EA043982B1 - IMMUNOGENIC COMPOSITIONS OF TERT AND METHODS OF TREATMENT USING THEIR - Google Patents

Description

Cross reference to related applications

This patent application claims priority to U.S. Provisional Application No. 62/402695, filed September 30, 2016, and U.S. Provisional Application No. 62/468124, filed March 7, 2017, both of which are incorporated herein by reference in their entirety.

Field of technology

Disclosed herein are compositions and methods for treating cancer, and in particular, immunogenic compositions for treating and providing protection against tumor growth.

State of the art

Cancer is a leading cause of death worldwide, and in the United States it is the second leading cause of death, accounting for nearly 1 in every 4 deaths. Cancer arises from a single cell that has changed from a normal cell to a tumor cell. This transformation is often a multistep process, progressing from precancerous lesions to malignant tumors. Several factors contribute to this progression, including aging, genetic influences, 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 (eg , 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), behavioral changes (eg, smoking, diet, and level of physical activity), and vaccination against viruses (eg, hepatitis B virus, human papillomavirus). Treatment may include chemotherapy, radiation therapy, and surgical removal of the tumor or cancerous tissue. Although numerous methods of prevention and treatment are available, such methods often have limited success in effectively preventing and/or treating cancer.

Accordingly, there is a need to identify and develop compositions and methods for the prevention and/or treatment of cancer to facilitate the 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.

Brief description of the invention

In one embodiment, the invention provides an immunogenic composition comprising a nucleic acid molecule encoding a TERT consensus antigen. In one embodiment, the TERT consensus antigen comprises an amino acid sequence selected from the group consisting of: a) an amino acid sequence selected from the group consisting of SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54 and SEQ ID NO: 56, b) an amino acid sequence that is 95% or more identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54 and SEQ ID NO: 56, and c) a fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54 and SEQ ID NO: 56, wherein the fragment contains at least 95% of the full-length amino acid sequence.

In one embodiment of the invention, the immunogenic composition further comprises one or more nucleotide sequences encoding one or more amino acid sequences selected from the group consisting of: a) an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO : 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20 , SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70; b) an amino acid sequence that is 95% or more identical to an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70 and c) a fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, wherein the fragment contains at least 95% of the full-length amino acid sequence.

In one embodiment of the invention, the immunogenic composition further comprises a nucleotide sequence encoding one or more antigens selected from the group consisting of: MAGE A1, gp100, viral antigen, and combinations thereof. In one embodiment, the viral antigen is a hepatitis B virus (HBV), hepatitis C virus (HCV) or human papilloma virus (HPV) antigen. In one embodiment, the HBV antigen is HBV core antigen or HBV surface antigen, or a combination thereof. In od

- 1043982 In this embodiment, the HCV antigen is HCV NS34A antigen, HCV NS5A antigen, HCV NS5B antigen, HCV NS4B antigen, or a combination thereof. In one embodiment, the HPV antigen is HPV type 6 E6 antigen, HPV type 6 E7 antigen, HPV type 11 E6 antigen, HPV type 11 E7 antigen, HPV type 16 E6 antigen, HPV type 16 E7 antigen, HPV type 18 E6 antigen , HPV type 18 E7 antigen, or a combination thereof.

In one embodiment of the invention, the immunogenic composition further comprises an immune checkpoint inhibitor.

In one embodiment of the invention, the nucleic acid molecule contains a nucleotide sequence selected from the group consisting of a) a nucleotide sequence selected from the group consisting of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO : 51, SEQ ID NO: 53 and SEQ ID NO: 55, b) a nucleotide sequence that is 95% or more identical to a nucleotide sequence selected from the group consisting of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53 and SEQ ID NO: 55, and c) a fragment of a nucleotide sequence selected from the group consisting of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO : 49, SEQ ID NO: 51, SEQ ID NO: 53 and SEQ ID NO: 55, wherein the fragment contains at least 95% of the full-length nucleotide sequence.

