to the developmental stage at which expression occurs, or in response to external stimuli such as physiological stress, pathogens, metal ions or inducing agents. Typical 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 SV40 late promoter and CMV IE promoter .

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Signal peptide and leader sequence are used interchangeably throughout this document and refer to an amino acid sequence that can be linked at the amino terminus of a protein described herein. Signal peptides/leader sequences typically control protein localization. The signal peptides/leader sequences used herein preferably facilitate the secretion of a protein from the cell in which it is produced. Signal peptides/leader sequences are often cleaved from the rest of the protein, often called the mature protein, upon secretion from the cell. Signal peptides/leader sequences are linked at the amino terminus (ie N-terminus) of the protein.

Stringent hybridization conditions, as used herein, mean conditions under which a first nucleotide sequence (eg, a probe) will hybridize to a second nucleotide sequence (eg, a target), such as in a complex mixture of nucleic acids. The stringent conditions depend on the sequence and will be different under different circumstances. Severe conditions can be selected to be lower than the melting temperature (Tm) by about 510°C for a particular sequence at a particular ionic strength and pH. Tm can be the temperature (at a certain ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize at equilibrium with the target sequence (since target sequences are present in excess, at Tm, at equilibrium 50 % probes occupied). Severe conditions may be conditions in which the salt concentration is less than about 1.0 M sodium ion concentration, for example, about 0.01-1.0 M sodium ion concentration (or other salts) at a pH of from 7.0 to 8.3, and the temperature is at least about 30°C for short probes (eg, about 10-50 nucleotides) and at least about 60°C for long probes (eg, more than about 50 nucleotides). Harsh conditions can also be achieved by adding destabilizing agents such as formamide. For selective or specific hybridization, the positive signal may be at least 2 to 10 times the background hybridization. Typical stringent hybridization conditions include the following: 50% formamide, 5x SSC and 1% SDS, incubation at 42°C or 5x SSC, 1% SDS, incubation at 65°C, with a wash in 0.2x SSC and 0.1% SDS at 65°C.

Subject, as used herein, can mean a mammal that wants or needs to be vaccinated with the vaccines described herein. The mammal may be a human, chimpanzee, dog, cat, horse, cow, mouse or rat.

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

Substantially identical, as used herein, means that the first and second 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 range of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 180, 270, 360, 450, 540 or more nucleotides or amino acids, or relative to nucleic acids, if the first sequence is substantially complementary to the complement of the second sequence.

Treat or treat, as used throughout this document, can mean protecting an animal from disease by preventing, suppressing, controlling, or eliminating the disease entirely. Prevention of a disease involves administering the vaccine of the present invention to an animal before the onset of the disease. Disease suppression involves administering a vaccine of the present invention to an animal after the induction of the disease but before its clinical manifestation. Disease control involves administering a vaccine of the present invention to an animal after clinical manifestation of the disease.

Option as used herein with respect to nucleic acids means (i) a region or fragment of a reference nucleotide sequence; (ii) complement of the reference nucleotide sequence or portion thereof; (iii) a nucleic acid substantially identical to the reference nucleic acid or its complement; or (iv) a nucleic acid that hybridizes under stringent conditions with a reference nucleic acid, its complement, or substantially identical sequences thereof.

Variant with respect to a peptide or polypeptide that differs in amino acid sequence by insertion, deletion, or conservative amino acid substitution, but retains at least one biological activity. A variant may also mean a protein with an amino acid sequence substantially identical to a reference protein with an amino acid sequence that retains at least one biological activity. Conservative substitution

- 7 045958 amino acids, that is, the replacement of an amino acid with another amino acid with similar properties (eg, hydrophilicity, degree and distribution of charged regions) is recognized in the art, usually as a substitution with a minor change. These minor changes can be determined, in particular, by considering the hydrophobicity index of the amino acids, as understood in the art. Kyte et al., J. Mol. Biol. 157:105-132 (1982). This index of hydrophobicity of an amino acid is based on consideration of its hydrophobicity and charge. It is known in the art that amino acids with similar hydrophobicity indices can be substituted and still retain protein function. In one aspect, amino acids having hydrophobicity indices of ±2 are substituted. Amino acid hydrophilicity can also be used to identify substitutions that could lead to proteins that retain biological function. Considering amino acid hydrophilicity in the context of a peptide allows the calculation of the highest local average hydrophilicity of that peptide, a useful measurement that has been reported to correlate well with antigenicity and immunogenicity. US Patent No. 4,554,101, which is incorporated herein by reference in its entirety. Substitution of amino acids having similar hydrophilicity values can result in peptides that retain biological activity, such as immunogenicity, as is known in the art. Substitutions can be made with amino acids having hydrophilicity values within ±2 relative to each other. Both the hydrophobicity index and the hydrophilicity value of an amino acid are influenced by the specific side chain of that amino acid. In accordance with this observation, it should be understood that amino acid substitutions that are compatible with biological function depend on the relative similarity of the amino acids, and especially the side chains of these amino acids, as measured by hydrophobicity, hydrophilicity, charge, size and other properties.

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

The amino acid sequence can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical over the entire length of the amino acid sequence or its fragment.

Vector, as used herein, means a nucleotide sequence containing an origin of replication. The vector may be a viral vector, a bacteriophage, a bacterial artificial chromosome, or a yeast artificial chromosome. The vector may be a DNA or RNA vector. The vector may be a self-replicating extrachromosomal vector and is preferably a DNA plasmid. A given vector may contain or include one or more heterologous nucleotide sequences.

2. Vaccine.

The present invention is aimed at creating a vaccine against cancer. This vaccine may contain one or more cancer antigens. The vaccine can prevent tumor development. The vaccine may reduce tumor development. The vaccine can prevent tumor cells from metastasizing. Depending on the cancer antigen, the vaccine may be used 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 recognize a cancer antigen that is not recognized by the immune system and is a self-antigen. A recognized cancer antigen changes from a self-antigen to a foreign antigen for recognition by the immune system. Reconstructing the nucleotide and amino acid sequence of a recombinant cancer antigen from a self-antigen to a foreign antigen breaks the tolerance of the antigen by the immune system. In order to break tolerance, several remodeling agents can be applied to the cancer antigen, as described below.

The vaccine's recombinant cancer antigen is not recognized as a self-antigen, thereby breaking tolerance. Breakdown of tolerance is capable of inducing antigen-specific T-cell and/or intense humoral immune responses, thereby inducing or triggering an immune response that targets or responds to the cancer or tumor by expressing an antigen. In some embodiments, the induced or evoked immune response may be cellular, humoral, or both cellular and humoral immune responses. In some embodiments, the induced or evoked cellular immune response may include the induction or secretion of interferon gamma (IFN-γ) and/or tumor necrosis factor alpha (TNF-α). In other embodiments, the induced or elicited immune response may reduce or inhibit one or more immune response suppressive factors that promote the development of a tumor or cancer by expressing an antigen, for example, without limitation,

- 8 045958 factors suppressing MHC presentation, factors activating 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 suppressor immune cells, CTLA-4, PD-1, MDSC, MCP-1 and immune checkpoint molecules.

In a specific embodiment, the vaccine may mediate the clearance or prevent the development of tumor cells by inducing (1) humoral immunity through B cell responses to produce antibodies that block the formation of monocyte chemoattractant protein-1 (MCP-1), thereby inhibiting suppressor cells myeloid-derived cells (MDSC) and suppressing tumor development; (2) increasing cytotoxic T lymphocytes such as CD8 ⁺ (CTL) to attack and kill tumor cells; (3) increasing T helper cell responses; (4) and increasing inflammatory responses through 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 can reduce tumor formation by 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44% , 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59% and 60% after immunization . The vaccine can prevent and block the increase in monocyte chemoattractant protein 1 (MCP-1), a cytokine secreted by myeloid-derived suppressor cells. The vaccine can increase 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 may increase the cellular immune response in the subject receiving the vaccine by up to 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 times, from about 100 times to about 6000 times, from about 150 times to about 6000 times, from about 200 times to about 6000 times, from about 250 times to 6000 times or from about 300-fold to about 6000-fold the size of the cellular immune response in a subject not receiving the vaccine. In some embodiments, the vaccine may increase the cellular immune response in a subject receiving the vaccine by up to about 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750 , 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000 , 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 5 100, 5200, 5300, 5400, 5500 , 5600, 5700, 5800, 5900 or 6000 times compared to the cellular immune response in a subject not receiving the vaccine.

The vaccine may increase interferon gamma (IFN-γ) levels in the subject receiving the vaccine by up to about 50-fold to about 6000-fold, about 50-fold to about 5500-fold, about 50-fold to about 5000-fold, about 50 times to about 4500 times, about 100 times to about 6000 times, about 150 times to about 6000 times, about 200 times to about 6000 times, about 250 times to about 6000-fold or about 300-fold to about 6000-fold compared to IFN-γ levels in a subject not receiving the vaccine. In some embodiments, the vaccine may increase IFN-γ levels in a subject receiving the vaccine by up to about 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700. 750,800,850,900,950,1000,1100,1200,1300,1400,1500,1600,1700,1800,1900,2000,2100,2200,2300,2400,2500,26 00, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 500 0, 5100, 5200, 5300, 5400, 5500, 5600, 5700, 5800, 5900, or 6000 times the IFN-γ levels in a subject not receiving the vaccine.

The vaccine may be a DNA vaccine. DNA vaccines are disclosed in US Pat. Nos. 5,593,972, 5739118, 5817637, 5830876, 5962428, 5,981,505, 5580859, 5703055, and 5676594, which are incorporated herein by reference in their entirety. The DNA vaccine may additionally contain elements or reagents that inhibit it from integrating into the chromosome.

The vaccine may be RNA of one or more cancer antigens. An RNA vaccine can be introduced into a cell.

The vaccine may be an attenuated live vaccine, a vaccine using recombinant antigen delivery vectors, subunit vaccines, and glycoprotein vaccines, such as, but not limited to, the vaccines described in US Pat. Nos.: 4,510,245; 4797368; 4722848; 4790987; 4920209; 5017487; 5077044; 5110587; 5112749; 5174993; 5223424; 5225336; 5240703; 5242829; 5294441; 5294548; 5310668; 5387744; 5389368; 5424065; 5451499; 5453364; 5462734; 5470734; 5474935; 5482713; 5591439; 5643579; 5650309; 5698202; 5955088; 6034298; 6042836; 6156319 and 6589529, which are incorporated herein by reference.

The vaccine of this invention may have the necessary characteristics of effective vaccines, such as safety, such that the vaccine itself does not cause disease or death; protection against disease; induction of neutralizing antibody; induction of protective T cell responses; as well as ensuring ease of administration, a small number of side effects

- 9 045958 effects, biological stability and low dose cost. The vaccine may perform some or all of these functions by containing the cancer antigen described below.

As described in more detail below, the vaccine may further comprise one or more inhibitors of one or more immune checkpoint molecules (ie, an immune checkpoint inhibitor). Immune checkpoint molecules are described in more detail below. An immune checkpoint inhibitor is any nucleic acid or protein that prevents the suppression of any component in the immune system, for example, MHC presentation, T cell presentation and/or differentiation, B cell presentation and/or differentiation, any cytokine, chemokine or signaling for proliferation and/or differentiation of immune cells. As also described in more detail below, the vaccine can be further combined with antibodies to checkpoint inhibitors, such as PD-1 and PDL-1, to enhance the stimulation of both cellular and humoral immune responses. The use of antibodies to PD-1 or PDL-1 prevents PD-1 or PDL-1 from suppressing T cell and/or B cell responses.

Cancer antigen.

This vaccine may contain one or more cancer antigens. The cancer antigen may be a nucleotide sequence, an amino acid sequence, or a combination thereof. The nucleotide sequence may be DNA, RNA, cDNA, a variant thereof, a fragment thereof, or a combination thereof. The nucleotide sequence may also contain additional sequences encoding linker or tag sequences that are linked to the cancer antigen by a peptide bond. The amino acid sequence may be a protein, a peptide, a variant thereof, a fragment thereof, or a combination thereof. The cancer antigen may be a recombinant cancer antigen.

One method for constructing a nucleic acid and its encoded recombinant cancer antigen amino acid sequence is to introduce mutations that alter specific amino acids throughout the amino acid sequence of the native cancer antigen. Introduction of mutations does not change the cancer antigen so much that it cannot be applied universally to mammals, preferably humans or dogs, but changes it enough that the resulting amino acid sequence breaks tolerance or is considered a foreign antigen to generate an immune response. Another method may be the creation of a consensus recombinant cancer antigen that has at least 85% to 99% amino acid sequence identity to its corresponding native cancer antigen; preferably at least 90% to 98% sequence identity; more preferably, at least 93% to 98% sequence identity; or even more preferably, at least 95% to 98% sequence identity. In some cases, the recombinant cancer antigen has 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to its corresponding native cancer antigen. This native cancer antigen is an antigen that is typically associated with a specific type of cancer or cancerous tumor. Depending on the cancer antigen, the cancer antigen consensus sequence may be present in mammals or within subtypes of a species, or in viral strains or serotypes. Some cancer antigens are not very different from the amino acid sequence of the wild type cancer antigen. Some cancer antigens have nucleotide/amino acid sequences that are so divergent among species that a consensus sequence cannot be obtained. In these cases, a recombinant cancer antigen is produced that will break tolerance and generate an immune response, an antigen that has at least 85% to 99% amino acid sequence identity to its corresponding native cancer antigen; preferably at least 90% to 98% sequence identity; more preferably, at least 93% to 98% sequence identity; or even more preferably, at least 95% to 98% sequence identity. In some cases, the recombinant cancer antigen has 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to its corresponding native cancer antigen. The above approaches can be combined such that the final recombinant cancer antigen has a percentage similarity to the amino acid sequence of the native cancer antigen as described above.

The cancer antigen may be one or more of the following antigens: tyrosinase (Tyr), tyrosinase-dependent protein 1 (TYRP1), tyrosinase-dependent protein 2 (TYRP2), melanoma-associated antigen 4 (MAGEA4), growth hormone-releasing hormone amino acid sequence (GHRH), 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 polynucleotide sequences encoding tyrosinase (Tyr), tyrosinase-dependent protein 1 (TYRP1), tyrosinase-dependent protein 2 (TYRP2), melanoma-associated antigen 4 (MAGEA4), the amino acid sequence of a hormone-releasing hormone growth hormone (GHRH), amino acid sequence of antigen

- 10 045958

MART-1/melan-A (MART-1/Melan-A), cancer-testis antigen (NY-ESO-1), cancer-testis antigen II (NY-ESO-1), PRAME, viral antigen, or combinations thereof. The viral antigen may be one or more of the following viruses: hepatitis B virus (eg, core and surface protein), hepatitis C virus (eg, nonstructural protein (NS) 34A (NS34A), NS5A, NS5B, NS4B), and also human papillomavirus (HPV) 6, HPV 11, HPV 16 and HPV 18.

(1) Tyrosinase (Tyr).

The vaccine of this invention may contain the cancer antigen tyrosinase (Tyr), a fragment thereof, or a variant thereof. Tyrosinase is a copper-containing enzyme with the catalytic activity of tyrosine hydroxylase and DOPA oxidase, which can be obtained from microorganisms and plant and animal tissues. In particular, tyrosinase catalyzes the formation 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 in the TYR gene contribute to changes in skin pigmentation.

Additionally, in cancer or tumors such as melanoma, tyrosinase can become dysregulated, leading to increased melanin synthesis. Accordingly, tyrosinase may be a cancer antigen associated with melanoma. In subjects suffering from melanoma, tyrosinase may be a target of cytotoxic T cell recognition. However, in some cases, the immune response to a cancer or tumor (including melanoma) may be suppressed, resulting in a microenvironment that supports tumor formation and/or progression and therefore disease progression.

Suppression of the immune response can be alleviated by myeloid-derived suppressor cells (MDSCs), which are a mixed population of immature macrophages, granulocytes, dendritic cells, and myeloid cells. Myeloid cells may be a heterogeneous population of myeloid progenitor cells and immature myeloid cells (IMC). Markers of MDSC may include expression of Gr-1 and CD11b (i.e., Gr-1 ⁺ and CD11b ⁺ cells).

MDSC circulation may increase in association with chronic infection, and increased MDSC populations may be associated with autoimmune response and inflammation. In particular, expansion of MDSCs (or presence in tumor or cancer tissue) may contribute to tumor development and evasion of immune detection and/or regulation, and thus MDSCs may influence immune responses to cancer vaccines.

MDSCs can be regulated by Regulator of G protein signaling 2 (Rgs2), and Rgs2 can be highly expressed in tumor-derived MDSCs. Rgs2 can also be widely expressed in various cells, such as myeloid cells. MDSCs derived from tumor-bearing mice may function differently than MDSCs derived from mice that are not tumor-bearing mice. One such difference may be the regulation of the production of the chemokine MCP-1, which is secreted by MDSCs. MCP-1 may promote cell migration by signaling through CCR2, a G protein-coupled receptor (GPCR) found on monocytes, endothelial cells and T cells. Accordingly, MCP-1 may lead to endothelial cell migration, thereby promoting vascularization. Blocking MCP-1 using neutralizing antibodies can inhibit angiogenesis and thus may lead to reduced tumor metastasis and increased survival. Thus, MCP-1 can be considered an angiogenic factor. In addition to secreting MCP-1, MDSCs can secrete growth factors, thereby further promoting tumor development.

The Tyr antigen is capable of inducing antigen-specific T cell and/or potent humoral immune responses, thereby inducing or triggering an immune response that targets or responds to the cancer or tumor by expression of the antigen. In some embodiments, the induced or evoked immune response may be cellular, humoral, or both cellular and humoral immune responses. In some embodiments, the induced or evoked cellular immune response may include the induction or secretion of interferon gamma (IFN-γ) and/or tumor necrosis factor alpha (TNF-α). In other embodiments, the induced or elicited immune response may reduce or inhibit one or more immune response suppressive factors that promote tumor or cancer development by expressing an antigen, such as, but not limited to, MHC presentation suppressive factors, antigen-specific activating factors regulatory T cells (Tregs), PDL1, FasL, cytokines such as IL-10 and TFG-β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by suppressor immune cells, CTLA-4, PD-1 , MDSC, MCP-1 and immune checkpoint molecules, as described in more detail below.

As demonstrated herein, the Tyr antigen induces antigen-specific T cell and potent humoral immune responses against cancer or neoplastic cells (e.g.

- 11 045958 melanoma). In particular, the Tyr antigen is an important target for immune-mediated clearance through the induction of (1) humoral immunity through B cell responses to produce antibodies that block the formation of monocyte chemoattractant protein-1 (MCP-1), thereby inhibiting myeloid suppressor cells origin (MDSC) and suppressing tumor development; (2) increasing cytotoxic T lymphocytes such as CD8 ⁺ (CTL) to attack and kill tumor cells; (3) increasing T helper cell responses; (4) and increasing inflammatory responses through IFN-γ and TFN-α, or preferably all of the above. Thus, a protective immune response against tumorigenesis and tumor development is provided by vaccines containing the Tyr antigen (e.g., the Tyr consensus antigen, which is described in more detail below), due to the fact that these vaccines prevent suppression of the immune response by reducing the MDSC population, found in cancerous or tumor tissue, and blocking vascularization of cancer or tumor tissue by reducing the formation or secretion of MCP-1. Accordingly, any user can create a vaccine of the present invention containing the Tyr antigen to provide broad immunity against tumorigenesis, tumor metastasis and tumor development.

Tyr antigen may contain protein epitopes that make them particularly effective as immunogens to which immune responses to Tyr can be induced. The Tyr antigen may contain a full-length translation product, a variant thereof, a fragment thereof, or a combination thereof. The Tyr antigen may contain a consensus protein.

The nucleotide sequence encoding the Tyr consensus antigen can be optimized for codon frequency and corresponding RNA transcripts. The nucleic acid encoding the Tyr consensus antigen may be codon- and RNA-optimized for expression. In some embodiments, the nucleotide sequence encoding the Tyr consensus antigen may contain a Kozak sequence (eg, GCC ACC) to increase translation efficiency. The nucleic acid encoding the Tyr consensus antigen may contain multiple stop codons (eg, TGA TGA) to increase translation termination efficiency.

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

The Tyr consensus antigen may be the nucleotide sequence of SEQ ID NO: 1, which encodes the amino acid sequence of SEQ ID NO: 2.

SEQ ID NO: 1 encodes a Tyr consensus protein associated with an IgE leader sequence. The Tyr consensus protein can be associated with an IgE leader sequence and an HA tag. In other embodiments, the Tyr consensus protein may not contain or be associated with an IgE leader sequence and/or an HA tag.

In some embodiments, the Tyr consensus antigen may be a nucleotide sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over the entire length of the nucleotide sequence given in SEQ ID NO: 1. In other embodiments of the invention, the Tyr consensus antigen may be a nucleotide sequence that encodes an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over the entire length of the amino acid sequence given in SEQ ID NO: 2. Tyr consensus antigen may be an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over the entire length of the amino acid sequence given in SEQ ID NO: 2.

Some embodiments of the invention relate to nucleotide sequences encoding proteins homologous to the Tyr consensus protein, an immunogenic fragment of the Tyr consensus protein, and immunogenic fragments of homologous proteins. Such nucleic acid molecules 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 can be provided . Likewise, nucleotide sequences encoding immunogenic fragments provided herein and immunogenic protein fragments that are homologous to proteins provided herein are also provided.

- 12 045958 document.

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

Some embodiments of the invention relate to nucleotide sequences encoding proteins with a certain percentage of identity to the full-length Tyr consensus protein, an immunogenic fragment of the Tyr consensus protein, and immunogenic protein fragments having identity to the Tyr consensus protein. Such nucleic acid molecules that encode immunogenic proteins having up to 80% identity with respect to the full-length Tyr consensus sequence, up to 85% identity with respect to the full-length consensus sequence, up to 90% identity with respect to the full-length Tyr consensus sequence, up to 91% identity with respect to the full-length Tyr consensus sequence, up to 92% identity with respect to the full-length Tyr consensus sequence, up to 93% identity with respect to the full-length Tyr consensus sequence, up to 94% identity with respect to the full-length Tyr consensus sequence, up to 95% identity with respect to the full-length Tyr consensus sequence, up to 96% identity with respect 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 can be provided. Likewise, nucleotide sequences encoding the immunogenic fragments described herein and immunogenic protein fragments with a similar percentage of identity as stated above to the Tyr proteins described herein are also provided.

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

Some embodiments of the invention relate to fragments of the sequence SEQ ID NO: 1. The fragments 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 sequence SEQ ID NO: 1. Fragments may be at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous to fragments of SEQ ID NO: 1 The fragments 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 fragments of SEQ ID NO: 1. In some embodiments The fragments of the invention contain sequences encoding 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 coding sequences that encode the leader sequence. In some embodiments, the fragments do not contain coding sequences that encode a leader sequence, such as, for example, an IgE leader sequence.

In addition, the amino acid sequence of the Tyr consensus protein is SEQ

- 13 045958

ID NO: 2.

The amino acid sequence of the Tyr consensus protein associated with an IgE leader sequence is SEQ ID NO: 2. The amino acid sequence of the Tyr consensus protein associated with an IgE leader sequence may be associated with an HA tag.

Some embodiments of the invention relate to proteins that are homologous to SEQ ID NO: 2. Some embodiments of the invention relate to immunogenic proteins that have 95% homology to the consensus protein sequences set forth in SEQ ID NO: 2. Some embodiments of the invention relate to immunogenic proteins that have 96% homology to the consensus protein sequences set forth in SEQ ID NO: 2. Some embodiments of the invention provide immunogenic proteins that have 97% homology to the consensus protein sequences set forth in SEQ ID NO: 2. Some embodiments embodiments of the invention relate to immunogenic proteins that have 98% homology to the consensus protein sequences set forth in SEQ ID NO: 2. Some embodiments of the invention relate to immunogenic proteins that have 99% homology to the consensus protein sequences set forth in SEQ ID NO: 2.

Some embodiments of the invention provide proteins that are identical to the sequence of SEQ ID NO: 2. Some embodiments of the invention provide immunogenic proteins having an amino acid sequence that is 80% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 2. Some embodiments embodiments of the invention provide immunogenic proteins having an amino acid sequence that is 85% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 2. Some embodiments of the invention provide immunogenic proteins having an amino acid sequence that is 90% identical to the full-length consensus amino acid sequence sequences set forth in SEQ ID NO: 2. Some embodiments of the invention provide immunogenic proteins having an amino acid sequence that is 91% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 2. Some embodiments of the invention provide immunogenic proteins having an amino acid sequence that is 92% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 2. Some embodiments of the invention relate to immunogenic proteins having an amino acid sequence that is 93% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: : 2. Some embodiments of the invention provide immunogenic proteins having an amino acid sequence that is 94% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 2. Some embodiments of the invention provide immunogenic proteins having an amino acid sequence that is 95% identical % identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 2. Some embodiments of the invention relate to immunogenic proteins having an amino acid sequence that is 96% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 2. Some embodiments of the invention refer to immunogenic proteins having an amino acid sequence that is 97% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 2. Some embodiments of the invention relate to immunogenic proteins having an amino acid sequence that is 98% identical to the full-length consensus amino acid sequences, set forth in SEQ ID NO: 2. Certain embodiments of the invention provide immunogenic proteins having an amino acid sequence that is 99% identical to the full-length consensus amino acid sequences set forth 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. The consensus protein fragments 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. Immunogenic fragments of the sequence SEQ ID NO: 2 can be provided. Immunogenic fragments may contain 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%,

- 14 045958 at least 96%, at least 97%, at least 98%, or at least 99% of the sequence of SEQ ID NO: 2. In some embodiments, the fragments contain a leader sequence, such as, for example, a leader sequence immunoglobulin, such as the 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 protein fragments with amino acid sequences homologous to the immunogenic fragments of SEQ ID NO: 2 may be provided. Such immunogenic fragments 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 proteins that are 95% or more homologous to the sequence SEQ ID NO: 2. Some embodiments of the invention provide immunogenic fragments having 96% homology to immunogenic fragments of the consensus protein sequences herein. Some embodiments of the invention provide immunogenic fragments having 97% homology to immunogenic fragments of the consensus protein sequences herein. Some embodiments of the invention provide immunogenic fragments having 98% homology to immunogenic fragments of the consensus protein sequences herein. Some embodiments of the invention provide immunogenic fragments having 99% homology to immunogenic fragments of the consensus protein sequences herein. In some embodiments, the fragments comprise 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 protein fragments may be provided with amino acid sequences identical to the immunogenic fragments of SEQ ID NO: 2. Such immunogenic fragments may contain 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 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 proteins that are 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical in amino acid sequence, as set forth in SEQ ID NO: 2. In some embodiments, the fragments comprise 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.