In one embodiment of the invention, the immunogenic composition contains one or more nucleotide sequences selected from the group consisting of: a) a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67 and SEQ ID NO: 69; b) a nucleotide sequence that is 95% or more identical to a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 , SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67 and SEQ ID NO: 69, and c) a fragment of a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67 and SEQ ID NO: 69, wherein the fragment contains at least 95% of the full-length nucleotide sequence.

In one embodiment of the invention, the nucleic acid molecule is a plasmid.

In one embodiment of the invention, the immunogenic composition contains one or more plasmids.

In one embodiment of the invention, the immunogenic composition further comprises an adjuvant. In one embodiment, the adjuvant is IL-12, IL-15, IL-28 or RANTES.

In one embodiment, the invention provides a method for treating or preventing cancer in a subject in need thereof, the method comprising administering to the subject an immunogenic composition comprising a nucleic acid molecule encoding a TERT consensus antigen. In one embodiment of the invention, the method of administration includes the step of electroporation. In one embodiment, the method further comprises administering an immune checkpoint inhibitor to the subject.

In one embodiment of the invention, the immunogenic composition and the immune checkpoint inhibitor are administered to a subject in one composition. In one embodiment of the invention, the immunogenic composition and the immune checkpoint inhibitor are administered separately to a subject.

In one embodiment, the cancer is selected from the group consisting of melanoma, head and neck cancer, prostate cancer, liver cancer, cervical cancer, recurrent respiratory papillomatosis (RRP), anal cancer, blood cancer, ovarian cancer, and combinations thereof .

In one embodiment, the invention provides a nucleic acid molecule comprising one or more nucleotide sequences selected from the group consisting of: a) a nucleotide sequence selected from the group consisting of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53 and SEQ ID NO: 55, b) a nucleotide sequence that is 95% or more identical to a nucleotide sequence selected from the group consisting of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53 and SEQ ID NO: 55, and c) a fragment of a nucleotide sequence selected from the group consisting of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53 and SEQ ID NO: 55, wherein the fragment contains at least 95% of the full-length nucleotide sequence.

In one embodiment of the invention, the nucleic acid molecule further comprises one or more nucleotide sequences selected from the group consisting of: a) nucleotide

- 2 043982 tide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67 and SEQ ID NO: 69; b) a nucleotide sequence that is 95% or more identical to a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 , SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67 and SEQ ID NO: 69, and c) a fragment of a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67 and SEQ ID NO: 69, wherein the fragment contains at least 95% of the full-length nucleotide sequence.

In one embodiment of the invention, the nucleic acid molecule is a plasmid.

In one embodiment, the invention provides an amino acid molecule comprising one or more amino acid sequences selected from the group consisting of: a) an amino acid sequence selected from the group consisting of SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO : 50, SEQ ID NO: 52, SEQ ID NO: 54 and SEQ ID NO: 56, b) an amino acid sequence that is 95% or more identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54 and SEQ ID NO: 56, and c) a fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 46, SEQ ID NO : 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54 and SEQ ID NO: 56, wherein the fragment contains at least 95% of the full-length amino acid sequence.

In one embodiment of the invention, the amino acid molecule further comprises one or more amino acid sequences selected from the group consisting of: a) an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70; b) an amino acid sequence that is 95% or more identical to an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70 and c) a fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, wherein the fragment contains at least 95% of the full-length amino acid sequence.

Brief description of graphic materials

In fig. 1 including FIG. 1A-1B, the pTyr construct is depicted.

In fig. 2, including FIG. 2A-2B depict the results of experiments demonstrating the immunization strategy and induction of cell-mediated immune responses by Tyr DNA vaccination, respectively.

In fig. 3 depicts the results of experiments demonstrating fluorescence-activated cell sorting (FACS) in control and immunized mice.

In fig. 4, including FIG. 4A-4B depict the results of experiments demonstrating the induction of tyrosinase-specific antibodies in immunized mice.

In fig. 5, including FIG. 5A-5B depict the results of experiments showing Kaplan-Meier survival curves and tumor volume curves, respectively, after tumor challenge of control and immunized mice.

In fig. 6, including FIG. 6A-6B depict the results of experiments demonstrating MDSC cell populations in immunized and non-immunized mice.

In fig. 7 depicts the results of experiments showing MDSC staining in mice immunized with pVax1 and pTyr.

In fig. 8, including FIG. 8A-8B show the results of experiments demonstrating MCP-1 secretion by MDSCs.

In fig. 9 depicts the results of experiments demonstrating the phylogenetic relationship of Tyr nucleotide sequences among these organisms.

In fig. 10, including FIG. 1OA-1OC depicts the results of experiments showing (A) a schematic representation of the pPRAME plasmid map (also known herein as pGX1411); (B) Staining of RD and 293T cells for nuclei with DAPI and for consen

- 3 043982 PRAME sustic antigen; and (C) Western blotting for the PRAME consensus antigen in lysates of untransfected cells (control), cells transfected with pVAX (pVAX), and cells transfected with pPRAME (PRAME-pVAX).

In fig. 11, including FIG. 11A-11B show the results of experiments showing graphs depicting the dependence of groups of mice on spot forming units (SFU)/10 ⁶ splenocytes in the case of gamma interferon (IFN-γ).

In fig. 12, including FIG. 12A-12C show the results of experiments showing (A) a schematic representation of the pNY-ESO-1 plasmid map (also referred to herein as pGX1409); (B) staining cells 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).

In fig. 13 is a graph of groups of mice versus spot forming units (SFU)/10 ⁶ splenocytes for gamma interferon (IFN-γ).

In fig. 14 is a graph of groups of mice versus spot forming units (SFU)/10 ⁶ splenocytes for gamma interferon (IFN-γ).

In fig. 15 is a schematic representation of various cancers with some of their associated cancer antigen(s).

In fig. 16 is a schematic representation of the TERT design. A synthetic hTERT consensus was developed for human TERT with 5 mutations to eliminate its function. Native RhTERT encodes rhesus TERT with 2 mutations. Both designs have been optimized. The Table shows the protein homology between optimized TERT and native TERT identified in human, rhesus and mouse.

In fig. 17, including FIG. 17A-17B depict the characterization of murine TERT-specific IFN-γ responses against native Rh peptides. (Fig. 17A) Immunization schedule. Mice (n=8 per group) were immunized at 0, 2 and 4 weeks with native RhTERT and synthetic consensus hTERT via IM/electroporation. (Fig. 17B) Frequency of TERT-specific IFN-γ spot-forming units (SFU) per million splenocytes isolated from vaccinated mice as determined by the IFN-γ ELISpot assay using Rh peptides.

In fig. 18, including FIG. 18A-18C depict the characteristics of TERT-specific rhesus IFN-γ responses. (Fig. 18A) Immunization schedule. Rhesus monkeys (n=5 per group) were immunized at 0, 4, 8, and 12 weeks with native RhTERT or synthetic consensus hTERT, and with rhIL-12 via IM/electroporation. (FIGS. 18B and 18C) Frequency of TERT-specific IFNγ spot-forming units (SFU) per million splenocytes isolated from vaccinated mice as determined by IFN-γ ELISpot assay using native rhesus peptides and synthetic hTERT consensus peptide.

In fig. 19 depicts the results of experiments demonstrating that synthetic consensus hTERT (pGX1434) induces robust T cell responses in mice and slows TC-1 tumor growth. (Left) Mice received 3 immunizations 2 weeks apart with 0, 20, 40, or 60 μg of pGX1434. T cell responses in mice were assessed after 5 weeks, demonstrating that pGX1434 induced a robust T cell response. (right) Mice were implanted with ^25x104 TC-1 cells. Mice then received 4 immunizations 1 week apart with 25 μg of pGX1434. Tumor volume was measured over 32 days and it was determined that mice treated with pGX1434 had reduced tumor volume compared to untreated mice.