As disclosed herein with respect to coupling 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, which is encoded by the start codon of the nucleotide sequence, then encodes the protein without the signal peptide-encoding sequences.

(2) Tyrosinase-dependent protein 1 (TYRP1).

The vaccine of the present invention may contain cancer antigen tyrosinase-dependent protein 1 (TYRP1), a fragment thereof, or a variant thereof. TYRP1, encoded by the TYRP1 gene, is a transmembrane glycoprotein with a molecular mass of 75 kDa and is expressed in both normal and malignant melanocytes and melanoma cells. Like tyrosinase, TYRP1 contains a modified M-box potential that can bind to microphthalmia transcription factor (MITF), which plays a central role in melanocyte structure to activate pigmentation, proliferation and differentiation of cells. TYRP1 may help stabilize tyrosinase and may form a heterodimer that may prevent premature melanocyte death by attenuating tyrosinase-mediated cytotoxicity.

As described above with respect to tyrosinase, tyrosinase-dependent protein 1 (TYRP-1) may also be involved in the synthesis of melanin and the pigment apparatus of melanocytes, and may also be recognized by the immune system in subjects suffering from melanoma. Accordingly, TYRP-1 may be a melanoma-associated antigen.

The TRYP-1 antigen is capable of inducing antigen-specific T-cell and/or intense humoral immune responses, thereby inducing or eliciting an immune response that targets

- 15 045958 flax for cancer or tumor or reacts to them with antigen expression. In some embodiments, the induced or evoked immune response may be cellular, humoral, or both cellular and humoral immune responses. In some embodiments, the induced or evoked cellular immune response may include the induction or secretion of interferon gamma (IFN-γ) and/or tumor necrosis factor alpha (TNF-α). In other embodiments, the induced or elicited immune response may reduce or inhibit one or more immune response suppressive factors that stimulate the development of a tumor or cancer by expressing an antigen, such as, but not limited to, MHC presentation suppressive factors, antigen-specific activating factors regulatory T cells (Tregs), PDL1, FasL, cytokines such as IL-10 and TFG-β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by suppressor immune cells, CTLA-4, PD-1 , MDSC, MCP-1 and immune checkpoint molecules, as described in more detail below.

TYRP-1 antigen may contain protein epitopes that make them particularly effective as immunogens to which immune responses to TYRP-1 can be induced. The TYRP-1 antigen may contain a full-length translation product, a variant thereof, a fragment thereof, or a combination thereof. The TYRP-1 antigen may contain a consensus protein.

The nucleotide sequence encoding the TYRP-1 consensus antigen can be optimized for codon frequency and corresponding RNA transcripts. The nucleic acid encoding the TYRP-1 consensus antigen may be codon- and RNA-optimized for expression. In some embodiments, the nucleotide sequence encoding the TYRP-1 consensus antigen may contain a Kozak sequence (eg, GCC ACC) to increase translation efficiency. The nucleic acid encoding the TYRP-1 consensus antigen may contain multiple stop codons (eg, TGA TGA) to increase translation termination efficiency.

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

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

In some embodiments, the TYRP-1 consensus antigen may be a nucleotide sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over the entire length of the nucleotide sequence given in SEQ ID NO: 3. In others In embodiments of the invention, the TYRP-1 consensus antigen may be a nucleotide sequence that encodes an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over the entire length of the amino acid sequence given in SEQ ID NO: 4 The TYRP-1 consensus antigen may be an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over the entire length of the amino acid sequence given in SEQ ID NO: 4.

Some embodiments of the invention relate to nucleotide sequences encoding proteins homologous to the TYRP-1 consensus protein, an immunogenic fragment of the TYRP-1 consensus protein, and immunogenic fragments of homologous proteins. Such nucleic acid molecules 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 can be provided . Likewise, nucleotide sequences encoding the immunogenic fragments set forth herein and immunogenic protein fragments that are homologous to the proteins set forth herein are also provided.

- 16 045958 document.

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

Some embodiments of the invention relate to nucleotide sequences encoding proteins with a certain percentage of identity to the full-length TYRP-1 consensus protein, an immunogenic fragment of the TYRP-1 consensus protein, and immunogenic protein fragments having identity to the TYRP1 consensus protein. Such nucleic acid molecules that encode immunogenic proteins that have up to 80% identity with respect to the full-length TYRP-1 consensus sequence, up to 85% identity with respect to the full-length TYRP-1 consensus sequence, up to 90% identity with respect to the full-length TYRP-1 consensus sequence, up to 91% identity relative to the full-length TYRP-1 consensus sequence, up to 92% identity relative to the full-length TYRP-1 consensus sequence, up to 93% identity relative to the full-length TYRP-1 consensus sequence, up to 94% identity relative to the full-length TYRP-1 consensus sequence, up to 95% identity relative to full-length TYRP-1 consensus sequence, up to 96% identity relative to the full-length TYRP-1 consensus sequence, up to 97% identity relative to the full-length TYRP-1 consensus sequence, up to 98% identity relative to the full-length TYRP-1 consensus sequence, and up to 99% identity relative to the full-length consensus sequence TYRP-1 sequences can be provided. Likewise, nucleotide sequences encoding the immunogenic fragments described herein and immunogenic protein fragments with a similar percentage of identity as stated above to the TYRP-1 proteins described herein are also provided.

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

Some embodiments of the invention relate to fragments of the sequence SEQ ID NO: 3. The fragments 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 sequence SEQ ID NO: 3. Fragments may be at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous to fragments of SEQ ID NO: 3 The fragments 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 fragments of SEQ ID NO: 3. In some embodiments The fragments of the invention contain sequences encoding 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 coding sequences that encode the leader sequence. In some embodiments, the fragments do not contain coding sequences that encode a leader sequence, such as, for example, an IgE leader sequence.

- 17 045958

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

Some embodiments of the invention relate to proteins that are homologous to SEQ ID NO: 4. Some embodiments of the invention relate to immunogenic proteins that have 95% homology to the consensus protein sequences shown in SEQ ID NO: 4. Some embodiments of the invention relate to immunogenic proteins that have 96% homology to the consensus protein sequences set forth in SEQ ID NO: 4. Some embodiments of the invention provide immunogenic proteins that have 97% homology to the consensus protein sequences set forth in SEQ ID NO: 4.

Some embodiments of the invention provide immunogenic proteins that have 98% homology to the consensus protein sequences set forth in SEQ ID NO: 4. Some embodiments of the invention provide immunogenic proteins that have 99% homology to the consensus protein sequences set forth in SEQ ID NO: 4.

Some embodiments of the invention provide proteins that are identical to the sequence of SEQ ID NO: 4. Some embodiments of the invention provide immunogenic proteins having an amino acid sequence that is 80% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 4. Some embodiments embodiments of the invention provide immunogenic proteins having an amino acid sequence that is 85% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 4. Some embodiments of the invention provide immunogenic proteins having an amino acid sequence that is 90% identical to the full-length consensus amino acid sequence sequences set forth in SEQ ID NO: 4. Some embodiments of the invention provide immunogenic proteins having an amino acid sequence that is 91% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 4. Some embodiments of the invention provide immunogenic proteins having an amino acid sequence that is 92% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 4. Some embodiments of the invention relate to immunogenic proteins having an amino acid sequence that is 93% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: : 4. Some embodiments of the invention provide immunogenic proteins having an amino acid sequence that is 94% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 4. Some embodiments of the invention provide immunogenic proteins having an amino acid sequence that is 95% identical % identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 4. Some embodiments of the invention relate to immunogenic proteins having an amino acid sequence that is 96% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 4. Some embodiments of the invention refer to immunogenic proteins having an amino acid sequence that is 97% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 4. Some embodiments of the invention relate to immunogenic proteins having an amino acid sequence that is 98% identical to the full-length consensus amino acid sequences, set forth in SEQ ID NO: 4. Certain embodiments of the invention provide immunogenic proteins having an amino acid sequence that is 99% identical to the full-length consensus amino acid sequences set forth 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. The consensus protein fragments 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 TYRP-1 consensus protein. Immunogenic fragments of the sequence SEQ ID NO: 4 can be provided. Immunogenic fragments may contain 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

- 18 045958 at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, according at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the sequence of SEQ ID NO: 4. In some embodiments, the fragments contain 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 protein fragments with amino acid sequences homologous to the immunogenic fragments of SEQ ID NO: 4 can be provided. Such immunogenic fragments 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 proteins that are 95% or more homologous to the sequence SEQ ID NO: 4. Some embodiments of the invention provide immunogenic fragments having 96% homology to immunogenic fragments of the consensus protein sequences herein. Some embodiments of the invention provide immunogenic fragments having 97% homology to immunogenic fragments of the consensus protein sequences herein. Some embodiments of the invention provide immunogenic fragments having 98% homology to immunogenic fragments of the consensus protein sequences herein. Some embodiments of the invention provide immunogenic fragments having 99% homology to immunogenic fragments of the consensus protein sequences herein. In some embodiments, the fragments comprise 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 protein fragments with amino acid sequences identical to the immunogenic fragments of SEQ ID NO: 4 can be provided. Such immunogenic fragments 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 proteins that are 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequences set forth in SEQ ID NO: 4. In some embodiments, the fragments contain 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.

As disclosed herein with respect to coupling 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, which is encoded by the start codon of the nucleotide sequence, then encodes the protein without the signal peptide-encoding sequences.

(3) Tyrosinase-dependent protein 2 (TYRP2).

The vaccine of the present invention may contain cancer antigen tyrosinase-dependent 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 519 amino acid protein expressed in both normal and malignant melanocytes and melanoma cells. TYRP2/DCT is a well-studied melanocyte-specific enzyme that, in combination with tyrosinase and TYRP1, is involved in the conversion of L-tyrosine to melanin in melanocytes. DCT specifically catalyzes the tautomerization of melanin precursors L-dopachrome into 5,6dihydroindole-2-carboxylic acid (DHICA), which is subsequently oxidized by TYRP1 (which is described above) to form eumelanin. Studies have shown that TYRP2/DCT may mediate drug resistance in melanoma cells, with specificity for DNA-damaging agents. Since TYRP2/DCT has been repeatedly reported to be highly expressed in melanomas, this melanocyte-specific enzyme plays an important role in promoting

- 19 045958 melanoma phenotype with true resistance to various anticancer DNA-damaging drugs.

As described above with respect to tyrosinase, tyrosinase-dependent protein 2 (TYRP-2) may also be involved in melanin synthesis and be recognized by the immune system in subjects suffering from melanoma. In addition, TYRP-2 may mediate drug resistance in melanoma cells. Accordingly, TYRP-2 may be a melanoma-associated antigen.

The TRYP-2 antigen is capable of inducing antigen-specific T-cell and/or potent humoral immune responses, thereby inducing or eliciting an immune response that targets or responds to the cancer or tumor by expression of the antigen. In some embodiments, the induced or evoked immune response may be cellular, humoral, or both cellular and humoral immune responses. In some embodiments, the induced or evoked cellular immune response may include the induction or secretion of interferon gamma (IFN-γ) and/or tumor necrosis factor alpha (TNF-α). In other embodiments, the induced or elicited immune response may reduce or inhibit one or more immune response suppressive factors that stimulate the development of a tumor or cancer by expressing an antigen, such as, but not limited to, MHC presentation suppressive factors, antigen-specific activating factors regulatory T cells (Tregs), PDL1, FasL, cytokines such as IL-10 and TFG-β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by suppressor immune cells, CTLA-4, PD-1 , MDSC, MCP-1 and immune checkpoint molecules, as described in more detail below.

TYRP2 antigen may contain protein epitopes that make them particularly effective as immunogens to which immune responses to TYRP2 can be induced. The TYRP2 antigen may contain a full-length translation product, a variant thereof, a fragment thereof, or a combination thereof. The TYRP2 antigen may contain a consensus protein.

The nucleotide sequence encoding the TYRP2 consensus antigen can be optimized for codon frequency and corresponding RNA transcripts. The nucleic acid encoding the TYRP2 consensus antigen may be codon- and RNA-optimized for expression. In some embodiments, the nucleotide sequence encoding the TYRP2 consensus antigen may contain a Kozak sequence (eg, GCC ACC) to increase translation efficiency. The nucleic acid encoding the TYRP2 consensus antigen may contain multiple stop codons (eg, TGA TGA) to increase translation termination efficiency.

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

The TYRP2 consensus antigen may be the nucleotide sequence of SEQ ID NO: 5, which encodes the amino acid sequence of SEQ ID NO: 6.

SEQ ID NO: 5 encodes the TYRP2 consensus protein associated with an IgE leader sequence.

The TYRP2 consensus protein can be associated with an IgE leader sequence and an HA tag. In other embodiments, the TYRP2 consensus protein may not contain or be associated with an IgE leader sequence and/or an HA tag.

In some embodiments, the TYRP2 consensus antigen may be a nucleotide sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over the entire length of the nucleotide sequence set forth in SEQ ID NO: 5. In other embodiments of the invention, the TYRP2 consensus antigen may be a nucleotide sequence that encodes an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over the entire length of the amino acid sequence given in SEQ ID NO: 6. TYRP2 consensus antigen may be an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical along the entire length

- 20 045958 acid sequence given in SEQ ID NO: 6.

Some embodiments of the invention relate to nucleotide sequences encoding proteins homologous to the TYRP2 consensus protein, an immunogenic fragment of the TYRP2 consensus protein, and immunogenic fragments of homologous proteins. Such nucleic acid molecules 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 can be provided . Likewise, nucleotide sequences encoding immunogenic fragments provided herein and immunogenic fragments of proteins that are homologous to proteins provided herein are also provided.

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

Some embodiments of the invention relate to nucleotide sequences encoding proteins with a certain percentage of identity to the full-length TYRP2 consensus protein, an immunogenic fragment of the TYRP2 consensus protein, and immunogenic protein fragments having identity to the TYRP2 consensus protein. Such nucleic acid molecules that encode immunogenic proteins that have 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 relative to the full-length TYRP2 consensus sequence, up to 93% identity relative to the full-length TYRP2 consensus sequence, up to 94% identity relative to the full-length TYRP2 consensus sequence, up to 95% identity relative to the full-length TYRP2 consensus sequence, up to 96% identity relative 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 can be provided. Likewise, nucleotide sequences encoding the immunogenic fragments described herein and immunogenic protein fragments with a similar percentage of identity as stated above to the TYRP2 proteins described herein are also provided.

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

Some embodiments of the invention relate to fragments of the sequence SEQ ID NO: 5. The fragments 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 sequence SEQ ID NO: 5. Fragments may be at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous to fragments of SEQ ID NO: 5 .Fragments can be

- 21 045958 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 fragments of the sequence SEQ ID NO: 5. In some embodiments, the fragments contain sequences encoding 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 coding sequences that encode the leader sequence. In some embodiments, the fragments do not contain coding sequences that encode a leader sequence, such as, for example, an IgE leader sequence.

In addition, the amino acid sequence of the TYRP2 consensus protein is SEQ ID NO: 6. The amino acid sequence of the TYRP2 consensus protein associated with the IgE leader sequence is SEQ ID NO: 6. The amino acid sequence of the TYRP2 consensus protein associated with the IgE leader sequence may be associated with NAmetka.

Some embodiments of the invention relate to proteins that are homologous to SEQ ID NO: 2. Some embodiments of the invention relate to immunogenic proteins that have 95% homology to the consensus protein sequences set forth in SEQ ID NO: 6. Some embodiments of the invention relate to immunogenic proteins that have 96% homology to the consensus protein sequences set forth in SEQ ID NO: 6. Some embodiments of the invention provide immunogenic proteins that have 97% homology to the consensus protein sequences set forth in SEQ ID NO: 6. Some embodiments embodiments of the invention relate to immunogenic proteins that have 98% homology to the consensus protein sequences set forth in SEQ ID NO: 6. Some embodiments of the invention relate to immunogenic proteins that have 99% homology to the consensus protein sequences set forth in SEQ ID NO: 6.

Some embodiments of the invention provide proteins that are identical to the sequence of SEQ ID NO: 6. Some embodiments of the invention provide immunogenic proteins having an amino acid sequence that is 80% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 6. Some embodiments embodiments of the invention provide immunogenic proteins having an amino acid sequence that is 85% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 6. Some embodiments of the invention provide immunogenic proteins having an amino acid sequence that is 90% identical to the full-length consensus amino acid sequence sequences set forth in SEQ ID NO: 6. Some embodiments of the invention provide immunogenic proteins having an amino acid sequence that is 91% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 6. Some embodiments of the invention provide immunogenic proteins having an amino acid sequence that is 92% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 6. Some embodiments of the invention relate to immunogenic proteins having an amino acid sequence that is 93% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: : 6. Some embodiments of the invention provide immunogenic proteins having an amino acid sequence that is 94% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 6. Some embodiments of the invention provide immunogenic proteins having an amino acid sequence that is 95% identical % identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 6. Some embodiments of the invention relate to immunogenic proteins having an amino acid sequence that is 96% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 6. Some embodiments of the invention refer to immunogenic proteins having an amino acid sequence that is 97% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 6. Some embodiments of the invention relate to immunogenic proteins having an amino acid sequence that is 98% identical to the full-length consensus amino acid sequences, set forth in SEQ ID NO: 6. Certain embodiments of the invention provide immunogenic proteins having an amino acid sequence that is 99% identical to the full-length consensus amino acid sequences set forth 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.

- 22 045958

The consensus protein fragments 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. Immunogenic fragments of the sequence SEQ ID NO: 6 can be provided. Immunogenic fragments may contain 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 sequence of SEQ ID NO: 6. In some embodiments, the fragments contain 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 protein fragments with amino acid sequences homologous to the immunogenic fragments of SEQ ID NO: 6 can be provided. Such immunogenic fragments 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 proteins that are 95% or more homologous to the sequence SEQ ID NO: 6. Some embodiments of the invention provide immunogenic fragments having 96% homology to immunogenic fragments of the consensus protein sequences herein. Some embodiments of the invention provide immunogenic fragments having 97% homology to immunogenic fragments of the consensus protein sequences herein. Some embodiments of the invention provide immunogenic fragments having 98% homology to immunogenic fragments of the consensus protein sequences herein. Some embodiments of the invention provide immunogenic fragments having 99% homology to immunogenic fragments of the consensus protein sequences herein. In some embodiments, the fragments comprise 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 protein fragments with amino acid sequences identical to the immunogenic fragments of SEQ ID NO: 6 can be provided. Such immunogenic fragments 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 proteins that are 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequences set forth in SEQ ID NO: 6. In some embodiments, the fragments contain 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.

As disclosed herein with respect to coupling 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, which is encoded by the start codon of the nucleotide sequence, then encodes the protein without the signal peptide-encoding sequences.

(4) Melanoma-associated antigen 4 (MAGEA4).

The vaccine of the present invention may contain a cancer antigen. Melanoma-associated antigen 4 (MAGEA4), fragment or variant thereof. MAGE-A4, encoded by the MAGE-A4 gene, is a 317 amino acid protein that is expressed in male germ cells and tumor cells of various histological types, such as gastrointestinal carcinomas.

- 23 045958 intestinal tract, esophagus and lung. MAGE-A4 binds 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 adhesion-independent growth of Gankyrin-overexpressing cells in vitro and suppress 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. It is likely that MAGE expression in tumor tissue is not a cause, but a consequence of oncogenesis, and MAGE genes take part in the immune process by targeting early tumor cells for destruction.

Melanoma-associated antigen 4 (MAGEA4) may be involved in embryonic development as well as tumor transformation and/or development. MAGEA4 is typically expressed in the testis and placenta. However, MAGEA4 can be expressed in various tumor types, such as melanoma, head and neck squamous cell carcinoma, lung carcinoma, and breast carcinoma. Accordingly, MAGEA4 may be an antigen associated with various tumors.

The MAGEA4 antigen is capable of inducing antigen-specific T-cell and/or potent humoral immune responses, thereby inducing or eliciting an immune response that targets or responds to the cancer or tumor by expression of the antigen. In some embodiments, the induced or evoked immune response may be cellular, humoral, or both cellular and humoral immune responses. In some embodiments, the induced or evoked cellular immune response may include the induction or secretion of interferon gamma (IFN-γ) and/or tumor necrosis factor alpha (TNF-α). In other embodiments, the induced or elicited immune response may reduce or inhibit one or more immune response suppressive factors that stimulate the development of a tumor or cancer by expressing an antigen, such as, but not limited to, MHC presentation suppressive factors, antigen-specific activating factors 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 suppressor immune cells, CTLA-4, PD-1 , MDSC, MCP-1 and immune checkpoint molecules, as described in more detail below.

MAGEA4 antigen may contain protein epitopes that make them particularly effective as immunogens to which immune responses to MAGEA4 can be induced. The MAGEA4 antigen may contain a full-length translation product, a variant thereof, a fragment thereof, or a combination thereof. The MAGEA4 antigen may contain a consensus protein.

The nucleotide sequence encoding the MAGEA4 consensus antigen can be optimized for codon frequency and corresponding RNA transcripts. The nucleic acid encoding the MAGEA4 consensus antigen may be codon- and RNA-optimized for expression. In some embodiments, the nucleotide sequence encoding the MAGEA4 consensus antigen may contain a Kozak sequence (eg, GCC ACC) to increase translation efficiency. The nucleic acid encoding the MAGEA4 consensus antigen may contain multiple stop codons (eg, TGA TGA) to increase translation termination efficiency.

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

The MAGEA4 consensus antigen may be the nucleotide sequence of SEQ ID NO: 7, which encodes the amino acid sequence of SEQ ID NO: 8. The sequence of SEQ ID NO: 7 encodes the MAGEA4 consensus protein linked to an IgE leader sequence. The MAGEA4 consensus protein can be associated with the IgE leader sequence and HATM. In other embodiments, the MAGEA4 consensus protein may not contain or be associated with an IgE leader sequence and/or an HA tag.

In some embodiments, the MAGEA4 consensus antigen may be a nucleotide sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity over the entire length of the nucleotide sequence given in SEQ ID NO: 7. In others

- 24 045958 embodiments of the invention, the MAGEA4 consensus antigen may be a nucleotide sequence that encodes an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88 %, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity over the entire length of the amino acid sequence given in SEQ ID NO: 8. The MAGEA4 consensus antigen may be an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over the entire length of the amino acid sequence given in SEQ ID NO: 8.

Some embodiments of the invention relate to nucleotide sequences encoding proteins homologous to the MAGEA4 consensus protein, an immunogenic fragment of the MAGEA4 consensus protein, and immunogenic fragments of homologous proteins. Such nucleic acid molecules 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 can be provided . Likewise, nucleotide sequences encoding immunogenic fragments provided herein and immunogenic fragments of proteins that are homologous to proteins provided herein are also provided.

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

Some embodiments of the invention relate to nucleotide sequences encoding proteins with a certain percentage of identity to the full-length MAGEA4 consensus protein, an immunogenic fragment of the MAGEA4 consensus protein, and immunogenic protein fragments having identity to the MAGEA4 consensus protein. Such nucleic acid molecules that encode immunogenic proteins that have 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 relative to the full-length MAGEA4 consensus sequence, up to 93% identity relative to the full-length MAGEA4 consensus sequence, up to 94% identity relative to the full-length MAGEA4 consensus sequence, up to 95% identity relative to the full-length MAGEA4 consensus sequence, up to 96% identity relative 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 can be provided. Likewise, nucleotide sequences encoding the immunogenic fragments described herein and immunogenic protein fragments with a similar percentage of identity as stated above to the MAGEA4 proteins described herein are also provided.

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

Some embodiments of the invention relate to fragments of the sequence SEQ ID NO: 7. The fragments may comprise at least 10%, at least 15%, at least 20%,

- 25 045958 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%, according at least 97%, at least 98%, or at least 99% of the sequence of SEQ ID NO: 7. Fragments may be at least 95%, at least 96%, at least 97%, at least are 98% or at least 99% homologous to fragments of SEQ ID NO: 7. Fragments may be at least 80%, at least 85%, at least 90%, at least 91%, by at least 92%, by at least 93%, by at least 94%, by at least 95%, by at least 96%, by at least 97%, by at least 98%, or by are at least 99% identical to the fragments of SEQ ID NO: 7. In some embodiments, the fragments contain sequences encoding 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 coding sequences that encode the leader sequence. In some embodiments, the fragments do not contain coding sequences that encode a leader sequence, such as, for example, an IgE leader sequence.

In addition, the amino acid sequence of the MAGEA4 consensus protein is SEQ ID NO: 8.

The amino acid sequence of the MAGEA4 consensus protein associated with the IgE leader sequence is SEQ ID NO: 8. The amino acid sequence of the MAGEA4 consensus protein associated with the IgE leader sequence can be associated with HA.

Some embodiments of the invention relate to proteins that are homologous to the sequence SEQ ID NO: 8. Some embodiments of the invention relate to immunogenic proteins that have 95% homology to the consensus protein sequences shown in SEQ ID NO: 8. Some embodiments of the invention relate to immunogenic proteins that have 96% homology to the consensus protein sequences set forth in SEQ ID NO: 8. Some embodiments of the invention provide immunogenic proteins that have 97% homology to the consensus protein sequences set forth in SEQ ID NO: 8. Some embodiments embodiments of the invention relate to immunogenic proteins that have 98% homology to the consensus protein sequences set forth in SEQ ID NO: 8. Some embodiments of the invention relate to immunogenic proteins that have 99% homology to the consensus protein sequences set forth in SEQ ID NO: 8.