In fig. 20 depicts the results of experiments demonstrating the immunogenicity of SynCon hTERT (pGX1434) in mice.

In fig. 21, including FIG. 21A-21B depict the results of experiments demonstrating that SynCon hTERT (pGX1434) and mut-hTERT (pGX1406) provide similar control of TC-1 tumor growth.

In fig. 22, including FIG. 22A-22B depict an experimental design demonstrating the immunogenicity of hTERT in non-human primates (NHP). In fig. 22A depicts a typical immunization schedule. In fig. 22B is a table showing the constructs that were administered to the various groups. pGX1434: hTERT; pGX1406: Mut-hTERT; pGX1447: Mut-rhTERT; pGX1404: WT-1; pGX1108: PSMA; pGX6006:rhIL-12.

In fig. 23, including FIG. 23A-23B depict the results of experiments demonstrating that SynCon hTERT disrupts self-tolerance in NHPs. In fig. 23A depicts the results of experiments demonstrating that responses to native rhesus TERT were detected by PD3 and PD4 in Group 1 animals. FIG. 23B depicts the results of experiments demonstrating that responses to native rhesus TERT were detected by PD3 and PD4 in Group 2 animals.

In fig. 24, including FIG. 24A-24C show the results of experiments demonstrating that the SynCon design allows self-tolerance to be broken. In fig. 24A shows a table where

- 4 043982 demonstrating the percentage of identity of various constructs relative to native sequences. In fig. 24B depicts the results of experiments demonstrating a TERT-specific IFNy response. In fig. 24C depicts the results of experiments demonstrating the TERT-specific IFNy response of Rhesus.

In fig. 25, including FIG. 25A-25B depict the results of experiments demonstrating the immunogenicity of NHP SynCon hTERT. In fig. 25A depicts the results of experiments demonstrating the TERT response induced using a combination of SynCon hTERT, PSMA, WT-1 and IL12. In fig. 25B depicts the results of experiments demonstrating the TERT response induced using a combination of mut-hTERT, PSMA, WT-1 and IL-12.

In fig. 26, including FIG. 26A-26C show the results of experiments demonstrating the immunogenicity of NHP SynCon hTERT. In fig. 26A depicts the results of experiments demonstrating the TERT response induced using a combination of TERT, PSMA, WT-1 and IL-12. In fig. 26B shows the results of experiments demonstrating the PSMA response induced using a combination of TERT, PSMA, WT-1 and IL-12. In fig. 26C depicts the results of experiments demonstrating the WT-1 response induced using a combination of TERT, PSMA, WT-1 and IL12.

In fig. 27, including FIG. 27A-27B depict the results of experiments demonstrating the coverage of multivalent SynCon vaccines. In fig. 27A depicts the immunization schedule used. In fig. 27B depicts the percentage of antigen-specific T cells that were identified after immunization.

In fig. 28, including FIG. 28A-28B depict the results of experiments demonstrating that the multivalent SynCon vaccine reduces tumor burden and increases survival. In fig. 28A depicts the immunization schedule used. In fig. 28B depicts the average tumor volume and % survival of animals immunized with the combination of WT1, PSMA and SynCon mTERT.

Detailed description of the invention

This invention relates to immunogenic compositions containing a synthetic consensus antigen TERT, alone or in combination with at least one additional cancer antigen or immune checkpoint protein inhibitor, and a method of using the compositions to treat diseases or disorders. In one embodiment, the immunogenic composition comprises TERT and at least one additional cancer antigen consensus sequence. Cancer antigen consensus sequences that may be included in an immunogenic composition include, but are not limited to, tyrosinase (Tyr), predominantly expressed in melanoma antigen (PRAME), tyrosinase-dependent protein 1 (Tyrp1), testicular cancer antigen (NY-ESO) -1), hepatitis B virus antigen, prostate-specific antigen (PSA), prostate-specific membrane antigen (PSMA), six-transmembrane epithelial prostate antigen (STEAP), prostate stem cell antigen (PSCA), fibroblast activation protein (FAP), receptor follicle stimulating hormone (FSHR) and Wilms tumor antigen 1 (WT-1). In one embodiment, the immunogenic composition of the invention may provide a combination of cancer antigens to prevent or treat cancer in a subject in need thereof.

One method for constructing a nucleic acid and corresponding encoded amino acid sequence for a recombinant cancer antigen is to introduce mutations that change specific amino acids in the overall amino acid sequence of the native cancer antigen. The introduction of mutations does not change the cancer antigen so much that it cannot be used universally in a mammalian subject, such as a human or dog, but does change the antigen enough that the resulting amino acid sequence breaks tolerance or is considered a foreign antigen in order to induce an immune response. Another method may be to generate a consensus recombinant cancer antigen that has at least 85 to 99% amino acid sequence identity, at least 90 to 98% sequence identity, at least 93 to 98% sequence identity, or at least at least 95% and up to 98% sequence identity relative to the corresponding native cancer antigen. In some cases, the recombinant cancer antigen has amino acid sequence identity that is 95, 96, 97, 98, or 99% relative to the corresponding native cancer antigen. A native cancer antigen is an antigen typically associated with a specific cancer or cancer. Depending on the cancer antigen, the cancer antigen consensus sequence may occur across different mammalian species, or within subtypes of a species, or among 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 nucleic acid/amino acid sequences that vary so widely among species that a consensus sequence cannot be determined. In these cases, a recombinant cancer antigen is generated, which

- 5 043982 breaks tolerance and induces an immune response that has at least 85 to 99% amino acid sequence identity; at least 90% and up to 98% sequence identity; at least 93% and up to 98% sequence identity; or at least 95% and up to 98% sequence identity to the corresponding native cancer antigen. In some cases, the recombinant cancer antigen has amino acid sequence identity that is 95, 96, 97, 98, or 99% relative to the 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 discussed above.

The recombinant cancer antigen can induce an antigen-specific T cell response and/or high titers of antibodies, thereby eliciting or inducing an immune response that targets or responds to the cancer or tumor expressing the antigen. In some embodiments, the induced or evoked immune response may be a cellular, humoral, or both cellular and humoral immune response. 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 evoked immune response may reduce or inhibit one or more immunosuppressive factors that promote the growth of a tumor or cancer expressing an antigen, such as, but not limited to, factors that suppress MHC presentation, factors that increase levels of antigen-specific regulatory T cells (Treg), PD-L1, FasL, cytokines such as IL-10 and TFG-β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by immunosuppressive cells, CTLA-4, PD-1, MDSCs , MCP-1 and immune checkpoint molecule.

The immunogenic composition 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 anti-PD-1 or anti-PDL-1 antibodies prevents PD-1 or PDL-1 from suppressing T cell and/or B cell responses. In general, the development of cancer antigens that are recognized by the immune system helps overcome other forms of immunosuppression by tumor cells, and these immunogenic compositions can be used in combination with suppressive or inhibitory therapies (eg, anti-PD-1 and anti-PDL-1 therapies) to further increasing 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 skilled in the art. In the event of a conflict, this document, including definitions, will control. Exemplary 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 this invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods and examples disclosed herein are illustrative only and should not be construed as limiting. The terminology used herein is intended to describe specific embodiments of the invention only and should not be construed as limiting.

The terms contains, includes, has, has, may, includes, and variations thereof, when used in the context of this document, are intended for use with open-ended transitional phrases, terms or words that do not exclude the possibility of additional actions or structures. Singular forms of nouns include plural forms of nouns unless the context clearly requires otherwise. This disclosure also discusses other embodiments comprising, consisting of, and consisting essentially of the embodiments or elements presented herein, whether explicitly presented or not.