Some embodiments of the invention provide proteins that are identical to the sequence of SEQ ID NO: 8. Some embodiments of the invention provide immunogenic proteins having an amino acid sequence that is 80% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 8. Some embodiments embodiments of the invention provide immunogenic proteins having an amino acid sequence that is 85% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 8. Some embodiments of the invention provide immunogenic proteins having an amino acid sequence that is 90% identical to the full-length consensus amino acid sequence sequences set forth in SEQ ID NO: 8. Some embodiments of the invention provide immunogenic proteins having an amino acid sequence that is 91% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 8. Some embodiments of the invention provide immunogenic proteins having an amino acid sequence that is 92% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 8. Some embodiments of the invention relate to immunogenic proteins having an amino acid sequence that is 93% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: : 8. Some embodiments of the invention provide immunogenic proteins having an amino acid sequence that is 94% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 8. Some embodiments of the invention provide immunogenic proteins having an amino acid sequence that is 95% identical % identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 8. Some embodiments of the invention relate to immunogenic proteins having an amino acid sequence that is 96% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 8. Some embodiments of the invention refer to immunogenic proteins having an amino acid sequence that is 97% identical to the full-length consensus amino acid sequences given in SEQ

- 26 045958

ID NO: 8. Some embodiments of the invention provide immunogenic proteins having an amino acid sequence that is 98% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 8. Some embodiments of the invention provide immunogenic proteins having an amino acid sequence that 99% identical to the full-length consensus amino acid sequences given 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. The consensus protein fragments 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. Immunogenic fragments of the sequence SEQ ID NO: 8 can be provided. Immunogenic fragments may contain 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 sequence of SEQ ID NO: 8. In some embodiments, the fragments contain 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 protein fragments with amino acid sequences homologous to the immunogenic fragments of SEQ ID NO: 8 can be provided. Such immunogenic fragments 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 proteins that are 95% or more homologous to the sequence SEQ ID NO: 8. Some embodiments of the invention provide immunogenic fragments having 96% homology to immunogenic fragments of the consensus protein sequences herein. Some embodiments of the invention provide immunogenic fragments having 97% homology to immunogenic fragments of the consensus protein sequences herein. Some embodiments of the invention provide immunogenic fragments having 98% homology to immunogenic fragments of the consensus protein sequences herein. Some embodiments of the invention provide immunogenic fragments having 99% homology to immunogenic fragments of the consensus protein sequences herein. In some embodiments, the fragments comprise 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 protein fragments with amino acid sequences identical to the immunogenic fragments of SEQ ID NO: 8 can be provided. Such immunogenic fragments 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 proteins that are 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequences set forth in SEQ ID NO: 8. In some embodiments, the fragments contain 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.

- 27 045958

As disclosed herein with respect to coupling 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, which is encoded by the start codon of the nucleotide sequence, then encodes the protein without the signal peptide-encoding sequences.

(5) Growth hormone releasing hormone (GHRH).

The vaccine of the present invention may contain the cancer antigen Growth hormone-releasing hormone (GHRH; also known as growth hormone-releasing factor (GRF or GHRF) or somatocrinin), a fragment thereof, or a variant thereof. GHRH is a 44 amino acid peptide hormone produced in the arcuate nucleus of the hypothalamus. GHRH is secreted by the hypothalamus and stimulates the release of growth hormone, a growth regulator, metabolism and somatic structure, from the pituitary gland. GHRH also stimulates growth hormone product. GHRH antagonists can inhibit the development of various types of cancer, such as osteosarcomas, glioblastomas, prostate cancer, kidney cancer, pancreatic cancer, colorectal cancer, and breast cancer. Accordingly, GHRH may be an antigen associated with various tumors.

The GHRH antigen is capable of inducing antigen-specific T-cell and/or potent humoral immune responses, thereby inducing or eliciting an immune response that targets or responds to the cancer or tumor by expression of the antigen. In some embodiments, the induced or evoked immune response may be cellular, humoral, or both cellular and humoral immune responses. In some embodiments, the induced or evoked cellular immune response may include the induction or secretion of interferon gamma (IFN-γ) and/or tumor necrosis factor alpha (TNF-α). In other embodiments, the induced or elicited immune response may reduce or inhibit one or more immune response suppressive factors that promote tumor or cancer development by expressing an antigen, such as, but not limited to, MHC presentation suppressive factors, antigen-specific activating factors regulatory T cells (Tregs), PDL1, FasL, cytokines such as IL-10 and TFG-β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by suppressor immune cells, CTLA-4, PD-1 , MDSC, MCP-1 and immune checkpoint molecules, as described in more detail below.

GHRH antigen may contain protein epitopes that make them particularly effective as immunogens to which immune responses to GHRH can be induced. The GHRH antigen may contain a full-length translation product, a variant thereof, a fragment thereof, or a combination thereof. The GHRH antigen may contain a consensus protein.

The nucleotide sequence encoding the GHRH consensus antigen can be optimized for codon frequency and corresponding RNA transcripts. The nucleic acid encoding the GHRH consensus antigen may be codon- and RNA-optimized for expression. In some embodiments, the nucleotide sequence encoding the GHRH consensus antigen may contain a Kozak sequence (eg, GCC ACC) to increase translation efficiency. The nucleic acid encoding the GHRH consensus antigen may contain multiple stop codons (eg, TGA TGA) to increase translation termination efficiency.

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

The GHRH consensus antigen may be the nucleotide sequence of SEQ ID NO: 9, which encodes the amino acid sequence of SEQ ID NO: 10.

The sequence of SEQ ID NO: 9 encodes the GHRH consensus protein associated with an IgE leader sequence. The GHRH consensus protein can be associated with an IgE leader sequence and an HA tag. In other embodiments, the GHRH consensus protein may not contain or be associated with an IgE leader sequence and/or an HA tag.

In some embodiments, the GHRH consensus antigen may be a nucleotide sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity over the entire length of the nucleotide sequence given in SEQ ID NO: 9. In other va

- 28 045958 In embodiments of the invention, the GHRH consensus antigen may be a nucleotide sequence that encodes an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88 %, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity over the entire length of the amino acid sequence given in SEQ ID NO: 10. The GHRH consensus antigen may be an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over the entire length of the amino acid sequence given in SEQ ID NO: 10.

Some embodiments of the invention relate to nucleotide sequences encoding proteins homologous to the GHRH consensus protein, an immunogenic fragment of the GHRH consensus protein, and immunogenic fragments of homologous proteins. Such nucleic acid molecules 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 can be provided . Likewise, nucleotide sequences encoding immunogenic fragments provided herein and immunogenic fragments of proteins that are homologous to proteins provided herein are also provided.

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

Some embodiments of the invention relate to nucleotide sequences encoding proteins with a certain percentage of identity to the full-length GHRH consensus protein, an immunogenic fragment of the GHRH consensus protein, and immunogenic protein fragments having identity to the GHRH consensus protein. Such nucleic acid molecules that encode immunogenic proteins that have 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 with respect to the full-length GHRH consensus sequence, up to 93% identity with respect to the full-length GHRH consensus sequence, up to 94% identity with respect to the full-length GHRH consensus sequence, up to 95% identity with respect to the full-length GHRH consensus sequence, up to 96% identity with respect 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 can be provided. Likewise, nucleotide sequences encoding the immunogenic fragments described herein and immunogenic protein fragments with a similar percentage of identity as stated above to the GHRH proteins described herein are also provided.

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

Some embodiments of the invention relate to fragments of the sequence SEQ ID NO: 9. The fragments may comprise at least 10%, at least 15%, at least 20%,

- 29 045958 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%, according at least 97%, at least 98%, or at least 99% of the sequence of SEQ ID NO: 9. Fragments may be at least 95%, at least 96%, at least 97%, at least are 98% or at least 99% homologous to fragments of SEQ ID NO: 9. Fragments may be at least 80%, at least 85%, at least 90%, at least 91%, by at least 92%, by at least 93%, by at least 94%, by at least 95%, by at least 96%, by at least 97%, by at least 98%, or by are at least 99% identical to the fragments of SEQ ID NO: 9. In some embodiments, the fragments contain sequences encoding 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 coding sequences that encode the leader sequence. In some embodiments, the fragments do not contain coding sequences that encode a leader sequence, such as, for example, an IgE leader sequence.

In addition, the amino acid sequence of the GHRH consensus protein is SEQ ID NO: 10. The amino acid sequence of the GHRH consensus protein associated with the IgE leader sequence is SEQ ID NO: 10. The amino acid sequence of the GHRH consensus protein associated with the IgE leader sequence may be associated with NAmetka.

Some embodiments of the invention relate to proteins that are homologous to SEQ ID NO: 10. Some embodiments of the invention relate to immunogenic proteins that have 95% homology to the consensus protein sequences set forth in SEQ ID NO: 10. Some embodiments of the invention relate to immunogenic proteins that have 96% homology to the consensus protein sequences set forth in SEQ ID NO: 10. Some embodiments of the invention provide immunogenic proteins that have 97% homology to the consensus protein sequences set forth in SEQ ID NO: 10. Some embodiments embodiments of the invention relate to immunogenic proteins that have 98% homology to the consensus protein sequences set forth in SEQ ID NO: 10. Some embodiments of the invention relate to immunogenic proteins that have 99% homology to the consensus protein sequences set forth in SEQ ID NO: 10.

Some embodiments of the invention relate to proteins that are identical to the sequence of SEQ ID NO: 10.

Some embodiments of the invention provide immunogenic proteins having an amino acid sequence that is 80% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 10. Some embodiments of the invention provide immunogenic proteins having an amino acid sequence that is 85% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 10. Some embodiments of the invention relate to immunogenic proteins having an amino acid sequence that is 90% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 10. Some embodiments of the invention relate to immunogenic proteins having an amino acid sequence that is 91% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 10. Some embodiments of the invention relate to immunogenic proteins having an amino acid sequence that is 92% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 10. Some embodiments of the invention provide immunogenic proteins having an amino acid sequence that is 93% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 10. Some embodiments of the invention provide immunogenic proteins having an amino acid sequence that is 94% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 10. Some embodiments of the invention provide immunogenic proteins having an amino acid sequence that is 95% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 10. Some embodiments embodiments of the invention provide immunogenic proteins having an amino acid sequence that is 96% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 10. Some embodiments of the invention provide immunogenic proteins having an amino acid sequence that is 97% identical to the full-length consensus amino acid sequence sequences given in SEQ

- 30 045958

ID NO: 10. Some embodiments of the invention provide immunogenic proteins having an amino acid sequence that is 98% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 10. Some embodiments of the invention provide immunogenic proteins having an amino acid sequence that 99% identical to the full-length consensus amino acid sequences 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. The consensus protein fragments 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. Immunogenic fragments of the sequence SEQ ID NO: 10 can be provided. Immunogenic fragments may contain 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 sequence of SEQ ID NO: 10. In some embodiments, the fragments contain 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 protein fragments with amino acid sequences homologous to the immunogenic fragments of SEQ ID NO: 10 can be provided. Such immunogenic fragments 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 proteins that are 95% or more homologous to the sequence SEQ ID NO: 10. Some embodiments of the invention provide immunogenic fragments having 96% homology to immunogenic fragments of the consensus protein sequences herein. Some embodiments of the invention provide immunogenic fragments having 97% homology to immunogenic fragments of the consensus protein sequences herein. Some embodiments of the invention provide immunogenic fragments having 98% homology to immunogenic fragments of the consensus protein sequences herein. Some embodiments of the invention provide immunogenic fragments having 99% homology to immunogenic fragments of the consensus protein sequences herein. In some embodiments, the fragments comprise 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 protein fragments with amino acid sequences identical to the immunogenic fragments of SEQ ID NO: 10 can be provided. Such immunogenic fragments 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 proteins that are 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequences set forth in SEQ ID NO: 10. In some embodiments, the fragments contain 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.

- 31 045958

As disclosed herein with respect to coupling 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, which is encoded by the start codon of the nucleotide sequence, then encodes the protein without the signal peptide-encoding sequences.

(6) MART-1/Melan-A.

The vaccine of the present invention may contain the cancer antigen MART-1 (also known 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 a structural protein and influences its expression, stability, directional migration, and processing, which is required for melanosome structure and maturation. Accordingly, MART-1 is essential for the regulation of mammalian pigmentation. Impairments in melanosome maturation have been associated with cancer susceptibility. MART-1 can be expressed in various types of cancer, including, but not limited to, melanomas.

Melan-A, also known as melanoma antigen recognized by T cells (MART-1), is a melanocyte differentiation antigen and can be found in normal skin, retina, and melanocytes. Melan-A can associate 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 types of tumors that are of melanocytic origin or differentiation (that is, the cells contain melanosomes), such as clear cell sarcoma and melanotic neurofibroma. Accordingly, Melan-A may be an antigen that is associated with various types of tumors derived from cells containing melanosomes.

The Melan-A antigen is capable of inducing antigen-specific T-cell and/or potent humoral immune responses, thereby inducing or eliciting an immune response that targets or responds to cancer or tumor by expression of the antigen. In some embodiments, the induced or evoked immune response may be cellular, humoral, or both cellular and humoral immune responses. In some embodiments, the induced or evoked cellular immune response may include the induction or secretion of interferon gamma (IFN-γ) and/or tumor necrosis factor alpha (TNF-α). In other embodiments, the induced or elicited immune response may reduce or inhibit one or more immune response suppressive factors that promote tumor or cancer development by expressing an antigen, such as, but not limited to, MHC presentation suppressive factors, antigen-specific activating factors regulatory T cells (Tregs), PDL1, FasL, cytokines such as IL-10 and TFG-β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by suppressor immune cells, CTLA-4, PD-1 , MDSC, MCP-1 and immune checkpoint molecules, as described in more detail below.

Melan-A antigen may contain protein epitopes that make them particularly effective as immunogens to which immune responses to Melan-A can be induced. The Melan-A antigen may contain a full-length translation product, a variant thereof, a fragment thereof, or a combination thereof. The Melan-A antigen may contain a consensus protein.

The nucleotide sequence encoding the Melan-A consensus antigen can be optimized for codon frequency and corresponding RNA transcripts.

The nucleic acid encoding the Melan-A consensus antigen may be codon- and RNA-optimized for expression. In some embodiments, the nucleotide sequence encoding the Melan-A consensus antigen may contain a Kozak sequence (eg, GCC ACC) to increase translation efficiency. The nucleic acid encoding the Melan-A consensus antigen may contain multiple stop codons (eg, TGA TGA) to increase translation termination efficiency.

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

The Melan-A consensus antigen may be the nucleotide sequence of SEQ ID NO: 11, which encodes the amino acid sequence of SEQ ID NO: 12. The sequence of SEQ ID NO: 11 encodes the MELAN-A consensus protein linked to an IgE leader sequence. Melan-A consensus protein may be associated with IgE and HA leader sequence

- 32 045958 label. In other embodiments, the Melan-A consensus protein may not contain or be associated with an IgE leader sequence and/or an HA tag.

In some embodiments, the Melan-A consensus antigen may be a nucleotide sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over the entire length of the nucleotide sequence given in SEQ ID NO: 11. In others In embodiments of the invention, the Melan-A consensus antigen may be a nucleotide sequence that encodes an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over the entire length of the amino acid sequence given in SEQ ID NO: 12 The Melan-A consensus antigen may be an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over the entire length of the amino acid sequence given in SEQ ID NO: 12.

Some embodiments of the invention relate to nucleotide sequences encoding proteins homologous to the Melan-A consensus protein, an immunogenic fragment of the Melan-A consensus protein, and immunogenic fragments of homologous proteins. Such nucleic acid molecules 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 can be provided . Likewise, nucleotide sequences encoding immunogenic fragments provided herein and immunogenic fragments of proteins that are homologous to proteins provided herein are also provided.

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

Some embodiments of the invention relate to nucleotide sequences encoding proteins with a certain percentage of identity to the full-length Melan-A consensus protein, an immunogenic fragment of the Melan-A consensus protein, and immunogenic protein fragments having identity to the Melan-A consensus protein. Such nucleic acid molecules that encode immunogenic proteins that have up to 80% identity with respect to the full-length Melan-A consensus sequence, up to 85% identity with respect to the full-length Melan-A consensus sequence, up to 90% identity with respect to the full-length Melan-A consensus sequence, up to 91% identity relative to the full-length Melan-A consensus sequence, up to 92% identity relative to the full-length Melan-A consensus sequence, up to 93% identity relative to the full-length Melan-A consensus sequence, up to 94% identity relative to the full-length Melan-A consensus sequence, up to 95% identity relative to full-length Melan-A consensus sequence, up to 96% identity relative to the full-length Melan-A consensus sequence, up to 97% identity relative to the full-length Melan-A consensus sequence, up to 98% identity relative to the full-length Melan-A consensus sequence, and up to 99% identity relative to the full-length consensus sequence Melan-A sequences can be provided.

Likewise, nucleotide sequences encoding the immunogenic fragments described herein and immunogenic protein fragments with a similar percentage of identity as stated above to the Melan-A proteins described herein are also provided.

- 33 045958 document.

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

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

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

Some embodiments of the invention relate to proteins that are homologous to SEQ ID NO: 12. Some embodiments of the invention relate to immunogenic proteins that have 95% homology to the consensus protein sequences set forth in SEQ ID NO: 12. Some embodiments of the invention relate to immunogenic proteins that have 96% homology to the consensus protein sequences set forth in SEQ ID NO: 12. Some embodiments of the invention provide immunogenic proteins that have 97% homology to the consensus protein sequences set forth in SEQ ID NO: 12. Some embodiments embodiments of the invention relate to immunogenic proteins that have 98% homology to the consensus protein sequences set forth in SEQ ID NO: 12. Some embodiments of the invention relate to immunogenic proteins that have 99% homology to the consensus protein sequences set forth in SEQ ID NO: 12.

Some embodiments of the invention provide proteins that are identical to the sequence of SEQ ID NO: 12. Some embodiments of the invention provide immunogenic proteins having an amino acid sequence that is 80% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 12. Some embodiments embodiments of the invention provide immunogenic proteins having an amino acid sequence that is 85% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 12. Some embodiments of the invention provide immunogenic proteins having an amino acid sequence that is 90% identical to the full-length consensus amino acid sequence sequences set forth in SEQ ID NO: 12. Some embodiments of the invention provide immunogenic proteins having an amino acid sequence that is 91% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 12. Some embodiments of the invention provide immunogenic proteins having an amino acid sequence that is 92% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 12. Some embodiments of the invention relate to immunogenic proteins having an amino acid sequence that is 93% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: : 12. Some embodiments of the invention provide immunogenic proteins having an amino acid sequence that is 94% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 12. Some embodiments of the invention provide immunogenic proteins having an amino acid sequence that is 95% identical % identical

- 34 045958 full-length consensus amino acid sequences set forth in SEQ ID NO: 12. Some embodiments of the invention relate to immunogenic proteins having an amino acid sequence that is 96% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 12. Some embodiments The inventions relate to immunogenic proteins having an amino acid sequence that is 97% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 12. Some embodiments of the invention relate to immunogenic proteins having an amino acid sequence that is 98% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 12. Certain embodiments of the invention provide immunogenic proteins having an amino acid sequence that is 99% identical to the full-length consensus amino acid sequences set forth 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. The consensus protein fragments 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. Immunogenic fragments of the sequence SEQ ID NO: 12 can be provided. Immunogenic fragments may contain 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 sequence of SEQ ID NO: 12. In some embodiments, the fragments contain 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 protein fragments may be provided with amino acid sequences homologous to immunogenic fragments of SEQ ID NO: 12. Such immunogenic fragments may contain 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% proteins that are 95% or more homologous to the sequence of SEQ ID NO: 12. Some embodiments of the invention provide immunogenic fragments having 96% homology to immunogenic fragments of the consensus protein sequences herein. Some embodiments of the invention provide immunogenic fragments having 97% homology to immunogenic fragments of the consensus protein sequences herein. Some embodiments of the invention provide immunogenic fragments having 98% homology to immunogenic fragments of the consensus protein sequences herein. Some embodiments of the invention provide immunogenic fragments having 99% homology to immunogenic fragments of the consensus protein sequences herein. In some embodiments, the fragments comprise 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 protein fragments may be provided with amino acid sequences identical to the immunogenic fragments of SEQ ID NO: 12. Such immunogenic fragments may contain 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 proteins that are 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%

- 35 045958 or 99% identical to the amino acid sequences shown in SEQ ID NO: 12. In some embodiments, the fragments contain 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.

As disclosed herein with respect to coupling 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, which is encoded by the start codon of the nucleotide sequence, then encodes the protein without the signal peptide-encoding sequences.

(7) NY-ESO-1.

The vaccine of the present invention may contain New York esophageal cancer antigen-1 (NY-ESO-1; also called CTAG1), a fragment thereof, or a variant thereof. NY-ESO-1, encoded by the CTAG1B gene, is a 180-amino acid protein with a glycine-rich N-terminal region and a superhydrophobic C-terminal region. NY-ESO-1 has limited expression in normal tissues but is common in cancer. NY-ESO-1 can be expressed in a variety of cancers, including, but not limited to, bladder, colorectal, esophageal, gastric, hepatocarcinoma, head and neck cancer, melanoma, non-small cell lung cancer, ovarian cancer, pancreatic cancer gland, synovial carcinoma and prostate cancer.

Cancer testis antigen (NY-ESO-1) can be expressed in the testes and ovaries. NY-ESO-1 may be associated with various types of cancer and is capable of inducing humoral immune responses. Subjects suffering from cancer or tumors may develop immunogenicity to NY-ESO-1. Accordingly, NY-ESO-1 may be an antigen associated with various tumors.

The NY-ESO-1 antigen is capable of inducing antigen-specific T-cell and/or potent humoral immune responses, thereby inducing or triggering an immune response that targets or responds to cancer or tumor by antigen expression. In some embodiments, the induced or evoked immune response may be cellular, humoral, or both cellular and humoral immune responses. In some embodiments, the induced or evoked cellular immune response may include the induction or secretion of interferon gamma (IFN-γ) and/or tumor necrosis factor alpha (TNF-α). In other embodiments, the induced or elicited immune response may reduce or inhibit one or more immune response suppressive factors that stimulate the development of a tumor or cancer by expressing an antigen, such as, but not limited to, MHC presentation suppressive factors, antigen-specific activating factors 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 suppressor immune cells, CTLA-4, PD-1 , MDSC, MCP-1 and immune checkpoint molecules, as described in more detail below.

The NY-ESO-1 antigen may increase the cellular immune response in a subject administered the NY-ESO-1 antigen by up to about 50-fold to about 6000-fold, about 50-fold to about 5500-fold, about 50-fold multiple to about 5000 times from about 50 times to about 4500 times, from about 100 times to about 6000 times, from about 150 times to about 6000 times, from about 200 times to about 6000 times, from about 250-fold to 6000-fold or about 300-fold to about 6000-fold the size of the cellular immune response in a subject not treated with NY-ESO-1 antigen. In some embodiments, the NYESO-1 antigen may increase the cellular immune response in a subject administered the NY-ESO-1 antigen by up to about 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550 , 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 230 0, 2400, 2500, 2600 , 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4 700, 4800, 4900, 5000, 5100 , 5200, 5300, 5400, 5500, 5600, 5700, 5800, 5900, or 6000 times the cellular immune response in a subject not administered the NY-ESO-1 antigen.

NY-ESO-1 antigen may increase gamma interferon (IFN-γ) levels in a subject administered NY-ESO-1 antigen by about 50-fold to about 6000-fold, from about 50-fold to about 5500-fold multiple, from about 50 times to about 5000 times, from about 50 times to about 4500 times, from about 100 times to about 6000 times, from about 150 times to about 6000 times, from about 200 times to about 6000-fold, about 250-fold to 6000-fold, or about 300-fold to about 6000-fold the IFN-γ levels of a subject not receiving NY-ESO-1 antigen. In some embodiments, the NY-ESO-1 antigen may increase the IFN-γ levels of a subject receiving the NY-ESO-1 antigen by up to about 50, 100, 150, 200, 250, 300, 350, 400, 450. 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400,

- 36 045958

1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 350 0, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 5100, 5200, 5300, 5400, 5500, 5600, 5700, 5800, 5900 or 600 0 times compared to IFN-γ levels a subject not injected with the NY-ESO-1 antigen.

The NY-ESO-1 antigen may contain protein epitopes that make them particularly effective as immunogens to which immune responses to NY-ESO-1 can be induced. The NYESO-1 antigen may contain a full-length translation product, a variant thereof, a fragment thereof, or a combination thereof. The NY-ESO-1 antigen may contain a consensus protein.

The nucleotide sequence encoding the NY-ESO-1 consensus antigen can be optimized for codon frequency and corresponding RNA transcripts. The nucleic acid encoding the NY-ESO-1 consensus antigen may be codon- and RNA-optimized for expression. In some embodiments, the nucleotide sequence encoding the NY-ESO-1 consensus antigen may contain a Kozak sequence (eg, GCC ACC) to increase translation efficiency. The nucleic acid encoding the NY-ESO-1 consensus antigen may contain multiple stop codons (eg, TGA TGA) to increase translation termination efficiency.

The nucleic acid encoding the NY-ESO-1 consensus antigen may also encode an immunoglobulin E (IgE) leader sequence. The nucleic acid encoding the NY-ESO-1 consensus 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 NY-ESO-1 consensus antigen by a peptide bond. The nucleic acid encoding the NY-ESO-1 consensus antigen may also contain a nucleotide sequence encoding an IgE leader sequence. In some embodiments, the nucleic acid encoding the NY-ESO-1 consensus antigen does not have or contain a nucleotide sequence encoding an IgE leader sequence.

The NY-ESO-1 consensus antigen may be the nucleotide sequence of SEQ ID NO: 13, which encodes the amino acid sequence of SEQ ID NO: 14. The sequence of SEQ ID NO: 13 encodes the NY-ESO-1 consensus protein linked to an IgE leader sequence. The NY-ESO-1 consensus protein can be associated with the IgE leader sequence and HAmarker. In other embodiments, the NY-ESO-1 consensus protein may not contain or be associated with an IgE leader sequence and/or an HA tag.

In some embodiments, the NY-ESO-1 consensus antigen may be a nucleotide sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over the entire length of the nucleotide sequence shown in SEQ ID NO: 13. In other embodiments, the NY-ESO-1 consensus antigen may be a nucleotide sequence that encodes an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87 %, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the entire length of the amino acid sequence given in SEQ ID NO: 14. The NY-ESO-1 consensus antigen may be an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over the entire length of the amino acid sequence shown in SEQ ID NO: 14.

Some embodiments of the invention relate to nucleotide sequences encoding proteins homologous to the NY-ESO-1 consensus protein, an immunogenic fragment of the NY-ESO-1 consensus protein, and immunogenic fragments of homologous proteins. Such nucleic acid molecules 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 can be provided . Likewise, nucleotide sequences encoding immunogenic fragments provided herein and immunogenic fragments of proteins that are homologous to proteins provided herein are also provided.

Some embodiments of the invention relate to nucleic acid molecules encoding immunogenic proteins having 95% homology to the nucleic acid coding sequences herein. Some embodiments of the invention provide nucleic acid molecules encoding immunogenic proteins having 96% homology to the nucleic acid coding sequences herein. Some embodiments of the invention provide nucleic acid molecules encoding immunogenic proteins having 97% homology to the nucleic acid coding sequences herein. Some embodiments of the invention relate to nucleic acid molecules encoding immunogenic proteins having 98% homology with respect to

- 37 045958 nucleic acid coding sequences in this document. Some embodiments of the invention relate to nucleic acid molecules encoding immunogenic proteins having 99% homology to the nucleic acid coding sequences herein. In some embodiments, nucleic acid molecules with coding sequences described herein that are homologous to a coding sequence of a consensus protein described herein include sequences encoding an IgE leader sequence linked to the 5' end of a coding sequence encoding homologous protein sequences described in this document.

Some embodiments of the invention relate to nucleotide sequences encoding proteins with a certain percentage of identity to the full-length NY-ESO-1 consensus protein, an immunogenic fragment of the NY-ESO-1 consensus protein, and immunogenic protein fragments having identity to the NY-ESO-1 consensus protein. Such nucleic acid molecules that encode immunogenic proteins that have up to 80% identity with respect to the full-length NY-ESO-1 consensus sequence, up to 85% identity with respect to the full-length consensus sequence, up to 90% identity with respect to the full-length NY-ESO-1 consensus sequence, up to 91 % identity relative to the full-length NY-ESO-1 consensus sequence, up to 92% identity relative to the full-length NY-ESO-1 consensus sequence, up to 93% identity relative to the full-length NY-ESO-1 consensus sequence, up to 94% identity relative to the full-length NY- consensus sequence ESO-1, up to 95% identity relative to the full-length NY-ESO-1 consensus sequence, up to 96% identity relative to the full-length NY-ESO-1 consensus sequence, up to 97% identity relative to the full-length NY-ESO-1 consensus sequence, up to 98% identity relative to the full-length NY-ESO-1 consensus sequence and up to 99% identity to the full-length NY-ESO-1 consensus sequence can be provided.

Likewise, nucleotide sequences encoding the immunogenic fragments described herein and immunogenic protein fragments with a similar percentage of identity as stated above to the NY-ESO-1 proteins described herein are also provided.

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

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

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

Some embodiments of the invention relate to proteins that are homologous to SEQ ID NO: 14. Some embodiments of the invention relate to immunogenic proteins that have 95% homology to the consensus protein sequences set forth in SEQ ID NO: 14. Some embodiments of the invention relate to immunogenic

- 38 045958 proteins that have 96% homology to the consensus protein sequences shown in SEQ ID NO: 14. Some embodiments of the invention relate to immunogenic proteins that have 97% homology to the consensus protein sequences shown in SEQ ID NO: 14. Some embodiments of the invention provide immunogenic proteins that have 98% homology to the consensus protein sequences set forth in SEQ ID NO: 14. Some embodiments of the invention provide immunogenic proteins that have 99% homology to the consensus protein sequences set forth in SEQ ID NO: 14.

Some embodiments of the invention provide proteins that are identical with respect to the sequence of SEQ ID NO: 14. Some embodiments of the invention provide immunogenic proteins having an amino acid sequence that is 80% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 14. Some Embodiments of the invention provide immunogenic proteins having an amino acid sequence that is 85% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 14. Some embodiments of the invention provide immunogenic proteins having an amino acid sequence that is 90% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 14. Some embodiments of the invention provide immunogenic proteins having an amino acid sequence that is 91% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 14. Some embodiments of the invention provide immunogenic proteins , having an amino acid sequence that is 92% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 14. Some embodiments of the invention relate to immunogenic proteins having an amino acid sequence that is 93% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 14. Some embodiments of the invention provide immunogenic proteins having an amino acid sequence that is 94% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 14. Some embodiments of the invention provide immunogenic proteins having an amino acid sequence that is 95% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 14. Some embodiments of the invention provide immunogenic proteins having an amino acid sequence that is 96% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 14. Some embodiments The inventions relate to immunogenic proteins having an amino acid sequence that is 97% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 14. Some embodiments of the invention relate to immunogenic proteins having an amino acid sequence that is 98% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 14. Certain embodiments of the invention provide immunogenic proteins having an amino acid sequence that is 99% identical to the full-length consensus amino acid sequences set forth 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. The consensus protein fragments 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. Immunogenic fragments of the sequence SEQ ID NO: 14 can be provided. Immunogenic fragments may contain 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 sequence of SEQ ID NO: 14. In some embodiments, the fragments contain 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 protein fragments with amino acid sequences homologous to them

- 39 045958 munogenic fragments of the sequence SEQ ID NO: 14 may be provided. Such immunogenic fragments 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 proteins that are 95% or more homologous to the sequence SEQ ID NO: 14. Some embodiments of the invention provide immunogenic fragments having 96% homology to immunogenic fragments of the consensus protein sequences herein. Some embodiments of the invention provide immunogenic fragments having 97% homology to immunogenic fragments of the consensus protein sequences herein. Some embodiments of the invention provide immunogenic fragments having 98% homology to immunogenic fragments of the consensus protein sequences herein. Some embodiments of the invention provide immunogenic fragments having 99% homology to immunogenic fragments of the consensus protein sequences herein. In some embodiments, the fragments comprise 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 protein fragments with amino acid sequences identical to the immunogenic fragments of SEQ ID NO: 14 can be provided. Such immunogenic fragments 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 proteins that are 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequences set forth in SEQ ID NO: 14. In some embodiments, the fragments contain 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.

As disclosed herein with respect to coupling 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, which is encoded by the start codon of the nucleotide sequence, then encodes the protein without the signal peptide-encoding sequences.

(8) NY-ESO-2.

The vaccine of the present invention may contain New York esophageal cancer antigen-2 (NY-ESO-2; also called cancer/testicular antigen 2, ESO2 and LAGE1), a fragment thereof, or a variant thereof. NY-ESO-2 is an autoimmunogenic tumor antigen that belongs to the ESO/LAGE family of cancer-testis antigens. NY-ESO-2 can be 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 melanomas, non-small cell lung carcinomas, bladder, prostate, and head and neck cancers. The gene encoding NY-ESO-2 also contains an alternative open reading frame that encodes a protein called CAMEL, a tumor antigen recognized by melanoma-specific cytotoxic T lymphocytes.

Like NY-ESO-1, NY-ESO-2 can be expressed in the testes and ovaries. NY-ESO-2 may also be associated with various types of cancer and immunogenicity in subjects suffering from cancer or tumors. Accordingly, NY-ESO-2 may be an antigen associated with various tumors.

The NY-ESO-2 antigen is capable of inducing antigen-specific T-cell and/or potent humoral immune responses, thereby inducing or eliciting an immune response that targets or responds to cancer or tumor by antigen expression. In some embodiments, the induced or evoked immune response may be cellular, humoral, or both cellular and humoral immune responses. In some embodiments, the induced or evoked cellular immune response may include the induction or secretion of interferon gamma (IFN-γ) and/or tumor necrosis factor alpha (TNF-α). In other embodiments, the induced or elicited immune response may

- 40 045958 reduce or inhibit one or more immune response suppressive factors that stimulate the development of a tumor or cancer by expressing an antigen, for example, but not limited to, factors that suppress MHC presentation, factors that activate 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 suppressor immune cells, CTLA-4, PD-1, MDSC, MCP-1 and molecules immune checkpoints, which is described in more detail below.

The NY-ESO-2 antigen may increase the cellular immune response in a subject administered the NY-ESO-2 antigen by up to about 50-fold to about 6000-fold, about 50-fold to about 5500-fold, about 50-fold multiple to about 5000 times, from about 50 times to about 4500 times, from about 100 times to about 6000 times, from about 150 times to about 6000 times, from about 200 times to about 6000 times, from about 250-fold to 6000-fold or about 300-fold to about 6000-fold the size of the cellular immune response in a subject not administered the NY-ESO-2 antigen. In some embodiments, the NYESO-2 antigen may increase the cellular immune response in a subject administered the NY-ESO-2 antigen by up to about 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550 , 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 230 0, 2400, 2500, 2600 , 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4 700, 4800, 4900, 5000, 5100 , 5200, 5300, 5400, 5500, 5600, 5700, 5800, 5900, or 6000 times the cellular immune response in a subject not administered the NY-ESO-2 antigen.

NY-ESO-2 antigen may increase interferon gamma (IFN-γ) levels in a subject administered NY-ESO-2 antigen by up to about 50-fold to about 6000-fold, about 50-fold to about 5500-fold multiple, from about 50 times to about 5000 times, from about 50 times to about 4500 times, from about 100 times to about 6000 times, from about 150 times to about 6000 times, from about 200 times to about 6000-fold, about 250-fold to 6000-fold, or about 300-fold to about 6000-fold the IFN-γ levels of a subject not receiving NY-ESO-2 antigen. In some embodiments, the NY-ESO-2 antigen may increase the IFN-γ levels of a subject receiving the NY-ESO-2 antigen by up to about 50, 100, 150, 200, 250, 300, 350, 400, 450. 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400,

1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200,

3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000,

5100, 5200, 5300, 5400, 5500, 5600, 5700, 5800, 5900, or 6000 times the IFN-γ levels of a subject not receiving NY-ESO-2 antigen.

The NY-ESO-2 antigen may contain protein epitopes that make them particularly effective as immunogens to which immune responses to NY-ESO-2 can be induced. The NYESO-2 antigen may contain a full-length translation product, a variant thereof, a fragment thereof, or a combination thereof. The NY-ESO-2 antigen may contain a consensus protein.

The nucleotide sequence encoding the NY-ESO-2 consensus antigen can be optimized for codon frequency and corresponding RNA transcripts. The nucleic acid encoding the NY-ESO-2 consensus antigen may be codon- and RNA-optimized for expression. In some embodiments, the nucleotide sequence encoding the NY-ESO-2 consensus antigen may contain a Kozak sequence (eg, GCC ACC) to increase translation efficiency. The nucleic acid encoding the NY-ESO-2 consensus antigen may contain multiple stop codons (eg, TGA TGA) to increase translation termination efficiency.

The nucleic acid encoding the NY-ESO-2 consensus antigen may also encode an immunoglobulin E (IgE) leader sequence. The nucleic acid encoding the NY-ESO-2 consensus 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 NY-ESO-2 consensus antigen by a peptide bond. The nucleic acid encoding the NY-ESO-2 consensus antigen may also contain a nucleotide sequence encoding an IgE leader sequence. In some embodiments, the nucleic acid encoding the NY-ESO-2 consensus antigen does not have or contain a nucleotide sequence encoding an IgE leader sequence.

The NY-ESO-2 consensus antigen may be the nucleotide sequence of SEQ ID NO: 15, which encodes the amino acid sequence of SEQ ID NO: 16.

SEQ ID NO: 1 encodes the NY-ESO-2 consensus protein associated with an IgE leader sequence.

The NY-ESO-2 consensus protein can be associated with the IgE leader sequence and HAmarker. In other embodiments, the NY-ESO-2 consensus protein may not contain or be associated with an IgE leader sequence and/or an HA tag.

- 41 045958

In some embodiments, the NY-ESO-2 consensus antigen may be a nucleotide sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over the entire length of the nucleotide sequence shown in SEQ ID NO: 15. In other embodiments, the NY-ESO-2 consensus antigen may be a nucleotide sequence that encodes an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87 %, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the entire length of the amino acid sequence given in SEQ ID NO: 16. The NY-ESO-2 consensus antigen may be an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over the entire length of the amino acid sequence shown in SEQ ID NO: 16.

Some embodiments of the invention relate to nucleotide sequences encoding proteins homologous to the NY-ESO-2 consensus protein, an immunogenic fragment of the NY-ESO-2 consensus protein, and immunogenic fragments of homologous proteins. Such nucleic acid molecules 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 can be provided . Likewise, nucleotide sequences encoding immunogenic fragments provided herein and immunogenic fragments of proteins that are homologous to proteins provided herein are also provided.

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

Some embodiments of the invention relate to nucleotide sequences encoding proteins with a certain percentage of identity to the full-length NY-ESO-2 consensus protein, an immunogenic fragment of the NY-ESO-2 consensus protein, and immunogenic protein fragments having identity to the NY-ESO-2 consensus protein. Such nucleic acid molecules that encode immunogenic proteins that have up to 80% identity with respect to the full-length NY-ESO-2 consensus sequence, up to 85% identity with respect to the full-length consensus sequence, up to 90% identity with respect to the full-length NY-ESO-2 consensus sequence, up to 91 % identity relative to the full-length NY-ESO-2 consensus sequence, up to 92% identity relative to the full-length NY-ESO-2 consensus sequence, up to 93% identity relative to the full-length NY-ESO-2 consensus sequence, up to 94% identity relative to the full-length NY- consensus sequence ESO-2, up to 95% identity relative to the full-length consensus sequence NY-ESO-2, up to 96% identity relative to the full-length consensus sequence NY-ESO-2, up to 97% identity relative to the full-length consensus sequence NY-ESO-2, 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 can be provided.

Likewise, nucleotide sequences encoding the immunogenic fragments described herein and immunogenic protein fragments with a similar percentage of identity as stated above to the NY-ESO-2 proteins described herein are also provided.

In some embodiments of the invention, the nucleotide sequence does not contain co

- 42 045958 leader sequence, which encodes the leader sequence. In some embodiments, the nucleotide sequence does not contain a coding sequence that encodes an IgE leader sequence.

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

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

Some embodiments of the invention relate to proteins that are homologous to the sequence SEQ ID NO: 16. Some embodiments of the invention relate to immunogenic proteins that have 95% homology to the consensus protein sequences shown in SEQ ID NO: 16. Some embodiments of the invention relate to immunogenic proteins that have 96% homology to the consensus protein sequences set forth in SEQ ID NO: 16. Some embodiments of the invention provide immunogenic proteins that have 97% homology to the consensus protein sequences set forth in SEQ ID NO: 16. Some embodiments embodiments of the invention relate to immunogenic proteins that have 98% homology to the consensus protein sequences set forth in SEQ ID NO: 16. Some embodiments of the invention relate to immunogenic proteins that have 99% homology to the consensus protein sequences set forth in SEQ ID NO: 16.

Some embodiments of the invention provide proteins that are identical with respect to the sequence of SEQ ID NO: 16. Some embodiments of the invention provide immunogenic proteins having an amino acid sequence that is 80% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 16. Some Embodiments of the invention provide immunogenic proteins having an amino acid sequence that is 85% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 16. Some embodiments of the invention provide immunogenic proteins having an amino acid sequence that is 90% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 16. Some embodiments of the invention provide immunogenic proteins having an amino acid sequence that is 91% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 16. Some embodiments of the invention provide immunogenic proteins having an amino acid sequence that is 92% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 16. Some embodiments of the invention relate to immunogenic proteins having an amino acid sequence that is 93% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 16. Some embodiments of the invention provide immunogenic proteins having an amino acid sequence that is 94% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 16. Some embodiments of the invention provide immunogenic proteins having an amino acid sequence that is 95% identical to the full-length consensus amino acid sequences shown in SEQ ID NO: 16. Some embodiments of the invention relate to immunogenic proteins having

- 43 045958 amino acid sequence that is 96% identical to the full-length consensus amino acid sequences shown in SEQ ID NO: 16. Some embodiments of the invention relate to immunogenic proteins having an amino acid sequence that is 97% identical to the full-length consensus amino acid sequences given in SEQ ID NO: 16. Some embodiments of the invention provide immunogenic proteins having an amino acid sequence that is 98% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 16. Some embodiments of the invention provide immunogenic proteins having an amino acid sequence which is 99% identical to the full-length consensus amino acid sequences 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. The consensus protein fragments 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. Immunogenic fragments of the sequence SEQ ID NO: 16 can be provided. Immunogenic fragments may contain 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 sequence of SEQ ID NO: 16. In some embodiments, the fragments contain 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 protein fragments with amino acid sequences homologous to the immunogenic fragments of SEQ ID NO: 16 can be provided. Such immunogenic fragments 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 proteins that are 95% or more homologous to the sequence SEQ ID NO: 16. Some embodiments of the invention provide immunogenic fragments having 96% homology to immunogenic fragments of the consensus protein sequences herein. Some embodiments of the invention provide immunogenic fragments having 97% homology to immunogenic fragments of the consensus protein sequences herein. Some embodiments of the invention provide immunogenic fragments having 98% homology to immunogenic fragments of the consensus protein sequences herein. Some embodiments of the invention provide immunogenic fragments having 99% homology to immunogenic fragments of the consensus protein sequences herein. In some embodiments, the fragments comprise 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 protein fragments with amino acid sequences identical to the immunogenic fragments of SEQ ID NO: 16 can be provided. Such immunogenic fragments 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 proteins that are 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequences set forth in SEQ ID NO: 16. In some embodiments, the fragments contain a leader after

- 44 045958 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.

As disclosed herein with respect to coupling 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, which is encoded by the start codon of the nucleotide sequence, then encodes the protein without the signal peptide-encoding sequences.

(9) PRAME.

The vaccine of the present invention may contain the PRAME cancer antigen, a fragment thereof, or a variant thereof. PRAME, encoded by the PRAME gene, is a 509 amino acid protein that is expressed in the testis, placenta, endometrium, ovary, adrenal gland, and in tissues derived from melanoma, lung, kidney, and head and neck carcinomas. PRAME is also expressed in acute leukemias in adults and children, as well as in multiple myeloma. PRAME contains an immunogenic nonapeptide capable of inducing a cytotoxic response when HLA-A24 is presented. Studies show that overexpression of PRAME in cultured cells induces caspase-independent cell death, responsible for lower proliferation rates. Other studies suggest that PRAME overexpression also provides growth or survival benefits by antagonizing retinoic acid receptor (RAR) signaling and is causally involved in the oncogenic process. Interference of RAR signaling results in loss of regulation of cell proliferation, differentiation, and development.

PRAME may have a gene expression profile similar to the cancer-testicular antigens MAGE, BAGE and GAGE, that is, expression in the testes. However, PRAME can be expressed in human melanomas and acute leukemias. PRAME can be recognized by cytolytic T lymphocytes.

Accordingly, PRAME may be an antigen associated with melanoma and leukemia.

The PRAME antigen is capable of inducing antigen-specific T-cell and/or potent humoral immune responses, thereby inducing or eliciting an immune response that targets or responds to the cancer or tumor by expression of the antigen. In some embodiments, the induced or evoked immune response may be cellular, humoral, or both cellular and humoral immune responses. In some embodiments, the induced or evoked cellular immune response may include the induction or secretion of interferon gamma (IFN-γ) and/or tumor necrosis factor alpha (TNF-α). In other embodiments, the induced or elicited immune response may reduce or inhibit one or more immune response suppressive factors that stimulate the development of a tumor or cancer by expressing an antigen, such as, but not limited to, MHC presentation suppressive factors, antigen-specific activating factors regulatory T cells (Tregs), PDL1, FasL, cytokines such as IL-10 and TFG-β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by suppressor immune cells, CTLA-4, PD-1 , MDSC, MCP-1 and immune checkpoint molecules, as described in more detail below.

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

PRAME antigen may increase gamma interferon (IFN-γ) levels in a subject receiving PRAME antigen by up to about 50-fold to about 6000-fold, about 50-fold to about 5500-fold, about 50-fold to about 5000-fold multiple, about 50 times to about 4500 times, about 100 times to about 6000 times, about 150 times to about 6000 times, about 200 times to about 6000 times, about 250 times fold to about 6000-fold or about 300-fold to about 6000-fold compared to IFN-γ levels in a non-injected subject

- 45 045958 antigen PRAME. In some embodiments, the PRAME antigen may increase IFN-γ levels in a subject receiving the PRAME antigen by up to about 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650. 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 250 0, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 490 0, 5000, 5100, 5200, 5300, 5400, 5500, 5600, 5700, 5800, 5900, or 6000 times the IFN-γ levels of a subject not receiving PRAME antigen.

PRAME antigen may contain protein epitopes that make them particularly effective as immunogens to which immune responses to PRAME can be induced. The PRAME antigen may contain a full-length translation product, a variant thereof, a fragment thereof, or a combination thereof. The PRAME antigen may contain a consensus protein.

The nucleotide sequence encoding the PRAME consensus antigen can be optimized for codon usage frequency and corresponding RNA transcripts. The nucleic acid encoding the PRAME consensus antigen may be codon- and RNA-optimized for expression. In some embodiments, the nucleotide sequence encoding the PRAME consensus antigen may contain a Kozak sequence (eg, GCC ACC) to increase translation efficiency. The nucleic acid encoding the PRAME consensus antigen may contain multiple stop codons (eg, TGA TGA) to increase translation termination efficiency.

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

The PRAME consensus antigen may be the nucleotide sequence of SEQ ID NO: 17, which encodes the amino acid sequence of SEQ ID NO: 18.

SEQ ID NO: 17 encodes the PRAME consensus protein associated with an IgE leader sequence.

The PRAME consensus protein can be associated with the IgE leader sequence and HATM. In other embodiments, the PRAME consensus protein may not contain or be associated with an IgE leader sequence and/or an HA tag.

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

Some embodiments of the invention relate to nucleotide sequences encoding proteins homologous to the PRAME consensus protein, an immunogenic fragment of the PRAME consensus protein, and immunogenic fragments of homologous proteins. Such nucleic acid molecules 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 can be provided . Likewise, nucleotide sequences encoding immunogenic fragments provided herein and immunogenic fragments of proteins that are homologous to proteins provided herein are also provided.

Some embodiments of the invention relate to nucleic acid molecules encoding immunogenic proteins having 95% homology to the nucleic acid coding sequences herein. Some embodiments of the invention relate to nucleic acid molecules encoding immunogenic proteins having 96% homology

- 46 045958 to her regarding the nucleic acid coding sequences in this document. Some embodiments of the invention provide nucleic acid molecules encoding immunogenic proteins having 97% homology to the nucleic acid coding sequences herein. Some embodiments of the invention relate to nucleic acid molecules encoding immunogenic proteins having 98% homology to the nucleic acid coding sequences herein. Some embodiments of the invention relate to nucleic acid molecules encoding immunogenic proteins having 99% homology to the nucleic acid coding sequences herein. In some embodiments, nucleic acid molecules with coding sequences described herein that are homologous to a coding sequence of a consensus protein described herein include sequences encoding an IgE leader sequence linked to the 5' end of a coding sequence encoding homologous protein sequences described in this document.

Some embodiments of the invention relate to nucleotide sequences encoding proteins with a certain percentage of identity to the full-length PRAME consensus protein, an immunogenic fragment of the PRAME consensus protein, and immunogenic protein fragments having identity to the PRAME consensus protein. Such nucleic acid molecules that encode immunogenic proteins that have up to 80% identity with respect to the full-length PRAME consensus sequence, up to 85% identity with respect to the full-length consensus sequence, up to 90% identity with respect to the full-length PRAME consensus sequence, up to 91% identity with respect to the full-length PRAME consensus sequence, up to 92% identity relative to the full-length PRAME consensus sequence, up to 93% identity relative to the full-length PRAME consensus sequence, up to 94% identity relative to the full-length PRAME consensus sequence, up to 95% identity relative to the full-length PRAME consensus sequence, up to 96% identity relative 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 can be provided. Likewise, nucleotide sequences encoding the immunogenic fragments described herein and immunogenic protein fragments with a similar percentage of identity as stated above to the PRAME proteins described herein are also provided.

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

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

In addition, the amino acid sequence of the PRAME consensus protein is SEQ ID NO: 18. The amino acid sequence of the PRAME consensus protein associated with the IgE leader sequence is SEQ ID NO: 18. The amino acid sequence of the PRAME consensus protein associated with the IgE leader sequence may be associated with the HA-tag.

- 47 045958

Some embodiments of the invention relate to proteins that are homologous to SEQ ID NO: 18. Some embodiments of the invention relate to immunogenic proteins that have 95% homology to the consensus protein sequences set forth in SEQ ID NO: 18. Some embodiments of the invention relate to immunogenic proteins that have 96% homology to the consensus protein sequences set forth in SEQ ID NO: 18. Some embodiments of the invention provide immunogenic proteins that have 97% homology to the consensus protein sequences set forth in SEQ ID NO: 18. Some embodiments embodiments of the invention relate to immunogenic proteins that have 98% homology to the consensus protein sequences set forth in SEQ ID NO: 18. Some embodiments of the invention relate to immunogenic proteins that have 99% homology to the consensus protein sequences set forth in SEQ ID NO: 18.