To list numeric ranges, this document explicitly treats each number in between with the same degree of precision. For example, for the range 6-9 the numbers 7 and 8 are included in addition to 6 and 9, and for the range 6.0-7.0 the numbers 6.0, 6.1, 6.2, 6.3, 6.4 are included , 6.5, 6.6, 6.7, 6.8, 6.9 and 7.0 are explicitly included.

The term adjuvant as used herein means any molecule added to the immunogenic compositions described herein to enhance the immunogenicity of the antigens encoded by the nucleic acid molecules and nucleic acid coding 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 and single chain antibodies, diabodies, bispecific antibodies, bifunctional antibodies and derivatives thereof . The antibody may

- 6 043982 be an antibody isolated from a mammalian serum sample, a polyclonal antibody, an affinity-purified antibody, or mixtures thereof that exhibit sufficient binding specificity to the desired epitope or sequence derived therefrom.

The term coding sequence or coding nucleic acid, as used herein, means nucleic acids (RNA or DNA molecules) that contain 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 directing expression in the cells of the individual or mammal to which the nucleic acid is administered.

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

Consensus or consensus sequence, as used in the context of 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. Immunogenic compositions containing proteins that contain consensus sequences and/or nucleic acid molecules that encode such proteins can be used to induce a broad immune response against an antigen.

Electroporation, electropermeabilization or electrokinetic enhancement (EP), used interchangeably herein, refers to the use of a transmembrane electric field pulse to induce microscopic pathways (pores) in a biomembrane; 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.

As used herein, the term fragment, with respect to nucleic acid sequences, means a nucleic acid sequence or portion thereof that encodes a polypeptide capable of eliciting an immune response in a mammal that cross-reacts with an antigen disclosed herein. The fragments may be fragments of DNA selected from at least one of various nucleotide sequences that encode protein fragments presented 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 nucleic acid sequences presented 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, at least 600 nucleotides or more, at least 650 nucleotides or more, at least 700 nucleotides or more, at least 750 nucleotides or more, at least 800 nucleotides or more, at least 850 nucleotides or more, at least 900 nucleotides or more, at least 950 nucleotides or more, or at least 1000 nucleotides or more of at least one of the nucleic acid sequences presented 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 disclosed herein. The fragments may be polypeptide fragments selected from at least one of the various amino acid sequences presented 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 comprise at least 20 amino acids or more, at least 30 amino acids or more, at least 40 amino acids or more, at least 50 amino acids or more, at least 60 amino acids or more, at least 70 amino acids or more, at least 80 amino acids or more, at least 90 amino acids or more, at least 100 amino acids or more, at least 110 amino acids or more, according to at least 120 amino acids or more, at least 130 amino acids or more, at least 140 amino acids or more,

- 7 043982 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 the protein sequence disclosed herein.

As used herein, the term genetic construct refers to DNA or RNA molecules that contain 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 controlling expression in the cells of the individual to whom the nucleic acid molecule is administered. As used herein, the term expressible form refers to gene constructs that contain necessary regulatory elements operably linked to a coding sequence encoding a protein such that, when present in a cell of an individual, the coding sequence will be expressed.

When used in the context of this document, the term homology 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 hybridization of a fully complementary sequence to a target nucleic acid is functionally termed substantially homologous. When used in reference to a double-stranded nucleic acid sequence, such as a cDNA or a genomic clone, the term substantially homologous, as used herein, refers to a probe that can hybridize to a strand of the double-stranded nucleic acid sequence under low stringency conditions. When used in reference to a single-stranded nucleic acid sequence, the term substantially homologous, as used herein, refers to a probe that can hybridize (ie is complementary) to a single-stranded template nucleic acid sequence under low stringency conditions.

Identical or identity, as used herein, with respect to two or more nucleic acid or polypeptide sequences means that the sequences have a certain percentage of residues that are the same in the specified region. The percentage can be calculated by optimally aligning two sequences, comparing the two sequences in a specified region, determining the number of positions at which the same residue occurs in both sequences, obtaining the number of matching positions, dividing the number of matching positions by the total number of positions in the specified region, and multiplying the result by 100 to obtain percent sequence identity. In cases where two sequences are of different lengths or the alignment results in one or more overhangs and the specified comparison region contains only one sequence, the residues of one sequence are included in the denominator but not in the numerator in the calculations. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. Identity can be calculated manually or using a computer sequence algorithm such as BLAST or BLAST 2.0.