Some embodiments of the invention provide proteins that are identical with respect to the sequence of SEQ ID NO: 18. Some embodiments of the invention provide immunogenic proteins having an amino acid sequence that is 80% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 18. Some Embodiments of the invention provide immunogenic proteins having an amino acid sequence that is 85% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 18. Some embodiments of the invention provide immunogenic proteins having an amino acid sequence that is 90% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 18. Some embodiments of the invention provide immunogenic proteins having an amino acid sequence that is 91% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 18. Some embodiments of the invention provide immunogenic proteins , having an amino acid sequence that is 92% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 18. Some embodiments of the invention relate to immunogenic proteins having an amino acid sequence that is 93% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 18. Some embodiments of the invention provide immunogenic proteins having an amino acid sequence that is 94% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 18. Some embodiments of the invention provide immunogenic proteins having an amino acid sequence that is 95% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 18. Some embodiments of the invention provide immunogenic proteins having an amino acid sequence that is 96% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 18. Some embodiments The inventions relate to immunogenic proteins having an amino acid sequence that is 97% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 18. Some embodiments of the invention relate to immunogenic proteins having an amino acid sequence that is 98% identical to the full-length consensus amino acid sequences set forth in SEQ ID NO: 18. Some embodiments of the invention provide immunogenic proteins having an amino acid sequence that is 99% identical to the full-length consensus amino acid sequences set forth 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. The consensus protein fragments 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. Immunogenic fragments of the sequence SEQ ID NO: 18 can be provided. Immunogenic fragments may contain 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 sequence of SEQ ID NO: 18. In some embodiments, the fragments contain a leader sequence, such as, for example, an immunoglobulin leader sequence, such as an IgE leader sequence. In some embodiments of the invention

- 48 045958 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 protein fragments with amino acid sequences homologous to the immunogenic fragments of SEQ ID NO: 18 can be provided. Such immunogenic fragments 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 proteins that are 95% or more homologous to the sequence SEQ ID NO: 18. Some embodiments of the invention provide immunogenic fragments having 96% homology to immunogenic fragments of the consensus protein sequences herein. Some embodiments of the invention provide immunogenic fragments having 97% homology to immunogenic fragments of the consensus protein sequences herein. Some embodiments of the invention provide immunogenic fragments having 98% homology to immunogenic fragments of the consensus protein sequences herein. Some embodiments of the invention provide immunogenic fragments having 99% homology to immunogenic fragments of the consensus protein sequences herein. In some embodiments, the fragments comprise 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 protein fragments with amino acid sequences identical to the immunogenic fragments of SEQ ID NO: 18 can be provided. Such immunogenic fragments 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 proteins that are 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequences set forth in SEQ ID NO: 18. In some embodiments, the fragments contain 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.

As disclosed herein with respect to coupling 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, which is encoded by the start codon of the nucleotide sequence, then encodes the protein without the signal peptide-encoding sequences.

(10) PSA.

The vaccine of the present invention may contain the cancer antigen prostate-specific antigen (PSA; also known as gamma seminoprotein or kallikrein-3 (KLK3)), a fragment thereof, or a variant thereof. PSA is an androgen-regulated serine protease produced by prostate epithelial cells and prostate cancer cells and encoded by the KLK3 gene. PSA is often used as a serum marker for prostate cancer. PSA is a member of the tissue kallikrein family and breaks down the semenogelins in the seminal clot after cleavage of the proenzyme, releasing the active enzyme, thereby liquefying the seminal fluid, allowing the sperm to swim freely. In addition, the enzymatic activity of PSA is regulated by zinc concentration, that is, high concentrations of zinc inhibit the enzymatic activity of PSA.

The PSA antigen is capable of inducing antigen-specific T-cell and/or potent humoral immune responses, thereby inducing or eliciting an immune response that targets or responds to the cancer or tumor by expression of the antigen. In some embodiments, the induced or evoked immune response may be cellular, humoral, or both cellular and humoral immune responses. In some embodiments, the induced or evoked cellular immune response may include the induction or secretion of interferon gamma (IFN-γ) and/or tumor necrosis factor alpha (TNF-α). In other embodiments, the induced or elicited immune response may reduce

- 49 045958 or inhibit one or more immune response suppressive factors that stimulate the development of a tumor or cancer by expressing an antigen, for example, but not limited to, factors that suppress MHC presentation, factors that activate antigen-specific regulatory T cells (Tregs), PDL1 , FasL, cytokines such as IL-10 and TFG-β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by immune suppressor cells, CTLA-4, PD-1, MDSC, MCP-1 and immune molecules control points, which is described in more detail below.

PSA antigen may contain protein epitopes that make them particularly effective as immunogens to which immune responses to PSA can be induced. The PSMA antigen may contain a full-length translation product, a variant thereof, a fragment thereof, or a combination thereof. The PSA antigen may contain a consensus protein.

The nucleotide sequence encoding the PSA consensus antigen can be optimized for codon frequency and corresponding RNA transcripts. The nucleic acid encoding the PSA consensus antigen may be codon- and RNA-optimized for expression. In some embodiments, the nucleotide sequence encoding the PSA consensus antigen may contain a Kozak sequence (eg, GCC ACC) to increase translation efficiency. The nucleic acid encoding the PSA consensus antigen may contain multiple stop codons (eg, TGA TGA) to increase translation termination efficiency.

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

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

(11) PSMA.

The vaccine of the present invention may contain the cancer antigen prostate-specific membrane antigen (PSMA; also known as Glutamate carboxypeptidase II (GCPII), H-acetyl-caspartyl-L-glutamate peptidase I (NAALADasa 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-containing metalloenzyme found in membranes and extracellular spaces. PSMA is highly expressed in the human prostate gland and is upregulated in prostate cancer. PSMA has also been found to be overexpressed in other types of cancer, such as solid tumors of the kidney, breast and colon.

The PSMA antigen is capable of inducing antigen-specific T-cell and/or potent humoral immune responses, thereby inducing or triggering an immune response that targets or responds to the cancer or tumor by expression of the antigen. In some embodiments, the induced or evoked immune response may be cellular, humoral, or both cellular and humoral immune responses. In some embodiments, the induced or evoked cellular immune response may include the induction or secretion of interferon gamma (IFN-γ) and/or tumor necrosis factor alpha (TNF-α). In other embodiments, the induced or elicited immune response may reduce or inhibit one or more immune response suppressive factors that promote tumor or cancer development by expressing an antigen, such as, but not limited to, MHC presentation suppressive factors, antigen-specific activating factors regulatory T cells (Tregs), PDL1, FasL, cytokines such as IL-10 and TFG-β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by suppressor immune cells, CTLA-4, PD-1 , MDSC, MCP-1 and immune checkpoint molecules, as described in more detail below.

PSMA antigen may contain protein epitopes that make them particularly effective as immunogens to which immune responses to PSMA can be induced. The PSMA antigen may contain a full-length translation product, a variant thereof, a fragment thereof, or a combination thereof. The PSMA antigen may contain a consensus protein.

The nucleotide sequence encoding the PSMA consensus antigen can be optimized for codon frequency and corresponding RNA transcripts. Nucleic

- 50 045958 acid encoding the PSMA consensus antigen may be codon- and RNA-optimized for expression. In some embodiments, the nucleotide sequence encoding the PSMA consensus antigen may contain a Kozak sequence (eg, GCC ACC) to increase translation efficiency. The nucleic acid encoding the PSMA consensus antigen may contain multiple stop codons (eg, TGA TGA) to increase translation termination efficiency.

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

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

(12) STEAP.

The vaccine of the present invention may contain the cancer antigen sixth transmembrane epithelial antigen of the prostate (STEAP), a fragment thereof, or a variant thereof. STEAP is a metal reductase encoded by the STEAP1 gene. STEAP is highly expressed in prostate tissue and is upregulated in cancer cells. STEAP is proposed to be a sixth transmembrane protein and is a cell surface antigen located at intercellular junctions.

The STEAP antigen is capable of inducing antigen-specific T-cell and/or potent humoral immune responses, thereby inducing or eliciting an immune response that targets or responds to the cancer or tumor by expression of the antigen. In some embodiments, the induced or evoked immune response may be cellular, humoral, or both cellular and humoral immune responses. In some embodiments, the induced or evoked cellular immune response may include the induction or secretion of interferon gamma (IFN-γ) and/or tumor necrosis factor alpha (TNF-α). In other embodiments, the induced or elicited immune response may reduce or inhibit one or more immune response suppressive factors that stimulate the development of a tumor or cancer by expressing an antigen, such as, but not limited to, MHC presentation suppressive factors, antigen-specific activating factors regulatory T cells (Tregs), PDL1, FasL, cytokines such as IL-10 and TFG-β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by suppressor immune cells, CTLA-4, PD-1 , MDSC, MCP-1 and immune checkpoint molecules, as described in more detail below.

STEAP antigen may contain protein epitopes that make them particularly effective as immunogens to which immune responses to STEAP can be induced. The STEAP antigen may contain a full-length translation product, a variant thereof, a fragment thereof, or a combination thereof. The STEAP antigen may contain a consensus protein.

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

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

- 51 045958

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

(13) PSCA.

The vaccine of the present invention may contain the cancer antigen prostate-specific stem cell antigen (PSCA), a fragment thereof, or a variant thereof. PSCA is a glycosylphosphatidylinositol (GPI)-anchored cell surface protein 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 types of cancer, including prostate, bladder, and pancreatic cancer.

The PSCA antigen is capable of inducing antigen-specific T-cell and/or potent humoral immune responses, thereby inducing or triggering an immune response that targets or responds to the cancer or tumor by expression of the antigen. In some embodiments, the induced or evoked immune response may be cellular, humoral, or both cellular and humoral immune responses. In some embodiments, the induced or evoked cellular immune response may include the induction or secretion of interferon gamma (IFN-γ) and/or tumor necrosis factor alpha (TNF-α). In other embodiments, the induced or elicited immune response may reduce or inhibit one or more immune response suppressive factors that promote tumor or cancer development by expressing an antigen, such as, but not limited to, MHC presentation suppressive factors, antigen-specific activating factors regulatory T cells (Tregs), PDL1, FasL, cytokines such as IL-10 and TFG-β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by suppressor immune cells, CTLA-4, PD-1 , MDSC, MCP-1 and immune checkpoint molecules, as described in more detail below.

PSCA antigen may contain protein epitopes that make them particularly effective as immunogens to which immune responses to PSCA can be induced. The PSCA antigen may contain a full-length translation product, a variant thereof, a fragment thereof, or a combination thereof. The PSCA antigen may contain a consensus protein.

The nucleotide sequence encoding the PSCA consensus antigen can be optimized for codon frequency and corresponding RNA transcripts. The nucleic acid encoding the PSCA consensus antigen may be codon- and RNA-optimized for expression. In some embodiments, the nucleotide sequence encoding the PSCA consensus antigen may contain a Kozak sequence (eg, GCC ACC) to increase translation efficiency. The nucleic acid encoding the PSCA consensus antigen may contain multiple stop codons (eg, TGA TGA) to increase translation termination efficiency.

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

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

(14) hTERT.

The vaccine of the present invention may contain the hTERT cancer antigen, a fragment thereof, or a variant thereof. hTERT is a human telomerase reverse transcriptase that synthesizes a TTAGGG tag at the end of telomeres to prevent cell death due to chromosome shortening. Hyperproliferative cells may have abnormally high levels of hTERT expression. Abnormal hTERT expression may also occur in hyperproliferative cells infected with HCV and HPV. Thus, immunotherapy for HPV and HCV can be enhanced by targeting cells that express hTERT at abnormal levels. The HPV and HCV antigens are discussed in more detail below. The cancer antigen hTERT may be further defined in US Patent Application No. 14/139660, filed December 23, 2013, the disclosure of which is incorporated herein by reference in its entirety.

In addition, hTERT expression in dendritic cells transfected with hTERT genes may

- 52 045958 induce cytotoxic CD8+ T cells and detect CD4+ T cells in an antigen-specific manner. Thus, the use of hTERT expression in antigen presenting cells (APCs) to delay aging and maintain their ability to present a preferred antigen can be used in immunotherapeutic methods, such as the methods described herein.

The hTERT antigen may be associated with or expressed by 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. Accordingly, a vaccine containing the hTERT antigen described herein can be used to treat subjects suffering from 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 hTERT antigen is capable of inducing antigen-specific T cell and/or potent humoral immune responses, thereby inducing or eliciting an immune response that targets or responds to the cancer or tumor by expression of the antigen. In some embodiments, the induced or evoked immune response may be cellular, humoral, or both cellular and humoral immune responses. In some embodiments, the induced or evoked cellular immune response may include the induction or secretion of interferon gamma (IFN-γ) and/or tumor necrosis factor alpha (TNF-α). In other embodiments, the induced or elicited immune response may reduce or inhibit one or more immune response suppressive factors that stimulate the development of a tumor or cancer by expressing an antigen, such as, but not limited to, MHC presentation suppressive factors, antigen-specific activating factors regulatory T cells (Tregs), PDL1, FasL, cytokines such as IL-10 and TFG-β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by suppressor immune cells, CTLA-4, PD-1 , MDSC, MCP-1 and immune checkpoint molecules, as described in more detail below.

hTERT antigen may contain protein epitopes that make them particularly effective as immunogens to which immune responses to hTERT can be induced. The hTERT antigen may contain a full-length translation product, a variant thereof, a fragment thereof, or a combination thereof. The hTERT antigen may contain a consensus protein.

The nucleotide sequence encoding the hTERT antigen or hTERT consensus antigen can be optimized with respect to codon usage frequency and corresponding RNA transcripts. The nucleic acid encoding the hTERT antigen or hTERT consensus antigen may be codon- and RNA-optimized for expression. In some embodiments, the nucleotide sequence encoding the hTERT antigen or hTERT consensus antigen may contain a Kozak sequence (eg, GCC ACC) to increase translation efficiency. The nucleic acid encoding the hTERT antigen or hTERT consensus antigen may contain multiple stop codons (eg, TGA TGA) to increase translation termination efficiency.

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

In some embodiments, the nucleic acid encoding an hTERT antigen or hTERT consensus antigen may be a heterologous nucleotide sequence and/or contain one or more heterologous nucleotide sequences. A nucleic acid encoding an hTERT antigen or hTERT consensus antigen may be mutated relative to a wild-type hTERT antigen such that one or more amino acids or residues in the amino acid sequence of the hTERT antigen or hTERT consensus antigen, respectively, are replaced or replaced by another amino acid or residue. A nucleic acid encoding an hTERT antigen or a hTERT consensus antigen may be mutated relative to a wild-type hTERT antigen such that one or more residues in the amino acid sequence of the hTERT antigen or hTERT consensus antigen, respectively, are replaced or replaced by another residue, thereby causing the immune system no longer be tolerant to hTERT in milk

- 53 045958 a feeder who is administered a nucleic acid encoding an hTERT antigen or a hTERT consensus antigen, an hTERT antigen or a hTERT consensus antigen, or combinations thereof. The nucleic acid encoding an hTERT antigen or hTERT consensus antigen may be mutated relative to a wild-type hTERT antigen such that arginine 589, aspartate 1005, or both arginine 589 and aspartate 1005 in the amino acid sequence of the hTERT antigen or hTERT consensus antigen are replaced or replaced by a residue tyrosine

The hTERT antigen may be the nucleotide sequence of SEQ ID NO: 23, which encodes the amino acid sequence of SEQ ID NO: 24.

SEQ ID NO: 23 encodes the hTERT protein linked to an IgE leader sequence. The hTERT protein can be associated with an IgE leader sequence and an HA tag. In other embodiments, the hTERT protein may not contain or be associated with an IgE leader sequence and/or an HA tag.

In some embodiments, the hTERT antigen may be a nucleotide sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over the entire length of the nucleotide sequence given in SEQ ID NO: 23. In other embodiments of the invention The hTERT antigen may be a nucleotide sequence that encodes an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over the entire length of the amino acid sequence shown in SEQ ID NO: 24. The hTERT antigen may be an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over the entire length of the amino acid sequence shown in SEQ ID NO: 24.

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

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

Some embodiments of the invention relate to nucleotide sequences encoding proteins with a certain percentage of identity to the full-length hTERT protein, an immunogenic fragment of the hTERT protein, and immunogenic protein fragments having identity to the hTERT protein. Such nucleic acid molecules that encode immunogenic proteins that have up to 80% identity with respect to the full-length hTERT sequence, up to 85% identity with respect to the full-length sequence, up to 90% identity with respect to the full-length hTERT sequence, up to 91% identity with respect to the full-length hTERT sequence, up to 92% identity relative to the full-length hTERT sequence, up to 93% identity relative to the full-length hTERT sequence, up to 94% identity relative to the full-length hTERT sequence, up to 95% identity relative to the full-length hTERT sequence, up to 96% identity relative to

- 54 045958 full-length hTERT sequence, up to 97% identity to the full-length hTERT sequence, up to 98% identity to the full-length hTERT sequence, and up to 99% identity to the full-length hTERT sequence, can be provided. Likewise, nucleotide sequences encoding the immunogenic fragments described herein and immunogenic protein fragments with a similar percentage of identity as stated above to the hTERT proteins described herein are also provided.

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

Some embodiments of the invention relate to fragments of the sequence SEQ ID NO: 23. The fragments 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 sequence SEQ ID NO: 23. The fragments may be at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous to fragments of SEQ ID NO: 23 The fragments 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 portions of SEQ ID NO: 23. In some embodiments In embodiments of the invention, the fragments contain sequences encoding 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 coding sequences that encode the leader sequence. In some embodiments, the fragments do not contain coding sequences that encode 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 associated with the IgE leader sequence is SEQ ID NO: 24. The amino acid sequence of the hTERT protein associated with the IgE leader sequence can be associated with HA -tag.

Some embodiments of the invention relate to proteins that are homologous to the sequence SEQ ID NO: 24. Some embodiments of the invention relate to immunogenic proteins that have 95% homology to the protein sequences shown in SEQ ID NO: 24. Some embodiments of the invention relate to immunogenic proteins that have 96% homology to the protein sequences set forth in SEQ ID NO: 24. Some embodiments of the invention relate to immunogenic proteins that have 97% homology to the protein sequences set forth in SEQ ID NO: 24. Some embodiments of the invention include to immunogenic proteins that have 98% homology to the protein sequences set forth in SEQ ID NO: 24. Some embodiments of the invention relate to immunogenic proteins that have 99% homology to the protein sequences set forth in SEQ ID NO: 24.

Some embodiments of the invention provide proteins that are identical with respect to the sequence of SEQ ID NO: 24. Some embodiments of the invention provide immunogenic proteins having an amino acid sequence that is 80% identical to the full-length amino acid sequences set forth in SEQ ID NO: 24. Some embodiments embodiments of the invention relate to immunogenic proteins having an amino acid sequence that is 85% identical to the full-length amino acid sequences set forth in SEQ ID NO: 24. Some embodiments of the invention relate to immunogenic proteins having an amino acid sequence that is 90% identical to the full-length amino acid sequences, set forth in SEQ ID NO: 24. Some embodiments of the invention provide immunogenic proteins having an amino acid sequence that is 91% identical to the full-length amino acid sequences set forth in SEQ ID NO: 24. Some embodiments of the invention provide immunogenic proteins having an amino acid sequence , which is 92% identical to the full-length amino acid sequences shown in SEQ ID NO: 24.

Some embodiments of the invention relate to immunogenic proteins having an amino acid sequence that is 93% identical to the full-length amino acid sequences set forth in SEQ ID NO: 24. Some embodiments of the invention from

- 55 045958 relate to immunogenic proteins having an amino acid sequence that is 94% identical to the full-length amino acid sequences set forth in SEQ ID NO: 24. Some embodiments of the invention relate to immunogenic proteins having an amino acid sequence that is 95% identical to the full-length amino acid sequences set forth in SEQ ID NO: 24. Some embodiments of the invention provide immunogenic proteins having an amino acid sequence that is 96% identical to the full-length amino acid sequences set forth in SEQ ID NO: 24. Some embodiments of the invention provide immunogenic proteins having an amino acid sequence sequence that is 97% identical to the full-length amino acid sequences set forth in SEQ ID NO: 24. Some embodiments of the invention provide immunogenic proteins having an amino acid sequence that is 98% identical to the full-length amino acid sequences set forth in SEQ ID NO: 24. Some Embodiments of the invention relate to immunogenic proteins having an amino acid sequence that is 99% identical to the full-length amino acid sequences set forth 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. Protein fragments 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% protein. Immunogenic fragments of the sequence SEQ ID NO: 24 can be provided. Immunogenic fragments may contain 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 sequence of SEQ ID NO: 24. In some embodiments, the fragments contain 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 protein fragments with amino acid sequences homologous to the immunogenic fragments of SEQ ID NO: 24 can be provided. Such immunogenic fragments 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 proteins that are 95% or more homologous to the sequence SEQ ID NO: 18. Some embodiments of the invention provide immunogenic fragments that have 96% homology to the immunogenic fragments of the protein sequences herein. Some embodiments of the invention provide immunogenic fragments that have 97% homology to the immunogenic fragments of the protein sequences herein. Some embodiments of the invention provide immunogenic fragments that have 98% homology to the immunogenic fragments of the protein sequences herein. Some embodiments of the invention provide immunogenic fragments that have 99% homology to the immunogenic fragments of the protein sequences herein. In some embodiments, the fragments comprise 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 protein fragments with amino acid sequences identical to the immunogenic fragments of SEQ ID NO: 24 can be provided. Such immunogenic fragments 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

- 56 045958 at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of proteins that are 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequences set forth in SEQ ID NO: 24. In some embodiments, the fragments contain 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.

As disclosed herein with respect to coupling 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, which is encoded by the start codon of the nucleotide sequence, then encodes the protein without the signal peptide-encoding sequences.

Fragments of SEQ ID NO: 23 may contain 30 or more nucleotides, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 may contain 45 or more nucleotides, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 may contain 60 or more nucleotides, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 may contain 75 or more nucleotides, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 may contain 90 or more nucleotides, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 may contain 120 or more nucleotides, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 may contain 150 or more nucleotides, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 may contain 180 or more nucleotides, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 may contain 210 or more nucleotides, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 may contain 240 or more nucleotides, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 may contain 270 or more nucleotides, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 may contain 300 or more nucleotides, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 may contain 360 or more nucleotides, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 may contain 420 or more nucleotides, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 may contain 480 or more nucleotides, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 may contain 540 or more nucleotides, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 may contain 600 or more nucleotides, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 may contain 300 or more nucleotides, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 may contain 660 or more nucleotides, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 may contain 720 or more nucleotides, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 may contain 780 or more nucleotides, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 may contain 840 or more nucleotides, preferably including sequences encoding an immunodominant epitope. In some embodiments of the invention, fragments of the sequence

- 57 045958 SEQ ID NO: 23 may contain 900 or more nucleotides, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 may comprise 960 nucleotides or more, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 may contain 1020 or more nucleotides, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 may contain 1080 or more nucleotides, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 may contain 1140 or more nucleotides, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 may contain 1200 or more nucleotides, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 may contain 1260 nucleotides or more, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 may contain 1320 or more nucleotides, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 may contain 1380 or more nucleotides, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 may contain 1440 or more nucleotides, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 may contain 1500 or more nucleotides, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 may contain 1560 or more nucleotides, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 may contain 1620 or more nucleotides, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 may contain 1680 or more nucleotides, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 may contain 1740 or more nucleotides, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 may contain 1800 or more nucleotides, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 may contain 1860 or more nucleotides, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 may contain 1920 or more nucleotides, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 34 may contain 1980 or more nucleotides, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 may contain 2040 or more nucleotides, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 may contain 2100 or more nucleotides, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 may contain 2160 or more nucleotides, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 may contain 2220 or more nucleotides, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 may contain 2280 or more nucleotides, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 may contain 2340 or more nucleotides, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 may contain 2400 or more nucleotides, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 may contain 2460 or more nucleotides, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 may contain 2520 or more nuclei

- 58 045958 leotides, including preferably sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 may contain 2580 or more nucleotides, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 may contain 2640 or more nucleotides, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 may contain 2,700 or more nucleotides, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 may contain 2760 or more nucleotides, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 may contain 2820 or more nucleotides, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 may contain 2880 or more nucleotides, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 may contain 2940 or more nucleotides, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 may contain 3000 or more nucleotides, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 may contain 3060 or more nucleotides, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 may contain 3120 or more nucleotides, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 may contain 3180 or more nucleotides, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 may contain 3240 or more nucleotides, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 may contain 3300 or more nucleotides, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 may contain 3360 or more nucleotides, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 may contain 3420 or more nucleotides, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 may contain 3480 or more nucleotides, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 may contain coding sequences for IgE leader sequences. In some embodiments, the fragments of SEQ ID NO: 23 do not contain coding sequences for IgE leader sequences.

The fragments may contain 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 of the invention - less than 180 nucleotides, in some embodiments of the invention - less than 210 nucleotides, in some embodiments of the invention less than 240 nucleotides, in some embodiments of the invention - less than 270 nucleotides, in some embodiments of the invention - less than 300 nucleotides, in some embodiments of the invention - 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 of the invention - less than 660 nucleotides, in some embodiments - less than 720 nucleotides, in some embodiments - less than 780 nucleotides, in some embodiments - less than 840 nucleotides, in some embodiments - less than 900 nucleotides, in some embodiments - less than 960 nucleotides, in some embodiments less than 1020 nucleotides, in some embodiments less than 1080 nucleotides, in some embodiments less than 1140 nucleotides, in some embodiments less than 1200 nucleotides, in some embodiments less than 1260 nucleotides, in some embodiments less than 1320 nucleotides, in some embodiments less than 1380 nucleotides, in some embodiments less than 1440 nucleotides, in some embodiments less than 1440 nucleotides

- 59 045958 tenia - less than 1500 nucleotides, in some embodiments of the invention - less than 1560 nucleotides, in some embodiments of the invention - less than 1620 nucleotides, in some embodiments of the invention less than 1680 nucleotides, in some embodiments of the invention - less than 1740 nucleotides, in some 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 implementations of the invention - less than 2100 nucleotides, in some embodiments of the invention - less than 2160 nucleotides, in some embodiments of the invention - less than 2220 nucleotides, in some embodiments of the invention less than 2280 nucleotides, in some embodiments of the invention - less than 2340 nucleotides, in some embodiments of the invention - less than 2400 nucleotides, in some embodiments of the invention - less than 2460 nucleotides, in some embodiments of the invention - less than 2520 nucleotides, in some embodiments of the invention - less than 2580 nucleotides, in some embodiments of the invention - less than 2640 nucleotides, in some embodiments of the invention - less than 2700 nucleotides, in some embodiments of the invention - less than 2760 nucleotides, in some embodiments of the invention - less than 2820 nucleotides, in some embodiments of the invention less than 2860 nucleotides, in some embodiments of the invention - less than 2940 nucleotides, in some embodiments of the invention - 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 contain 15 or more amino acids, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 24 may contain 18 or more amino acids, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 24 may contain 21 or more amino acids, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 24 may contain 24 or more amino acids, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 24 may contain 30 or more amino acids, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 24 may contain 36 or more amino acids, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 24 may contain 42 or more amino acids, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 24 may contain 48 or more amino acids, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 24 may contain 54 or more amino acids, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 24 may contain 60 or more amino acids, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 24 may contain 66 or more amino acids, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 24 may contain 72 or more amino acids, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 24 may contain 90 or more amino acids, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 24 may contain 120 or more amino acids, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 24 may contain 150 or more amino acids, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 24 may contain 180 or more amino acids, preferably including sequences encoding an immunodominant epitope. In some embodiments, the iso

- 60 045958 fragments of the sequence SEQ ID NO: 24 may contain 210 or more amino acids, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 24 may contain 240 or more amino acids, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 24 may contain 270 or more amino acids, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 24 may contain 300 or more amino acids, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 24 may contain 330 or more amino acids, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 24 may contain 360 or more amino acids, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 24 may contain 390 or more amino acids, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 24 may contain 420 or more amino acids, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 24 may contain 450 or more amino acids, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 24 may contain 480 or more amino acids, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 24 may contain 510 or more amino acids, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 24 may contain 540 amino acids or more, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 24 may contain 570 or more amino acids, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 24 may contain 600 or more amino acids, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 24 may contain 630 or more amino acids, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 24 may contain 660 or more amino acids, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 24 may contain 690 or more amino acids, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 24 may contain 720 or more amino acids, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 24 may contain 750 or more amino acids, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 24 may contain 780 or more amino acids, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 24 may contain 810 or more amino acids, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 24 may contain 840 or more amino acids, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 24 may contain 870 or more amino acids, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 24 may contain 900 or more amino acids, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 24 may contain 930 or more amino acids, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 24 may contain 960 or more amino acids, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 24 may contain 990 or more amino acids, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 24 may contain 1020 or

- 61 045958 more amino acids, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 24 may contain 1050 or more amino acids, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 24 may contain 1080 or more amino acids, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 24 may contain 1110 or more amino acids, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 24 may contain 1140 or more amino acids, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 24 may contain 1170 or more amino acids, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 24 may contain 1200 or more amino acids, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 24 may contain 1230 or more amino acids, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 24 may contain 1260 or more amino acids, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 24 may contain 1290 or more amino acids, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 24 may contain 1320 or more amino acids, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 24 may contain 1350 or more amino acids, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 24 may contain 1380 or more amino acids, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 24 may contain 1410 or more amino acids, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 24 may contain 1440 or more amino acids, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 24 may contain 1470 or more amino acids, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 24 may contain 1500 or more amino acids, preferably including sequences encoding an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 24 may contain coding sequences for IgE leader sequences. In some embodiments, the fragments of SEQ ID NO: 24 do not contain coding sequences for IgE leader sequences.

The fragments may contain 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 implementations of the invention - less than 54 amino acids, in some embodiments of the invention - less than 60 amino acids, in some embodiments of the invention - less than 72 amino acids, in some embodiments of the invention - less than 90 amino acids, in some embodiments of the invention - less than 120 amino acids, in some embodiments implementations of the invention - less than 150 amino acids, in some embodiments of the invention - less than 180 amino acids, in some embodiments of the invention - less than 210 amino acids, in some embodiments of the invention - less than 240 amino acids, in some embodiments of the invention - less than 260 amino acids, in some embodiments implementations of the invention - less than 290 amino acids, in some embodiments of the invention - less than 320 amino acids, in some embodiments of the invention - less than 350 amino acids, in some embodiments of the invention - less than 380 amino acids, in some embodiments of the invention - less than 410 amino acids, in some embodiments implementations of the invention - less than 440 amino acids, in some embodiments of the invention - less than 470 amino acids, in some embodiments of the invention - less than 500 amino acids, in some embodiments of the invention - less than 530 amino acids, in some embodiments of the invention - less than 560 amino acids, in some embodiments implementation of the invention - less than 590 amino acids, in some embodiments of the invention - less than 620 amino acids, in some embodiments of the invention - less than 650 amino acids, in some embodiments

- 62 045958 embodiments of the invention - less than 680 amino acids, in some embodiments of the invention - less than 710 amino acids, in some embodiments of the invention - less than 740 amino acids, in some embodiments of the invention - less than 770 amino acids, in some embodiments of the invention - less than 800 amino acids , in some embodiments, less than 830 amino acids, in some embodiments, less than 860 amino acids, in some embodiments, less than 890 amino acids, in some embodiments, less than 920 amino acids, in some embodiments, less than 950 amino acids , in some embodiments, less than 980 amino acids, in some embodiments, less than 1010 amino acids, in some embodiments, less than 1040 amino acids, in some embodiments, less than 1070 amino acids, in some embodiments, 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 amino acids.

(15) MAGE A1.

The vaccine of the present invention may contain the cancer antigen melanoma associated antigen 1 (MAGE A1), a fragment thereof, or a variant thereof. MAGE A1, encoded by the MAGEA1 gene, is a 280-amino acid protein and has been found to be expressed only by tumor cells and germ cells. MAGE A1 depends on DNA methylation for its repression in normal somatic tissues. These genes are activated in many types of tumors during the process of genome-wide demethylation that often accompanies tumorigenesis. In particular, during malignant transformation, these genes are activated, expressed and can become antigenic targets that are recognized and attacked by the immune system. Thus, MAGE genes participate in the immune process by targeting some early tumor cells for immune damage. MAGE A1 can be expressed in a variety of cancers, including, but not limited to, melanomas, lung carcinomas, and esophageal squamous cell carcinomas.

The MAGE A1 antigen is capable of inducing antigen-specific T-cell and/or potent humoral immune responses, thereby inducing or triggering an immune response that targets or responds to the cancer or tumor by antigen expression. In some embodiments, the induced or evoked immune response may be cellular, humoral, or both cellular and humoral immune responses. In some embodiments, the induced or evoked cellular immune response may include the induction or secretion of interferon gamma (IFN-γ) and/or tumor necrosis factor alpha (TNF-α). In other embodiments, the induced or elicited immune response may reduce or inhibit one or more immune response suppressive factors that promote tumor or cancer development by expressing an antigen, such as, but not limited to, MHC presentation suppressive factors, antigen-specific activating factors 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 suppressor immune cells, CTLA-4, PD-1 , MDSC, MCP-1 and immune checkpoint molecules, as described in more detail below.

(16) WT1.

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

Mutation of WT1 can lead to the formation of a tumor or cancer, such as Wilms tumor or tumors expressing WT1. Wilms tumor often forms in one or both kidneys before metastasizing to other tissues, such as, but not limited to, liver tissue, urinary tissue, lymphatic tissue, and lung tissue. Accordingly, Wilms tumor can be considered a metastatic tumor. Wilms tumor typically occurs in young children (eg, less than 5 years of age) and can be either sporadic or hereditary. The WT1 cancer antigen may be further defined in PCT/US13/75141, filed December 23, 2013, which is incorporated herein by reference in its entirety.

- 63 045958

The WT-1 antigen is capable of inducing antigen-specific T cell and/or potent humoral immune responses, thereby inducing or eliciting an immune response that targets or responds to the cancer or tumor by expression of the antigen. In some embodiments, the induced or evoked immune response may be cellular, humoral, or both cellular and humoral immune responses. In some embodiments, the induced or evoked cellular immune response may include the induction or secretion of interferon gamma (IFN-γ) and/or tumor necrosis factor alpha (TNF-α). In other embodiments, the induced or elicited immune response may reduce or inhibit one or more immune response suppressive factors that promote tumor or cancer development by expressing an antigen, such as, but not limited to, MHC presentation suppressive factors, antigen-specific activating factors regulatory T cells (Tregs), PDL1, FasL, cytokines such as IL-10 and TFG-β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by suppressor immune cells, CTLA-4, PD-1 , MDSC, MCP-1 and immune checkpoint molecules, as described in more detail below.

Accordingly, this vaccine can be used to treat subjects suffering from Wilms tumor. The vaccine can be used to treat subjects suffering from 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, as well as blood cancer. The vaccine may also be used to treat subjects suffering from cancer or tumors that express WT1 to prevent the subjects from developing such tumors. The WT1 antigen may be different from the native, normal WT1 gene, and thus provide therapy or prophylaxis against a tumor expressing the WT1 antigen. Accordingly, WT1 antigen sequences that differ from the native WT1 gene (eg, mutated genes or WT1 sequences) are provided herein.

The native WT1 gene transcripts are converted into a series of mRNAs, and the resulting proteins are not all equally effective in inducing an immune response. The mutated WT1 genes described herein do not allow alternative processing that produces a single full-length transcript and results in greater induction of effector T and B cell responses. The first mutated WT1 sequence is called CON WT1 with modified zinc fingers or ConWT1-L. SEQ ID NO: 19 is a nucleotide sequence encoding the zinc finger modified WT1 CON WT1 antigen. SEQ ID NO: 20 is the amino acid sequence encoding the WT1 CON WT1 modified zinc finger antigen. The second mutated WT1 sequence is called CON WT1 without zinc fingers or ConWT1-S. SEQ ID NO: 21 is a nucleotide sequence encoding the WT1 CON WT1 zinc finger antigen. SEQ ID NO: 22 is the amino acid sequence of the WT1 CON WT1 antigen without the modified zinc fingers.

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

A WT1 consensus antigen may contain one or more mutations in one or more zinc fingers, thereby eliciting stronger and broader cellular and/or humoral immune responses than the corresponding codon-optimized WT1 antigen. One or more mutations may be a substitution of one or more amino acids that coordinate a zinc ion in one or more zinc fingers. One or more amino acids that coordinate the zinc ion may be a CCHH motif. Accordingly, in some embodiments, one or more mutations may replace 1, 2, 3, or all 4 amino acids of the CCH motif.

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

- 64 045958 sequences 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 may be removed from the WT1 consensus antigen. One, two, three, or all four zinc fingers may be removed from the WT1 consensus antigen.

The WT1 consensus antigen may be a nucleotide sequence of SEQ ID NO: 19, which encodes the sequence of SEQ ID NO: 20. In some embodiments, the WT1 consensus antigen may be a nucleotide 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 nucleotide sequence set forth in SEQ ID NO: 19. In other embodiments, the WT1 consensus antigen may be a nucleotide sequence that encodes 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 along the entire length of the amino acid sequence given in SEQ ID NO: 20.

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

The WT1 consensus antigen may be a nucleic acid of SEQ ID NO: 21 that encodes the sequence of SEQ ID NO: 22. In some embodiments, the WT1 consensus antigen may be a nucleotide 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 nucleotide sequence set forth in SEQ ID NO: 21. In other embodiments, the WT1 consensus antigen may be a nucleotide sequence that encodes 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% identical over the entire length of the amino acid sequence given 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 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 given in SEQ ID NO: 22.

Immunogenic fragments of the sequences SEQ ID NO: 20 and SEQ ID NO: 22 can be provided. The immunogenic fragments may contain at least 60%, at least 65%,

- 65 045958 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 sequence of SEQ ID NO: 20 and/or SEQ ID NO: 22. In some embodiments, the immunogenic fragments may comprise at least 60%, at least 65%, at least 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 sequence of SEQ ID NO: 20, provided that if residues 312, 317, 342 and 347 of SEQ ID NO: 20 are present in the immunogenic fragment, then these residues are any residue other than cysteine ( C), and with the proviso that if residues 330, 334, 360 and 364 of SEQ ID NO: 20 are present in the immunogenic fragment, those residues are any residue other than histidine (H). In other embodiments, 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 the sequence of SEQ ID NO: 20, provided that if residues 312, 317 are present in the immunogenic fragment , 330, 334, 342, 347, 360 and 364 of SEQ ID NO: 20, then these residues are glycine (G).

In some embodiments, the immunogenic fragments comprise a leader sequence, for example, an immunoglobulin leader sequence, such as an immunoglobulin E (IgE) leader sequence. In some embodiments, the immunogenic fragments do not contain a leader sequence.

Immunogenic protein fragments with amino acid sequences identical to the immunogenic fragments of SEQ ID NOs: 20 and 22 can be provided. Such fragments may contain 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 proteins having 95% or greater identity to the sequence of SEQ ID NO: 20 and/or SEQ ID NO: 22. Some embodiments of the invention provide immunogenic fragments that have 96% or greater identity to the immunogenic fragments of the WT1 protein sequences herein. Some embodiments of the invention provide immunogenic fragments that have 97% or greater identity to the immunogenic fragments of the WT1 protein sequences herein. Some embodiments of the invention provide immunogenic fragments that have 98% or greater identity to the immunogenic fragments of the WT1 protein sequences herein. Some embodiments of the invention provide immunogenic fragments that have 99% or greater identity to the immunogenic fragments of the WT1 protein sequences herein. In some embodiments, the immunogenic fragments comprise a leader sequence, for example, an immunoglobulin leader sequence, such as an IgE leader sequence. In some embodiments, the immunogenic fragments do not contain a leader sequence.

Some embodiments of the invention relate to immunogenic fragments of the sequences SEQ ID NO: 19 and SEQ ID NO: 21. The immunogenic fragments may contain 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 sequence SEQ ID NO : 19 and/or SEQ ID NO: 21. In some embodiments, the immunogenic fragments comprise sequences that encode a leader sequence, for example, an immunoglobulin leader sequence, such as an IgE leader sequence. In some embodiments, the immunogenic fragments do not contain coding sequences that encode a leader sequence.

Immunogenic nucleic acid fragments with nucleotide sequences identical to the immunogenic fragments of SEQ ID NO: 19 and SEQ ID NO: 21 can be provided. Such fragments may contain 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 nucleic acids having 95% or greater identity to the sequence of SEQ ID NO: 19 and/or SEQ ID NO: 21. Some embodiments of the invention provide immunogenic fragments that have 96% or greater identity to the immunogenic fragments of the WT1 nucleotide sequences herein. Some embodiments of the invention provide immunogenic fragments that have 97% or greater identity to the immunogenic

- 66 045958 gene fragments of WT1 nucleotide sequences in this document. Some embodiments of the invention provide immunogenic fragments that have 98% or greater identity to the immunogenic fragments of the WT1 nucleotide sequences herein. Some embodiments of the invention provide immunogenic fragments that have 99% or greater identity to the immunogenic fragments of the WT1 nucleotide sequences herein. In some embodiments, the immunogenic fragments comprise sequences that encode a leader sequence, for example, an immunoglobulin leader sequence, such as an IgE leader sequence. In some embodiments, the immunogenic fragments do not contain coding sequences that encode a leader sequence.

(17) gp100.

The vaccine of the present invention may contain cancer antigen glycoprotein 100 (gp100; also known as Trp2 and premelanosomal protein (PMEL)), a fragment thereof, or a variant thereof. gp100 is encoded by the PMEL gene. It is a type 1 transmembrane glycoprotein with a molecular weight of 70 kDa, consisting of 661 amino acids, which plays a central role in melanosome biogenesis, as it is involved in the maturation of melanosomes from stage I to stage II. gp100 determines the formation of strata from multivesicular bodies and is directly involved in the biogenesis of premelanosomes. gp100 is enriched in premelanosomes compared to mature melanosomes but is overexpressed in neonatal proliferating melanocytes and during tumor development. The gp100 protein contains a number of immunogenic epitopes that are recognized by peripheral blood cytotoxic T lymphocytes of melanoma patients and tumor infiltrating lymphocytes.

The gp100 antigen is capable of inducing antigen-specific T cell and/or potent humoral immune responses, thereby inducing or eliciting an immune response that targets or responds to the cancer or tumor by expression of the antigen. In some embodiments, the induced or evoked immune response may be cellular, humoral, or both cellular and humoral immune responses. In some embodiments, the induced or evoked cellular immune response may include the induction or secretion of interferon gamma (IFN-γ) and/or tumor necrosis factor alpha (TNF-α). In other embodiments, the induced or elicited immune response may reduce or inhibit one or more immune response suppressive factors that stimulate the development of a tumor or cancer by expressing an antigen, such as, but not limited to, MHC presentation suppressive factors, antigen-specific activating factors regulatory T cells (Tregs), PDL1, FasL, cytokines such as IL-10 and TFG-β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by suppressor immune cells, CTLA-4, PD-1 , MDSC, MCP-1 and immune checkpoint molecules, as described in more detail below.

(18) Viral antigens.

The cancer antigen may be a viral antigen, a fragment thereof, or a variant thereof. The viral antigen may be a hepatitis B virus, hepatitis C virus or human papilloma virus (HPV) antigen. The HPV may be HPV 6, HPV 11, HPV 16 or HPV 18, described below.

The viral antigen is capable of inducing antigen-specific T-cell and/or potent humoral immune responses, thereby inducing or eliciting an immune response that targets or responds to the cancer or tumor by expression of the antigen. In some embodiments, the induced or evoked immune response may be cellular, humoral, or both cellular and humoral immune responses. In some embodiments, the induced or evoked cellular immune response may include the induction or secretion of interferon gamma (IFN-γ) and/or tumor necrosis factor alpha (TNF-α). In other embodiments, the induced or elicited immune response may reduce or inhibit one or more immune response suppressive factors that promote tumor or cancer development by expressing an antigen, such as, but not limited to, MHC presentation suppressive factors, antigen-specific activating factors 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 suppressor immune cells, CTLA-4, PD-1 , MDSC, MCP-1 and immune checkpoint molecules, as described in more detail below.

Hepatitis B virus antigen.

The viral antigen may be an antigen from the hepatitis B virus (HBV), a fragment thereof, or a variant thereof. HBV antigen may be associated with or cause liver cancer. In some embodiments, the HBV antigen may be a heterologous nucleic acid molecule(s), such as plasmid(s), that encode one or more

- 67 045958 antigens from HBV. HBV antigen can be full-length or immunogenic fragments of full-length proteins.

The HBV antigen may contain consensus sequences and/or one or more modifications for improved expression. Genetic modifications, including codon optimization, RNA optimization, and addition of a high-performance immunoglobulin leader sequence to increase the immunogenicity of the constructs, can be included in the modified consensus sequences. The HBV consensus antigen may contain a signal peptide, such as an immunoglobulin signal peptide, such as an IgE or IgG signal peptide, and in some embodiments may contain an HA tag. Immunogens can be designed to induce stronger and broader cellular immune responses than corresponding codon-optimized immunogens.

The HBV antigen may be HBV core protein, HBV surface protein, HBV DNA polymerase, HBV gene X-encoded protein, a fragment thereof, a variant thereof, or a combination thereof. The HBV antigen may be genotype HBV core protein, genotype HBV core protein, genotype HBV core protein, genotype HBV core protein, genotype HBV core protein, genotype HBV core protein, genotype HBV core protein, genotype HBV core protein, genotype HBV core protein. genotype H, HBV surface protein genotype A, HBV surface protein genotype B, HBV surface protein genotype C, HBV surface protein genotype D, HBV surface protein genotype E, HBV surface protein genotype F, HBV surface protein genotype G, HBV surface protein genotype H , a fragment thereof, a variant thereof, or a combination thereof. The HBV antigen may be a consensus HBV core protein or a consensus HBV surface protein.

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

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

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

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

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

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

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

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

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

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

In other embodiments, the HBV antigen may be a consensus

- 68 045958 surface construct of an HBV genotype C DNA sequence, an IgE leader sequence linked to a consensus sequence for the surface protein of HBV genotype C, or a consensus surface sequence of the HBV genotype C protein.

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

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

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

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

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

Hepatitis C virus antigen.

The viral antigen may be an antigen from the hepatitis C virus (HCV), a fragment thereof, or a variant thereof. HCV antigen may be associated with or cause liver cancer. In some embodiments, an HCV antigen may be a heterologous nucleic acid molecule(s), such as a plasmid(s), that encodes one or more antigens from HCV. HCV antigen can be full-length or immunogenic fragments of full-length proteins.

The HCV antigen may contain consensus sequences and/or one or more modifications for improved expression. Genetic modifications, including codon optimization, RNA optimization, and addition of a high-performance immunoglobulin leader sequence to increase the immunogenicity of the constructs, can be included in the modified consensus sequences. The HCV consensus antigen may contain a signal peptide, such as an immunoglobulin signal peptide, such as an IgE or IgG signal peptide, and in some embodiments may contain an HA tag. Immunogens can be designed to induce stronger and broader cellular immune responses than corresponding codon-optimized immunogens.

An HCV antigen may 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.

Human papillomavirus.

The viral antigen may be an antigen from HPV, a fragment thereof, or a variant thereof. The HPV antigen can be from HPV types 16, 18, 31, 33, 35, 45, 52 and 58, which cause cervical cancer, colorectal cancer and/or other types of cancer. The HPV antigen may be from HPV types 6 and/or 11, which cause genital warts and are known to cause head and neck cancer. The HPV antigen may be from HPV types 16 and/or 18, which cause cervical cancer. The HPV antigen can be from HPV types 6, 11 and/or 16, which cause RRP and anal cancer. HPV cancer antigen can be further defined in US Patent No. 8168769, filed July 30, 2007, US Patent No. 8389706, filed January 21, 2010, US Patent Application No. 13/271576, filed October 21, 2011, and US Patent Application No. 61/777198, filed March 12, 2013, each of which is incorporated by reference in its entirety.

HPV antigens can be HPV Eb or E7 domains from each HPV type. For example, for HPV type 16 (HPV16), the HPV16 antigen may include HPV16 E6 antigen, HPV16 E7 antigen, fragments, variants, or combinations thereof. Likewise, the HPV antigen may 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.

Herpes viruses.

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,

- 69 045958

EBV, roseolovirus, Kaposi's sarcoma-associated herpesvirus, or Muhv, and preferably CMV, HSV1, HSV2, CeHV1 and VZV.

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

Nucleotide sequences, including 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 were obtained. Vaccines may contain one or more nucleotide sequences that encode one or more consensus immunogenic protein variants selected from this group of sequences designed to optimize stability and expression in humans. Nucleotide sequence encoding the HCMV-gB consensus protein (SEQ ID NO: 25), nucleotide sequence encoding the consensus protein

HCMV-gM (SEQ ID NO: 27), nucleotide sequence encoding consensus protein

HCMV-gN (SEQ ID NO: 29), nucleotide sequence encoding consensus protein

HCMV-gH (SEQ ID NO: 31), nucleotide sequence encoding consensus protein

HCMV-gL (SEQ ID NO: 33), nucleotide sequence encoding consensus protein

HCMV-gO (SEQ ID NO: 35), nucleotide sequence encoding consensus protein

HCMV-UL128 (SEQ ID NO: 37), nucleotide sequence encoding the consensus protein HCMV-UL130 (SEQ ID NO: 39), nucleotide sequence encoding the consensus protein HCMV-UL-131A (SEQ ID NO: 41), nucleotide sequence, encoding the HCMV-UL-83 consensus protein (pp65) (SEQ ID NO: 43). The nucleotide sequence may additionally have an IgE coding leader sequence associated with the 5' end.

In light of evolutionary divergence from clinical strains and large genetic differences among common circulating human strains, consensus amino acid sequences were obtained for each of the immunogenic proteins. Consensus amino acid sequences for gB, gM, gH, gL, gE, gI, gK, gC, gD, UL128, UL130, UL-131A and UL-83 (pp65) were based on sequences from human clinical strains. Because of the large evolutionary divergence of the gN protein, a consensus sequence was obtained from only one (gN-4c) of the seven serotypes, which is the most seroprevalent (gN-4). Similarly, for gO, consensus amino acid sequences were derived from one (gO-5) of the eight serotypes due to the confirmed association of these specific serotypes with serotype gN-4c.

As described above, the herpes virus antigen may be a consensus herpes virus antigen. The herpes virus consensus antigen may be provided with a signal peptide. In some embodiments, the IgE leader sequence is linked to the N-terminus. As described herein, when referring to a signal peptide associated with the N-terminus of a consensus sequence, it is intended to specifically include embodiments of the invention in which the N-terminal Xa residue of the consensus sequences is replaced by a signal peptide. That is, as used throughout this document, Haa is intended to refer to any amino acid or lack of amino acid. Proteins that contain the consensus sequence given herein by SEQ ID NO: 26, 28, 30, 32, 34, 36, 38, 40, 42, 44 may contain these sequences without the N-terminal Xaa.

Amino acid sequences were obtained that contained, in each case, the IgE leader sequence at the N-terminus of the consensus sequences of the immunogenic protein of the herpes virus. Some embodiments of the invention provide nucleic acid constructs in which two or more herpes virus antigens are expressed as fusion proteins linked to each other by proteolytic cleavage sites. The furin proteolytic cleavage site is an example of a proteolytic cleavage site that can bind herpes virus antigens in the fusion protein expressed by the construct. The herpes family viral cancer antigen may further be any antigen described in US Patent Application No. 13/982,457, the contents of which are incorporated herein by reference in their entirety.

3. Vaccine in combination with an immune checkpoint inhibitor.

The vaccine may further comprise one or more inhibitors of one or more immune checkpoint molecules (ie, immune checkpoint inhibitors). Immune checkpoint molecules are described in more detail below. An immune checkpoint inhibitor is any nucleic acid or protein that prevents the suppression of any component in the immune system, for example, MHC presentation, T cell presentation and/or differentiation, B cell presentation and/or differentiation, any cytokine, chemokine or alarm

- 70 045958 for proliferation and/or differentiation of immune cells.

Such an inhibitor may be a nucleotide sequence, an amino acid sequence, a small molecule, or a combination thereof. The nucleotide sequence may be DNA, RNA, cDNA, a variant thereof, a fragment thereof, or a combination thereof. The nucleic acid may also contain additional sequences encoding linker or marker sequences that are linked to the immune checkpoint inhibitor by a peptide bond. A small molecule may be a low molecular weight, e.g., less than 800 dalton, organic or inorganic compound that may serve as an enzyme substrate, a ligand (or analog thereof) bound to a protein or nucleic acid, or a biological process regulator. The amino acid sequence may represent is a protein, a peptide, a variant thereof, a fragment thereof, or a combination thereof.

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

Immune checkpoint molecule.

The immune checkpoint molecule may be a nucleotide sequence, an amino acid sequence, a small molecule, or a combination thereof. The nucleotide sequence may be DNA, RNA, cDNA, a variant thereof, a fragment thereof, or a combination thereof. The nucleic acid may also contain additional sequences encoding linker or marker sequences that are linked to the immune checkpoint inhibitor by a peptide bond. A small molecule may be a low molecular weight, eg, less than 800 daltons, organic or inorganic compound that can serve as an enzyme substrate, a ligand (or analog thereof), bound to a protein or nucleic acid, or a regulator of a biological process. The amino acid sequence may be a protein, a peptide, a variant thereof, a fragment thereof, or a combination thereof.

(1) PD-1 and PD-L1.

The immune checkpoint molecule may be programmed cell death protein 1 (PD-1), programmed cell death ligand 1 (PD-L1), a fragment thereof, a variant thereof, or a combination 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 pro-B cells, and thus is involved in the fate and/or differentiation of these cells. Specifically, 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) signaling, thereby negatively regulating immune responses. PD-1 can negatively regulate CD8+ T cell responses, and thus inhibits CD8-mediated cytotoxicity and enhances tumor development.

PD-1 has two ligands, PD-L1 and PD-L2, which are members of the B7 family. PD-L1 is activated on macrophages and dendritic cells (DC) in response to LPS and GM-CSF treatment, and on T cells and B cells by TCR and B cell receptor signaling. PD-L1 is expressed by many tumor cell lines, including myelomas, mastocytomas, and melanomas.

Antibody to immune checkpoint molecule.

As described above, the immune checkpoint inhibitor may be an antibody. The antibody may bind or react with an antigen (ie, the immune checkpoint molecule described above). Accordingly, an antibody may be considered an anti-immune checkpoint molecule antibody or an immune checkpoint molecule antibody. The antibody may be encoded by a nucleotide sequence.

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

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

The heavy chain polypeptide may contain a set of complementarity determining regions (CDRs). The CDR set may contain three VH region hypervariable regions. Based on the N-terminus of the heavy chain polypeptide, these CDRs are designated CDR1, CDR2 and CDR3, respectively. CDR1, CDR2 and CDR3 of a heavy chain polypeptide may contribute to antigen binding or recognition.

The light chain polypeptide may comprise a light chain variable region (VL) and/or a light chain constant region (CL). The light chain polypeptide may contain a set of complementarity determining regions (CDRs). The CDR set may contain three VL hypervariable regions

- 71 045958 region. Based on the N-terminus of the light chain polypeptide, these CDRs are designated CDR1, CDR2 and CDR3, respectively. CDR1, CDR2 and CDR3 of a light chain polypeptide may contribute to antigen binding or recognition.

The antibody may comprise a set of heavy chain and light chain complementarity determining regions (CDRs), respectively located between a set of heavy chain and light chain frameworks (FRs), which provide support for the CDRs and define the spatial arrangement of the CDRs relative to each other. The CDR set may contain three heavy or light chain V region hypervariable regions. Based on the N-terminus of the heavy or light chain, these regions are designated CDR1, CDR2 and CDR3, respectively. The antigen binding region may therefore comprise six CDRs comprising a set of CDRs from each of the heavy and light chain V regions.

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

In addition, the proteolytic enzyme papain preferentially cleaves IgG molecules into several fragments, two of which (F(ab) fragments) each contain a covalent heterodimer containing an intact antigen-binding site. The enzyme pepsin is capable of cleaving IgG molecules to produce several fragments, including the F(ab') ₂ fragment, which contains both antigen-binding sites. Accordingly, the antibody may be Fab or F(ab') ₂ . The Fab may contain a heavy chain polypeptide and a light chain polypeptide. A Fab heavy chain polypeptide may contain a VH region and a CH1 region. The Fab light chain may contain a VL region and a CL region.

The antibody may be a polyclonal or monoclonal antibody. The antibody may be a chimeric antibody, a single chain antibody, an affinity mature antibody, a human antibody, a humanized antibody, or a fully human antibody. A humanized antibody may be an antibody other than a non-human species that binds a desired antigen having one or more complementarity determining regions (CDRs) from a non-human species and framework regions from a human immunoglobulin molecule.

(1) PD-1 antibody.

The anti-immune checkpoint molecule antibody may 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 may be Nivolumab. An anti-PD-1 antibody is capable of inhibiting the activity of PD-1, thereby inducing, triggering or enhancing an immune response to a tumor or cancer and reducing tumor development.

(2) PD-L1 antibody.

The anti-immune checkpoint molecule antibody may 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. An anti-PD-L1 antibody is capable of inhibiting the activity of PD-L1, thereby inducing, triggering or enhancing an immune response to a tumor or cancer and reducing tumor development.

4. Vaccine constructs and plasmids.

The vaccine may contain nucleic acid constructs or plasmids that encode the antigens and/or antibodies described above. Nucleic acid constructs or plasmids may include or contain one or more heterologous nucleotide sequences. Provided herein are genetic constructs capable of containing a nucleotide sequence that encodes the antigens and/or antibodies described above. The genetic construct may be present in the cell as a functioning extrachromosomal molecule. The genetic construct may be a linear mini-chromosome, including a centromere, telomere, or plasmid or cosmid. Genetic constructs may include or contain one or more heterologous nucleotide sequences.

Genetic constructs can be in the form of plasmids expressing the antigens and/or antibodies described above in any order.

The genetic construct may also be part of the genome of a recombinant viral vector, including a recombinant adenovirus, a recombinant adenovirus-associated virus, and a recombinant vaccinia virus. The genetic construct may be part of the genetic material in attenuated living microorganisms or recombinant microbial vectors that live in cells.

Genetic constructs may contain regulatory elements for the expression of genes coding nucleic acid sequences. Regulatory elements may be a promoter, enhancer, start codon, stop codon or polyadenylation signal.

Nucleotide sequences may constitute a genetic construct that can

- 72 045958 represent a vector. The vector may be capable of expressing the antigens and/or antibodies described above in a mammalian cell in an amount effective to elicit an immune response in the mammal. The vector may be recombinant. The vector may contain heterologous nucleic acid encoding the antigens and/or antibodies described above. The vector may be a plasmid. The vector can serve to transfect cells with nucleic acid encoding the above-described antigens and/or antibodies, while the transformed host cell is cultured and maintained under conditions under which the above-described antigens and/or antibodies are expressed.

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

The vector may contain a heterologous nucleic acid encoding the antigens and/or antibodies described above, and may further contain a start codon, which may be located upstream of one or more cancer antigen-encoding sequences, as well as a stop codon, which may be located upstream of one or more cancer antigen-encoding sequences. the direction of transcription from the coding sequence(s) of the antigens and/or antibodies described above. The start and stop codon may be in frame with the coding sequence(s) of the antigens and/or antibodies described above. The vector may also contain a promoter that is operably linked to the coding sequence(s) of the antigens and/or antibodies described above. The promoter operably linked to the coding sequence(s) of the antigens and/or antibodies described above may be a simian virus 40 (SV40) promoter, a murine mammary tumor virus (MMTV) promoter, a human immunodeficiency virus (HIV) promoter such as bovine immunodeficiency virus (BIV) long terminal repeat (LTR) promoter, Moloney virus promoter, avian leukemia virus (ALV) promoter, cytomegalovirus (CMV) promoter such as the CMV immediate early promoter, Epstein Barr virus (EBV) promoter, or sarcoma virus promoter Rousa (RSV). The promoter may also be a promoter from a human gene, such as human actin, human myosin, human hemoglobin, human muscle creatine or human metallothionein. The promoter may also be a tissue-specific promoter, such as a muscle-specific or skin-specific promoter, natural or synthetic. Examples of such promoters are described in US Patent Application Publication No. US20040175727, the contents of which are incorporated herein in their entirety.

The vector may also contain a polyadenylation signal, which may be located downstream of the coding sequence(s) of the antigens and/or antibodies described above. 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 the pCEP4 vector (Invitrogen, San Diego, Calif.).

The vector may also contain an enhancer upstream of the antigens and/or antibodies described above. The enhancer may be required for DNA expression. The enhancer may be human actin, human myosin, human hemoglobin, human muscle creatine, or a viral enhancer such as one of CMV, HA, RSV, or EBV. Enhancers of function polynucleotides are described in US Patent Nos. 5,593,972, 5,962,428 and WO94/016737, the contents of which are incorporated herein by reference in their entirety.

The vector may also contain a mammalian origin of replication for the purpose of maintaining the extrachromosomal vector and producing a number of copies of the vector in the cell. The vector may be pVAX1, pCEP4 or pREP4 from Invitrogen (San Diego, Calif.), which may contain the Epstein-Barr virus origin of replication and the EBNA-1 nuclear antigen coding region, which can result in multicopy episomal replication without integration. The vector may be pVAXl or a modified variant of pVaxl, such as the plasmid variant described herein. The pVax1 plasmid variant is a 2998 base pair variant of the pVAX1 plasmid vector backbone (Invitrogen, Carlsbad, Calif.). The CMV promoter is located at bases 137-724. The T7 promoter/priming region is located at bases 664-683. A number of insertion sites are 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, which was used as the backbone for plasmids 1-6 shown here:

C>G241 in the CMV promoter.

C>r1942 backbone, downstream of the bovine growth hormone polyadenylation signal (bGHpolyA).

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A> - 2876 backbones, downstream of the kanamycin gene.

C>T 3277 multicopy mutations (see Nucleic Acid Research 1985) at the pUC origin of replication (Ori).

G>C 3753 at the very end of pUC Ori upstream of the RNase H site.

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

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

The vector may also contain a regulatory sequence that may be well suited for gene expression in mammalian or human cells into which the vector is introduced. One or more cancer antigen sequences described herein may contain a codon that may enable more efficient transcription of the coding sequence in a host cell.

The vector may be pSE420 (Invitrogen, San Diego, Calif.), which can be used to produce protein in Escherichia coli (E. coli). The vector may also be pYES2 (Invitrogen, San Diego, Calif.), which can be used to produce protein in Saccharomyces cerevisiae yeast strains. The vector can also be from the complete baculovirus expression system MAHBAS™ (Invitrogen, San Diego, Calif.), which can be used to produce protein in insect cells. The vector may also be plasmid DNA pcDNA I or pcDNA3 (Invitrogen, San Diego, Calif.), which can be used to produce the protein in mammalian cells, such as Chinese hamster ovarian (CHO) cells. The vector may be expression vectors or systems for producing protein using conventional techniques and readily available starting materials, including Sambrook at al., Molecular Cloning and Laboratory Manual, Second Ed., Cold Spring Harbor (1989), which is incorporated herein document by reference in its entirety.

In some embodiments, the vector may contain one or more nucleotide sequences of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15 and/or 17.

5. Pharmaceutical compositions of the vaccine.

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

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In some embodiments, the pharmaceutical compositions of the present invention contain at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 nanograms of DNA vaccine. In some embodiments, the pharmaceutical compositions may contain at least 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 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 DNA vaccine. In some embodiments, the pharmaceutical composition may contain 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 DNA vaccine.

In other embodiments, the pharmaceutical composition may contain up to 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 contain up to 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 , 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 2 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, 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 DNA vaccine. In some embodiments, the pharmaceutical composition may contain up to 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 DNA vaccine.

The pharmaceutical composition may further contain other agents for dosage forms in accordance with the route of administration to be used. In cases where the pharmaceutical compositions are injectable pharmaceutical compositions, they are sterile, pyrogen-free and free of contaminants. Preferably, an isotonic dosage form is used. Typically, additives for isotonicity may include sodium chloride, dextrose, 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 composition.

The vaccine may additionally contain a pharmaceutically acceptable excipient.

The pharmaceutically acceptable excipient may be functional molecules such as excipients, adjuvants, carriers or diluents.

The pharmaceutically acceptable excipient may be a transfection facilitating agent, which may include surfactants such as immunostimulating complexes (ISCOMS), Freund's incomplete adjuvant, LPS analog including monophosphoryl lipid A, muramyl peptides, quinone analogs, vesicles such as squalane and squalene, hyaluronic acid, lipids, liposomes, calcium ions, viral proteins, polyanions, polycations or nanoparticles, or other known transfection facilitating agents.

The transfection facilitating agents are a polyanion, a polycation, including poly-Lglutamate (LGL), or a lipid. The transfection facilitating agent is poly-L-glutamate, and more preferably, poly-L-glutamate is present in the vaccine at a concentration of less than 6 mg/ml. In combination with the genetic construct, a transfection facilitating agent can also be administered, which may also include surfactants such as immunostimulating complexes (ISCOMS), Freund's incomplete adjuvant, LPS analogue, including monophosphoryl lipid A, muramyl peptides, quinone analogues and vesicles, such as squalane and squalane, as well as hyaluronic acid. In some embodiments, DNA vector vaccines may also contain a transfection facilitating agent such as lipids, liposomes, including lecithin-based liposomes or other liposomes known in the art, such as a DNA liposome mixture (see, for example, W09324640), calcium ions, viral proteins, polyanions, polycations or nanoparticles, or other known transfection facilitating agents.

Preferably, the transfection facilitating agent is a polyanion, polycation, including poly-L-glutamate (LGL), or 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.

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The pharmaceutically acceptable excipient may be an adjuvant. The adjuvant may be other genes that are expressed in an alternative plasmid or delivered as proteins in combination with the above plasmid in the vaccine. The adjuvant may be selected from the group consisting of: interferon α (IFN-α), interferon β (IFN-β), interferon γ, platelet-derived growth factor (PDGF), TNFa, TNFe, GM-CSF, epidermal growth factor (EGF) , skin T-lymphocyte-attracting chemokine (CTACK), epithelial thymus-expressed chemokine (TECK), mucosal-associated epithelial chemokine (MEC), IL-12, IL-15, MHC, CD80, CD86, including IL- 15 with the signal sequence removed and, optionally, with a signal peptide from IgE. The adjuvant may be IL-12, IL-15, IL-28, CTACK, TECK, platelet-derived growth factor (PDGF), TNFa, TNFe, 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 of the invention, the adjuvant is IL-12.

Other genes that may serve as adjuvants include those encoding: MCP-1, MIP-ln. 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 growth factor, fibroblast growth factor, IL- 7, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Flt, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL- R2, TRICK2, DR6, caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, inactive NIK, SAP K, SAP-1, JNK , interferon-responsive genes, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, Ox40, Ox40 LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1, TAP2 and their functional fragments.

6. Combination vaccines for the treatment of certain types of cancer.

The vaccine may be in the form of various combinations of cancer antigens, as described above, to treat certain types of cancer or tumors. Depending on the combination of one or more cancer antigens, the vaccine may target different types of cancer or other types of tumors. These cancers may include melanoma, blood cancers (eg, leukemia, lymphoma, myeloma), lung carcinomas, squamous cell carcinomas of the esophagus, bladder cancer, colorectal cancer, esophageal cancer, gastrointestinal cancer, hepatocarcinoma, head and neck cancer , brain, anal cancer, non-small cell lung carcinoma, pancreatic cancer, synovial carcinoma, prostate cancer, testicular cancer, liver cancer, cervical cancer, recurrent respiratory papillomatosis, skin cancer and stomach cancer. Fig. 15 illustrates examples of certain combinations of antigens that may be used to treat certain types of cancer.

Melanoma.

The vaccine may combine one or more cancer antigens such as tyrosinase, PRAME or gp100-Trp2 to treat or prevent melanoma (see FIG. 15). The vaccine may further combine one or more tyrosinase, PRAME, or gp100-Trp2 cancer antigens with any one or more hTERT, NY-ESO-1, MAGE-A1, or WT1 cancer antigens to treat or prevent melanoma. Other combinations of cancer antigens may also be used to treat or prevent melanoma.

Head and neck cancer.

The vaccine may contain the HPV 16 E6/E7 cancer antigen for the treatment or prevention of head and neck cancer (see FIG. 15). The vaccine may further combine the HPV 16 E6/E7 cancer antigen with any one or more hTERT, NY-ESO-1, MAGE-A1 or WT1 cancer antigens for the treatment or prevention of head and neck cancer. Other combinations of cancer antigens may also be used to treat or prevent head and neck cancer.

Recurrent respiratory papillomatosis/anal cancer.

The vaccine may combine one or more cancer antigens, such as HPV 6, HPV 11 or HPV 16, to treat or prevent recurrent respiratory papillomatosis or anal cancer (see FIG. 15). The vaccine may further combine one or more HPV 6, HPV11 or HPV16 cancer antigens with one or more hTERT, NY-ESO-1, MAGE-A1 or WT1 cancer antigens to treat or prevent recurrent respiratory papillomatosis or anal cancer. Other combinations of cancer antigens may also be used to treat or prevent recurrent respiratory papillomatosis or anal cancer.

Cervical cancer.

The vaccine may combine one or more cancer antigens, such as HPV 16 E6/E7 or HPV 18 E6/E7, to treat or prevent cervical cancer (see FIG. 15). The vaccine may further combine one or more cancer antigens, such as HPV 16 E6/E7 or HPV 18 E6/E7 with one or more hTERT, NY-ESO-1, MAGE-A1 or WT1 cancer antigens for the treatment or prevention of cervical cancer. Other combinations of cancer antigens may also be used to treat or prevent cervical cancer.

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Liver cancer.

The vaccine may combine one or more cancer antigens, such as HBV core antigen, HBV surface antigen, HCVNS34A, HCVNS5A, HCV NS5B, or HCVNS4B, to treat or prevent liver cancer (see FIG. 15). The vaccine may further combine one or more cancer antigens, such as HBV core antigen, HBV surface antigen, HCVNS34A, HCVNS5A, HCV NS5B or HCVNS4B with one or more hTERT, NY-ESO-1, MAGE-A1 or WT1 cancer antigens to treat or prevention of liver cancer. Other combinations of cancer antigens may also be used to treat or prevent liver cancer.

Glioblastoma.

The vaccine may contain CMV for the treatment or prevention of glioblastoma (see Fig. 15). The vaccine may further combine CMV with one or more hTERT, NY-ESO-1, MAGE-A1 or WT1 cancer antigens to treat or prevent glioblastoma. Other combinations of cancer antigens may also be used to treat or prevent glioblastoma.

Prostate cancer.

The vaccine may combine one or more cancer antigens, such as PSA, PSMA or STEAP, to treat or prevent prostate cancer (see FIG. 15). The vaccine may further combine one or more PSA, PSMA, or STEAP cancer antigens with one or more hTERT, NY-ESO-1, MAGE-A1, or WT1 cancer antigens to treat or prevent prostate cancer. Other combinations of cancer antigens may also be used to treat or prevent prostate cancer.

Blood cancer (eg, leukemia, lymphoma, myeloma).

The vaccine may 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 FIG. 51). The vaccine may further combine one or more PRAME, WT-1, hTERT cancer antigens with one or more NY-ESO-1 or MAGE-A1 cancer antigens to treat or prevent blood cancers such as leukemia, lymphoma and myeloma. Other combinations of cancer antigens may also be used to treat or prevent blood cancers such as leukemia, lymphoma and myeloma.

7. Method of vaccination.

Provided is a method of treating or preventing cancer using pharmaceutical compositions to provide genetic constructs and proteins of one or more cancer antigens described above that contain epitopes making them particularly effective immunogens against which an immune response to one or more cancer antigens can be induced. A vaccine administration method, or vaccination, may be provided to induce a therapeutic and/or prophylactic immune response. The vaccination process can induce an immune response in a mammal to one or more of the cancer antigens described herein. The vaccine may be administered to an individual to modulate the activity of the mammal's immune system and enhance the immune response. Vaccine administration may involve the transfection of one or more of the cancer antigens described herein into a nucleic acid molecule that is expressed in the cell and thereby delivered to the cell surface, whereupon the immune system recognizes and induces cellular, humoral, or cellular humoral response. Vaccine administration can be used to induce or cause an immune response in mammals to one or more cancer antigens described herein by administering the vaccine described herein to the mammals.

Upon administration of the vaccine to a mammal and subsequently the vector into cells of the mammal, the transfected cells will express and secrete one or more of the cancer antigens described herein. These secreted proteins, or synthetic antigens, will be recognized as foreign by the immune system, which will mount an immune response that may include: antibodies to one or more cancer antigens and a T cell response specifically to one or more cancer antigens. In some examples, a mammal vaccinated with the vaccines described herein will have a primed immune system and, when stimulated by one or more of the cancer antigens described herein, the primed immune system will enable rapid clearance of additional cancer antigens described herein, either by humoral, cellular, or both cellular and humoral immune responses. The vaccine may be administered to an individual to modulate the activity of the individual's immune system, thereby enhancing the immune response.

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

The dose of the vaccine can range from 1 μg to 10 mg of the active component/kg body weight

- 77 045958 la/time and can range from 20 mcg to 10 mg of component/kg body weight/time. The vaccine can be administered 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 vaccine doses for effective treatment may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more doses.

A method of creating an immune response using a vaccine.

The vaccine can be used to generate an immune response in a mammal, including a therapeutic or prophylactic immune response. The immune response may generate antibodies and/or killer T cells that are directed to one or more of the cancer antigens described herein. Such antibodies and T cells can be isolated.

Some embodiments of the invention provide methods for generating immune responses to one or more cancer antigens described herein, which include administering a vaccine to an individual. Some embodiments of the invention provide methods for prophylactically vaccinating an individual against a cancer or tumor expressing one or more cancer antigens described above, which includes administering the vaccine. Some embodiments of the invention provide methods for therapeutically vaccinating an individual suffering from a cancer or tumor expressing one or more cancer antigens, which includes administering the vaccine. Diagnosis of a cancer or tumor expressing one or more of the cancer antigens described herein may be routinely performed prior to administration of the vaccine.

A method of treating cancer using a vaccine.

The vaccine may be used to create or induce an immune response in a mammal that responds to or is directed at a cancer or tumor (eg, melanoma, head and neck, cervical, liver, prostate, blood cancer, squamous cell carcinoma of the esophagus, cancer gastrointestinal tract) of a mammal or subject in need thereof. The immune response generated can prevent cancer or tumor development.

The elicited immune response can prevent and/or reduce metastasis of cancer or tumor cells. Accordingly, the vaccine can be used in a method that treats and/or prevents cancer or tumors in a mammal or subject to which the vaccine is administered. Depending on the antigen used in the vaccine, the growth based cancer or tumor being treated may be any type of cancer, such as, but not limited to, melanoma, blood cancers (eg, leukemia, lymphoma, myeloma), lung carcinomas, squamous cell carcinomas esophagus, bladder cancer, colorectal cancer, esophageal cancer, gastrointestinal cancer, hepatocarcinoma, head and neck cancer, brain cancer, anal cancer, non-small cell lung carcinoma, pancreatic cancer, synovial carcinoma, prostate cancer, testicular cancer, liver cancer, cervical cancer, recurrent respiratory papillomatosis, skin cancer and stomach cancer.

In some embodiments, the vaccine may mediate the clearance or prevent the development of tumor cells by inducing (1) humoral immunity through B cell responses to produce antibodies that block the formation of monocyte chemoattractant protein-1 (MCP-1), thereby inhibiting suppressor cells myeloid-derived cells (MDSC) and suppressing tumor development; (2) increasing cytotoxic T lymphocytes such as CD8 ⁺ (CTL) to attack and kill tumor cells; (3) increasing T helper cell responses; (4) and increasing inflammatory responses through IFN-γ and TFN-α, or preferably all of the above.

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

In some embodiments, the administered vaccine may increase tumor-free survival, reduce tumor formation, increase tumor survival, or a combination thereof in a subject. The administered vaccine can increase tumor-free survival 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% in the subject. The administered vaccine can reduce tumor formation 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 the subject after immunization.

The administered vaccine can prevent and block the increase of monocyte chemoattractant protein 1 (MCP-1), a cytokine secreted by myeloid-derived suppressor cells, in a subject. In some embodiments, the administered vaccine may prevent and block the growth of MCP-1 in cancer or tumor tissue in a subject, thereby reducing the vascularization of the cancer or tumor tissue in the subject.

The administered vaccine can increase tumor survival 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%,

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64%, 65%, 66%, 67%, 68%, 69% and 70% in the subject. In some embodiments, the vaccine may be administered peripherally (as described in more detail below) to establish an antigen-specific immune response directed at cancer or tumor cells or tissues to clear or eliminate cancer or tumor expressing one or more cancer antigens. without harming or causing illness or death in the subject receiving the vaccine.

The administered vaccine may enhance the cellular immune response in a subject by up to 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 4500x, about 100x to about 6000x, about 150x to about 6000x, about 200x to about 6000x, about 250x to about 6000x or about 300x multiples to about 6000 times the size. In some embodiments, the administered vaccine can increase the cellular immune response in a subject by up to about 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850 , 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 280 0, 2900, 3000, 3100, 3200 , 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 5100, 5200, 5 300, 5400, 5500, 5600, 5700 , 5800, 5900 or 6000 times.

The administered vaccine may increase interferon gamma (IFN-γ) levels in a subject by up to about 50-fold to about 6000-fold, about 50-fold to about 5500-fold, about 50-fold to about 5000-fold, from about 50x to about 4500x, about 100x to about 6000x, about 150x to about 6000x, about 200x to about 6000x, about 250x to about 6000x multiple or from about 300 times to about 6000 times the size. In some embodiments, the administered vaccine may increase IFN-γ levels in a subject by up to about 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800. 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 5100, 520 0, 5300, 5400, 5500, 5600, 5700, 5800, 5900 or 6000 times.

The vaccine dose may range from 1 μg to 10 mg of active ingredient/kg body weight/time and may range from 20 μg to 10 mg of ingredient/kg body weight/time. The vaccine can be administered 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 vaccine doses for effective treatment may be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

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

The present invention also relates to a method of enhancing the immune response in a mammal using the vaccine described above. The vaccine described above may contain a cancer antigen as well as a PD1 antibody and/or a PDL1 antibody described above. The combination may be a single composition or may be separate and administered sequentially (either the cancer antigen first and then the PD1 antibody or PDL1 antibody, or the PD1 antibody or PDL1 antibody first and then the cancer antigen). In some embodiments, the cancer antigen may be administered to the subject in 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 o'clock, 9 o'clock, 10 o'clock, 11 o'clock, 12 o'clock, 13 o'clock, 14 o'clock, 15 o'clock, 16 o'clock, 17 o'clock, 18 o'clock, 19 o'clock, 20 o'clock, 21 o'clock, 22 o'clock, 23 o'clock, 24 o'clock , 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 before administering the PD-1 antibody or PD-L1 antibody to the subject. In other embodiments, the PD-1 antibody or PD-L1 antibody may be administered to the subject in 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 o'clock, 7 o'clock, 8 o'clock, 9 o'clock, 10 o'clock, 11 o'clock, 12 o'clock, 13 o'clock, 14 o'clock, 15 o'clock, 16 o'clock, 17 o'clock, 18 o'clock, 19 o'clock, 20 o'clock, 21 o'clock, 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 before administering the cancer antigen to the subject.

The combination of cancer antigen and PD1 antibody or PDL1 antibody induces the immune system more effectively than a vaccine containing only cancer antigen. This more effective immune response provides increased effectiveness in the treatment and/or prevention of certain cancers. Depending on the antigen used in the vaccine in combination with PDL1 antibody or antibody

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PD1, growth-based cancer or tumor being treated may be any type of cancer, such as, but not limited to, melanoma, blood cancers (eg, leukemia, lymphoma, myeloma), lung carcinomas, squamous cell carcinomas of the esophagus, bladder cancer, cancer colorectal cancer, esophageal cancer, gastrointestinal cancer, hepatocarcinoma, head and neck cancer, brain cancer, anal cancer, non-small cell lung carcinoma, pancreatic cancer, synovial carcinoma, prostate cancer, testicular cancer, liver cancer, cancer cervix, recurrent respiratory papillomatosis, skin cancer and stomach cancer.

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

In other alternative embodiments, the immune response in a subject receiving the vaccine may be increased by about 50% to about 1500%, from about 50% to about 1000%, or from about 50% to about 800%. In other embodiments, the immune response in a subject receiving the vaccine may be increased 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 by about 550%, by at least about 600%, by at least about 650%, by at least about 700%, by at least about 750%, by at least about 800%, by 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 dose may range from 1 μg to 10 mg of active ingredient/kg body weight/time and may range from 20 μg to 10 mg of ingredient/kg body weight/time. The vaccine can be administered 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 vaccine doses for effective treatment may be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

(2) Melanoma.

The vaccine can be used to create or induce in a mammal an immune response that is responsive to or directed to melanoma in the mammal or subject in need thereof. The immune response generated may prevent the development of melanoma. The induced immune response may reduce the development of melanoma. The elicited immune response may prevent and/or reduce metastasis of cancerous or melanoma tumor cells. Accordingly, the vaccine can be used in a method that treats and/or prevents melanoma in a mammal or subject to which the vaccine is administered.

In some embodiments, the administered vaccine may mediate the clearance or prevent the development of melanoma cells by inducing (1) humoral immunity through B cell responses to produce antibodies that block the formation of monocyte chemoattractant protein-1 (MCP-1), thereby slowing suppressive myeloid-derived cells (MDSC) and suppressing the development of melanoma; (2) expansion of cytotoxic T lymphocytes such as CD8' (CTL) to attack and kill melanoma cells; (3) increasing T helper cell responses; (4) and increasing inflammatory responses through IFN-γ and TFN-α, or preferably all of the above.

In some embodiments, the administered vaccine may increase melanoma-free survival, reduce melanoma formation, increase melanoma survival, or a combination thereof in a subject. The administered vaccine can increase melanoma-free survival by 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44% and 45% in the subject. The administered vaccine can reduce melanoma formation 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%,

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54%, 55%, 56%, 57%, 58%, 59% and 60% in the subject after immunization. The administered vaccine can prevent and block the increase of monocyte chemoattractant protein 1 (MCP-1), a cytokine secreted by myeloid-derived suppressor cells, in a subject. In some embodiments, the administered vaccine may prevent and block the growth of MCP-1 in melanoma tissue in a subject, thereby reducing the vascularization of the melanoma tissue in the subject. The administered vaccine can increase melanoma 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% subject.

8. Methods of administration.

The vaccine or pharmaceutical composition can be administered by various routes, including orally, parenterally, sublingually, intradermally, rectally, transmucosally, topically, by inhalation, by buccal administration, intrapleurally, intravenously, intraarterially, intraperitoneally, subcutaneously, intramuscularly, intranasally, intrathecally, and intra-articularly or through a combination of both. For veterinary use, the composition may be administered in a properly selected acceptable dosage form in accordance with normal veterinary practice. The veterinarian can easily determine the dosage schedule and route of administration that is most appropriate for a particular animal. The vaccine can be administered using traditional syringes, needleless injectors, ballistic transfection (gene guns) or through other physical methods such as electroporation (EP), hydrodynamic method or ultrasound.

The vaccine vector can be administered to a mammal using several well-known technologies, including DNA injection (also called DNA vaccination) with and without in vivo electroporation, liposome-mediated, nanoparticle-accelerated, recombinant vectors such as recombinant adenovirus associated with recombinant adenovirus virus and recombinant vaccinia virus. One or more cancer vaccine antigens can be administered via DNA injection as well as in vivo electroporation.

Electroporation.

The vaccine or pharmaceutical composition can be administered by electroporation. Vaccine administration by electroporation can be achieved using electroporation devices that can be configured to deliver to the desired mammalian tissue a pulse of energy sufficient to cause the formation of reversible pores in cell membranes, and preferably the pulse of energy is a constant current value similar to the user-specified input current. The electroporation device may include an electroporation component and an electrode assembly or a handle assembly. The electroporation component may include and combine one or more different electroporation device elements, including: a controller, a current signal generator, an impedance tester, a signal recorder, an input element, a status reporting element, a communication port, a memory component, a power supply and a switch. Electroporation can be performed using an in vivo electroporation device, such as the CELLECTRA® EP system (Inovio Pharmaceuticals, Inc., Blue Bell, PA) or the Elgen electroporator (Inovio Pharmaceuticals, Inc.) to facilitate transfection of cells with the plasmid.

Examples of electroporation devices and methods of electroporation that can facilitate the administration of DNA vaccines of the present invention include those described in US Pat. No. 7,245,963 to Draghia-Akli, et al., US Patent Publication No. 2005/0052630 to Smith et al. , the contents of which are incorporated herein by reference in their entirety. Other electroporation devices and methods that may be used to facilitate the administration of a DNA vaccine include the devices and methods provided in co-pending U.S. Patent Application Serial No. 11/874072, filed October 17, 2007 year for which priority is claimed under 35 USC § 119(e) based on US Provisional Applications Serial No. 60/852149, filed October 17, 2006, and No. 60/978982, filed October 10, 2007 years, all of which are included in this document in full.

US Patent No. 7245963 Draghia-Akli, et al. describes modular electrode systems and their use to facilitate the introduction of a biomolecule into the cells of a selected tissue in the body or plant. Modular electrode systems may contain a number of needle electrodes; hypodermic needle; an electrical connection element that provides a conductive connection from the constant current programmable pulse controller to the array of needle electrodes; and power supply. The operator can grip a series of needle electrodes that are mounted on a support structure and insert them firmly into the selected tissue in the body or plant. The biomolecules are then injected through a hypodermic needle into the selected tissue. The programmable constant current pulse controller is activated and a constant current electrical pulse is applied to a series of needle electrodes. Applied electrical impulse with DC current

- 81 045958 of this magnitude facilitates the introduction of a biomolecule into a cell between a series of electrodes. The entire contents of US Pat. No. 7,245,963 are incorporated herein by reference in their entirety.

US Patent Publication No. 2005/0052630 by Smith, et al. describes an electroporation device that can be used to effectively facilitate the introduction of a biomolecule into the cells of a selected tissue in the body or plant. The electroporation device contains an electrokinetic device (EKD device), the operation of which is determined by software or firmware. The EKD device produces a series of programmable constant current pulse trains between electrodes in an array based on user control and pulse parameter input, and provides the ability to store and retrieve live signal data. The electroporation device also includes a replaceable electrode disk with an array of needle electrodes, a central injection channel for the injection needle, and a removable guide disk. The entire contents of US Patent Publication No. 2005/0052630 are incorporated herein by reference in their entirety.

The electrode arrays and methods described in US Patent No. 7245963 and US Patent Publication No. 2005/0052630 can be adapted to penetrate deeply 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 (into the neurological system of the preferred biomolecule) is also fully inserted into the target organ and injected perpendicular to the target tissue, in an area that is previously delineated by the electrodes. The electrodes described in US Patent No. 7245963 and US Patent Publication No. 2005/005263 are preferably 20 mm long and 21 gauge.

It is further contemplated that in certain embodiments of the invention involving electroporation devices and their use, the electroporation devices are those described in the following patents: US Pat. No. 5,273,525, issued December 28, 1993, US Pat. No. 6,110,161, issued August 29, 2000, No. 6261281, issued July 17, 2001, and No. 6958060, issued October 25, 2005, and US Patent No. 6,939862, issued September 6, 2005. Also discussed herein are patents covering the subject matter of U.S. Patent No. 6,697,669, issued February 24, 2004, which relates to the introduction of DNA using any of a variety of devices, and U.S. Patent No. 7,328,064, issued February 5, 2008. , which refers to the DNA injection method. The above patents are incorporated by reference in their entirety.

9. Method of obtaining the vaccine.

Presented herein are methods for producing DNA plasmids that contain the vaccines discussed herein. The DNA plasmids, after a final step of subcloning into mammalian expression plasmids, can be used to inoculate a cell culture in a large fermentation tank using methods known in the art.

DNA plasmids for use with the EP devices of the present invention can be prepared or manufactured using a combination of known devices and technologies, but are preferably prepared using the optimized plasmid production technology described in US Patent Application Published No. 20090004716, which was filed May 23, 2007 of the year. In some examples, the DNA plasmids used in these studies can be prepared in concentrations that are greater than or equal to 10 mg/ml. The production technologies also include or combine various devices and operating standards generally known to those skilled in the art in addition to those described in US Patent Application Serial No. 60/939,792, including those described in the patent , subject to license, US Patent No. 7238522, issued July 3, 2007. The above application and patent, US Patent Application Serial No. 60/939792 and US Patent No. 72 3 8 522, respectively, are incorporated herein in their entirety.

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

10. Examples.

Example 1.

Construction of pTyr.

As illustrated in FIG. 1A and 9, tyrosinase (Tyr) can be found in various organisms. Accordingly, a consensus Tyr was obtained by aligning sequences corresponding to Tyr from the organisms illustrated in FIG. 1A, and selecting the most common amino acid and/or nucleotide for the consensus Tyr. The corresponding Tyr sequences for each organism were obtained from GenBank (NCBI). Thus, the consensus Tyr reflected conserved Tyr sequence elements across species.

The nucleotide sequence encoding the consensus Tyr was adapted to include an IgE leader sequence. Specifically, the IgE leader sequence was fused in frame upstream of the Tyr consensus nucleotide sequence (Fig. 1B). The resulting sequence was then inserted into the expression vector pVax1 to obtain the thyro construct

- 82 045958 zinase or plasmid (pTyr) such that the Kozak sequence precedes the nucleotide sequence encoding the IgE leader sequence and the consensus Tyr.

The insertion of the Tyr consensus nucleotide sequence into pVax1 was confirmed by restriction analysis. As illustrated in FIG. 1C, the Tyr consensus nucleotide sequence was separated from the pVax1 plasmid on an agarose DNA gel (ie, lane designated BamH1/Xho1), thereby confirming that the pVax1 vector contained the Tyr consensus nucleotide sequence.

Consensus Tyr expression was confirmed by transfecting HeLa cells with pTyr. Western blotting with anti-human Tyr antibody confirmed the expression of the Tyr consensus protein in HeLa cells (Fig. 1D). GPF staining further showed the expression of the Tyr consensus protein in transfected HeLa cells (Fig. 1E). In both Western blot and staining experiments.

Example 2.

pTyr vaccination with induced cell-mediated immune response.

The pTyr described above was used to vaccinate mice to assess whether a cellular immune response was induced by pTyr. C57/B6 mice were immunized using the immunization strategy illustrated in FIG. 2A. Some mice were immunized with pVax1, while other mice were immunized with pTyr. Mice immunized with pTyr were further divided into the following groups: (1) pTyr dosage 5 μg; (2) pTyr dosage 20 μg; (3) pTyr dosage 30 μg; and (4) pTyr dosage 60 μg.

On day 35 of the immunization strategy, splenocytes were isolated from C57B/6 mice and assessed for interferon-γ (IFN-γ) induction using an IFN-γ ELISPOT assay. As illustrated in FIG. 2B, 20 μg pTyr induced the highest levels of IFN-γ.

Additionally, the cellular immune response to pTyr was assessed in immunized Balb/c and C57B/6 mice. Mice were immunized with either pVax1 or pTyr. Splenocytes were isolated two weeks after the third immunization and stimulated with the Tyr consensus peptide. After stimulation, the number of splenocytes secreting IFN-γ was calculated as the average number of spots in triplicate stimulated wells. This analysis showed that C57/B6 mice were suitable for pTyr vaccination (data not shown).

Example 3.

pTyr vaccination with increased cytokines IFN-γ and TNF-α.

Cytokine production was studied in mice immunized with pTyr and pVax1. Mice were immunized using the strategy illustrated in FIG. 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 using FACS. In particular, the analysis examined CD8' and CD4' T cells. FACS allowed the identification of T cells positive for the cytokines IL-2, TNF-α, and IFN-γ. Of CD44 hi cells, a significant percentage of CD8+ T cells produced IFN-γ in mice immunized with pTyr compared with mice immunized with pVax1 (Fig. 3).

Example 4.

Tyr specific antibodies are produced in response to pTyr vaccination.

The humoral immune response was studied in mice vaccinated with pTyr. Specifically, C57BI/6 mice (n=4) were immunized with either pTyr or pVax1 three times at 2-week intervals. Each immunization consisted of a 20 μg/intramuscular injection followed by electroporation of MID-EP. After the third immunization (ie, day 35), serum was collected from mice and antibody titers were measured by ELISA using all IgG-specific HRP-labeled secondary antibodies. The serum was diluted as shown in Fig. 4A. As illustrated in FIG. 4A, Tyr-specific antibodies were produced by mice immunized with pTyr. Mice immunized with pTyr are represented by the filled circle in FIG. 4A, while mice immunized with pVax1 are represented by an open triangle in FIG. 4A.

In addition, sera from immunized mice were serially diluted at 1:20, 1:40, 1:80, 1:160, 1:320, and 1:640. Each serum dilution was added in triplicate to separate wells (50 μl/well) containing Tyr peptides. A peak increase in Tyr-specific titer, compared with non-immune serum, was found in all immunized groups of mice when retested on days 90 and 120. Typical results from three independent experiments at each serial dilution step are illustrated in FIG. 4B. These data further suggested that pTyr immunization induced the production of Tyr-specific antibodies in pTyr-immunized mice.

Example 5.

pTyr Vaccinated Mice Have Increased Survival When Injected with a pTyr Tumor was further analyzed to determine whether pTyr vaccination could provide protection against tumors. Specifically, C57BI/6 mice (10 per group) were immunized with either pTyr or pVax1 with 2

- 83 045958 in weekly intervals. Each immunization consisted of a 20 μg/intramuscular injection followed by electroporation of MID-EP. One week after the third immunization (ie, day 35), immunized mice were challenged intradermally with B16 melanoma until the tumor diameter exceeded 200 mm ² .

Subsequently, tumor-free survival and tumor volume were assessed in the immunized groups of mice. As illustrated in FIG. 5A (Kaplan-Meier survival curve), mice immunized with pTyr had improved tumor-free survival (i.e., 40% at day 40 and after tumor challenge) compared with mice vaccinated with pVax1 (p=0.05), all of them who died on day 20 after tumor injection. Mice immunized with pTyr also had reduced tumor volume (i.e., about 50%) compared with mice immunized with pVax1 (Fig. 5B). As in fig. 5A and in FIG. 5B, mice immunized with pVax1 are represented by filled squares, while mice immunized with pTyr are represented by filled circles. Accordingly, these data showed that pTyr vaccination provided protection against melanoma, namely increased tumor-free survival and reduced tumor volume.

Example 6.

The MDSC population is reduced in tumors from mice vaccinated with pTyr.

In pTyr-immunized mice and non-immunized mice, MDSC populations were examined to examine whether pTyr vaccination alters MDSC levels in tumors from the respective groups of mice. In particular, the percentage of Gr-1+ and CD11b+ cells in immunized and non-immunized mice was studied.

As illustrated in FIG. 6 and 7, MDSC levels were significantly reduced in tumors from pTyr-immunized mice compared with non-immunized mice (p=0.0004). The percentage of MDSC population in non-immunized mice was 40.00±4.826. The percentage of MDSC population in mice immunized with pTyr was 5.103 ± 0.7718. Accordingly, these data showed that immunization with pTyr reduced the MDSC population in tumors of pTyr-vaccinated mice.

Example 7.

pTyr vaccination reduced MCP-1 levels.

MDSCs can secrete the cytokine MCP-1, which stimulates angiogenesis or vascularization through endothelial cell migration. Given the above-described effects of pTyr vaccination on MDSC levels in tumors, MCP-1 levels were examined after pTyr vaccination.

As illustrated in FIG. 8A, MDSCs in B16 melanoma can secrete MCP-1. Therefore, pTyr-immunized mice and pVax1-immunized mice were challenged with B16 melanoma to investigate whether pTyr immunization alters MCP-1 levels. Intact mice were included as an additional control group. Following infection, MDSCs were isolated directly from tumor tissue and MCP-1 cytokine levels were analyzed by ELISA. The experiment was performed in triplicate and repeated twice.

As illustrated in FIG. 8B, MDSC in B16 melanoma or tumor tissue significantly secreted MCP-1 (see pVax1-immunized mice). However, there was no significant increase in MCP-1 levels in mice immunized with pTyr. Rather, MCP-1 levels in mice immunized with pTyr were approximately 3-fold lower than in mice immunized with pVax1. Accordingly, these data showed that pTyr vaccination reduced the level of MCP-1 secretion by MDSCs in the tumor of pTyr-immunized mice.

Example 8.

Construction of pPRAME.

A consensus sequence for PRAME was generated and the nucleotide sequence encoding the PRAME consensus 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 known herein as pPRAME) (see Fig. 10A).

To confirm that pPRAME resulted in expression of the PRAME consensus antigen, pVAX and pPRAME were transfected into RD cells and 293T cells. DAPI was used to stain nuclei, and the PRAME consensus antigen was also fluorescently stained. This staining, along with a fusion of the DAPI staining and the PRAME consensus antigen, is shown in FIG. 10V. These stainings demonstrated that the PRAME consensus antigen was expressed from pPRAME, and the PRAME consensus antigen was not detected in cells transfected with pVAX (ie, negative control).

In addition, Western blot analysis of lysates from transfected cells was used to confirm expression of the PRAME consensus antigen in transfected cells (Fig. 10C). Untransfected cells and cells transfected with pVAX were used as negative controls (see lanes labeled control and pVAX, respectively, in Fig. 10C). In fig. 10C beta-actin detection was used as a loading control. Thus, staining of transfected cells and Western blotting of lysates from transfected cells show

- 84 045958 whether the pPRAME vector ensured the expression of the PRAME consensus antigen in cells.

Example 9.

Interferon gamma response to pPRAME vaccination.

The pPRAME described above was used to vaccinate mice to assess whether a cellular immune response was induced by pPRAME. C57BL/6 mice were divided into groups. The first group was intact 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.

Following immunization, splenocytes were isolated from C57BL/6 mice and assessed for interferon-gamma (IFN-γ) induction by IFN-γ ELISPOT assay. As illustrated in FIG. 11A and 11B, each dose of pPRAME induced IFN-γ production or secretion in contrast to naïve negative control mice. Specifically, in vaccinated mice, compared with unvaccinated mice, IFN-γ levels were increased to about 3000-fold to about 4500-fold.

Accordingly, these data showed that vaccination with pPRAME, which encodes the PRAME consensus antigen, induced a cellular immune response, as evidenced by increased levels of IFN-γ, compared with no vaccination.

Example 10.

Construction of pNY-ESO-1.

A consensus sequence for NY-ESO-1 was generated, and the nucleotide sequence encoding the NY-ESO-1 consensus 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 yield pGX1409 (also known herein as pNY-ESO-1) (see FIG. 12A).

To confirm that pNY-ESO-1 resulted in expression of the NY-ESO-1 consensus antigen, cells were transfected with pVAX and pNY-ESO-1. DAPI was used to stain nuclei, and the consensus antigen NY-ESO-1 was also fluorescently stained. This staining, along with the fusion staining of DAPI and the NY-ESO-1 consensus antigen, is illustrated in FIG. 12V. These stainings demonstrated that the NY-ESO-1 consensus antigen was expressed from pNY-ESO-1, and the NY-ESO-1 consensus antigen was not detected in cells transfected with pVAX (ie, negative control).

In addition, Western blot analysis of lysates from transfected 293T and RD cells was used to confirm the expression of the NY-ESO-1 consensus antigen in the transfected cells (Fig. 12C). Untransfected cells and cells transfected with pVAX were used as negative controls (see lanes labeled control and pVAX, respectively, in Fig. 12C). In fig. 12C alpha-actin detection was used as a loading control. Thus, staining of transfected cells and Western blotting of lysates from transfected cells showed that the pNY-ESO-1 vector provided expression of the NYESO-1 consensus antigen in the cells.

Example 11.

Interferon gamma response to pNY-ESO-1 vaccination.

The pNY-ESO-1 described above was used to vaccinate mice to assess whether a cellular immune response was induced by pNY-ESO-1. C57BL/6 mice were divided into groups. The first group was intact and did not receive pNY-ESO-1. The second and third groups of mice received 25 μg and 50 μg of pNY-ESO-1, respectively.

Following immunization, splenocytes were isolated from C57BL/6 mice and assessed for interferon-gamma (IFN-γ) induction by IFN-γ ELISPOT assay. As illustrated in FIG. 13, each dose of pPRAME induced IFN-γ production or secretion in contrast to naïve negative control mice. Specifically, in vaccinated mice, compared with unvaccinated mice, IFN-γ levels were increased to about 700-fold to about 1100-fold.

Accordingly, these data showed that vaccination with pNY-ESO-1, which encodes the NY-ESO-1 consensus antigen, induced a cellular immune response, as evidenced by increased levels of IFN-γ, compared with no vaccination.

Example 12.

Interferon gamma response to pNY-ESO-2 vaccination.

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

This pNY-ESO-2 was used to vaccinate mice to assess whether a cellular immune response was induced by pNYESO-2. C57BL/6 mice were divided into groups. The first group was intact and did not receive pNY-ESO-2. The second and third groups of mice received 25 μg and 50 μg of pNY-ESO-2, respectively.

Following immunization, splenocytes were isolated from C57BL/6 mice and assessed for gamma induction.

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Claims (18)

interferon (IFN-γ) using the IFN-γ ELISPOT assay. As illustrated in FIG. 14, each dose of pNY-ESO-2 induced the production or secretion of IFN-γ in contrast to naïve negative control mice. Specifically, in vaccinated mice, compared with unvaccinated mice, IFN-γ levels were increased to about 400-fold to about 500-fold. Accordingly, these data showed that vaccination with pNY-ESO-2, which encodes the NY-ESO-2 consensus antigen, induced a cellular immune response, as evidenced by increased levels of IFN-γ, compared with no vaccination. It is understood that the foregoing detailed description and the accompanying examples are illustrative only and are not to be construed as limiting the scope of the present invention, which is defined 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, including without limitation those relating to the chemical structures, substituents, derivatives, intermediates, compositions, formulations or methods of use of the present invention, may be made without departing from the spirit and scope thereof. CLAIM 1. A nucleic acid molecule comprising (a) a growth hormone releasing hormone (GHRH) nucleotide sequence (SEQ ID NO: 10); (b) a nucleotide sequence encoding an amino acid sequence that is 95% or more identical to the amino acid sequence of growth hormone releasing hormone (GHRH) (SEQ ID NO: 10); or (c) a combination of (a) and (b). 2. The nucleic acid molecule of claim 1, wherein the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 9, a nucleotide sequence that is 95% or more identical to SEQ ID NO: 9, or a combination thereof. 3. A nucleic acid molecule according to any one of claims 1-2, which is a plasmid. 4. A vaccine for the treatment of a malignant neoplasm in a patient, where the vaccine contains a nucleic acid molecule according to any one of claims 1-3. 5. The vaccine according to claim 4, further containing a nucleic acid molecule encoding one or more antigens selected from the group consisting of: PSA, PSMA, STEAP, PSCA, MAGE A1, gp100, viral antigen and combinations thereof. 6. The vaccine according to claim 5, where the viral antigen is an antigen of hepatitis B virus (HBV), hepatitis C virus (HCV) or human papillomavirus (HPV). 7. The vaccine according to claim 6, wherein the HBV antigen is HBV core antigen or HBV surface antigen, or a combination thereof. 8. The vaccine according to claim 6, wherein the HCV antigen is HCV NS34A antigen, HCV NS5A antigen, HCV NS5B antigen, HCV NS4B antigen, or a combination thereof. 9. The vaccine according to claim 6, where the HPV antigen is E6 type 6 HPV, E7 type 6 HPV, E6 type 11 HPV, E7 type 11 HPV, E6 type 16 HPV, E7 type 16 HPV, E6 type 18 HPV, E7 antigen type 18 or a combination thereof. 10. The vaccine according to any one of claims 4-5, further comprising an immune checkpoint inhibitor selected from the group consisting of: anti-PD-1 antibody, anti-PD-L1 antibody and combinations thereof. 11. The vaccine according to any one of claims 4-10, additionally containing an adjuvant. 12. The vaccine according to claim 11, where the adjuvant is IL-12, IL-15, IL-28 or RANTES. 13. Use of a vaccine according to any one of claims 4-12 in a method for treating a malignant neoplasm in a patient. 14. Use of a vaccine according to any one of claims 4 to 13, where the vaccine is intended to be administered by electroporation. 15. Use of a vaccine according to any one of claims 4 to 14, wherein the vaccine is to be administered together with an immune checkpoint inhibitor. 16. Use according to claim 15, wherein the vaccine and the immune checkpoint inhibitor are intended to be administered to a patient in one formulation, or wherein the vaccine and the immune checkpoint inhibitor are intended to be administered to a patient separately. 17. Use according to claim 13, wherein the malignancy is selected from the group consisting of: melanoma, head and neck cancer, prostate cancer, liver cancer, cervical cancer, recurrent respiratory papillomatosis (RRP), anal cancer, hematologic malignancy, and their combinations. 18. An amino acid molecule comprising one or more amino acid sequences selected from the group consisting of: SEQ ID NO: 10, an amino acid sequence that -