An immune response, as used herein, means the activation of the immune system of a host, for example a mammal, in response to administration 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, when used in the context of this document, means that at least two nucleotides are covalently linked to each other. The description of one strand also determines the sequence of the complementary strand. Thus, the nucleic acid also spans the complementary strand of the represented single strand. Many nucleic acid variants can be used for the same purpose as a given nucleic acid. Thus, a nucleic acid also includes substantially identical nucleic acids and their complementary sequences. A single strand can produce a probe that can hybridize to a target sequence under stringent hybridization conditions. Thus, the nucleic acid also includes a probe that 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 and combinations of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine and isoguanine. Nucleic acids can be produced by chemical synthesis methods or by recombinant methods.

The term operably linked when used in the context of this document means that the expression of a gene is under the control of a promoter to which it is spatially linked. A promoter can be located 5' (before) or 3' (after) the gene under its control. The distance between a promoter and a gene can be approximately the same as the distance between that promoter and the gene it controls in the gene from which the promoter is derived. As is known in

- 8 043982 of the art, changing this distance can be carried out without loss of promoter function.

As used herein, the term peptide, protein, or polypeptide can refer to a linked sequence of amino acids and may 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 imparting, activating, or enhancing the expression of a nucleic acid in a cell. A promoter may contain one or more specific transcriptional regulatory sequences to further enhance expression and/or to alter the spatial expression and/or temporal expression of the sequence. The promoter may also contain distal enhancer or repressor elements, which may be located up to several thousand base pairs from the transcription start site. The promoter can be obtained from sources including viruses, bacteria, fungi, plants, insects and animals. A promoter may regulate expression of a gene component constitutively or differentially depending on the cell, tissue or organ in which expression occurs, or depending on the developmental stage at which expression occurs, or in response to external stimuli such as physiological stresses, pathogens, ions metals or inducing agents. Typical examples of promoters include bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac promoter operator, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, SV40 early promoter or SV40 late promoter and CMV promoter I.E.

The terms signal peptide and leader sequence are used interchangeably herein and refer to an amino acid sequence that may be attached at the amino terminus of a protein provided herein. Signal peptides/leader sequences typically direct protein localization. Signal peptides/leader sequences used herein can facilitate secretion of a protein from the cell in which it is produced. Signal peptides/leader sequences are often cleaved from a remnant of the protein, often called the mature protein, upon secretion from the cell. Signal peptides/leader sequences are attached at the amino terminus (ie N-terminus) of the protein.

The term stringent hybridization conditions as used herein means conditions under which a first nucleic acid sequence (eg, a probe) will hybridize to a second nucleic acid sequence (eg, a target), for example, in a multicomponent nucleic acid mixture. The strict conditions depend on the sequence and will be different in different circumstances. Severe conditions can be selected to be about 5-10°C lower than the melting temperature (Tm) for a particular sequence at a particular pH and ionic strength. 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 to the target sequence in equilibrium (since target sequences are present in excess, at Tm, 50% of the probes are in equilibrium). Severe conditions may be those in which the salt concentration is less than about 1.0 M sodium ion, for example, a concentration of about 0.01-1.0 M sodium ion (or other salts) at a pH of 7.0-8.3, 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 with the addition of destabilizing agents such as formamide. For selective or specific hybridization, the positive signal may be at least 2-10 times the background hybridization. Typical stringent hybridization conditions include the following: 50% formamide, 5x SSC and 1% SDS, incubated at 42°C or 5x SSC, 1% SDS, incubated at 65°C, washed in 0.2x SSC and 0.1% SDS at 65°C.

The term subject, when used in the context of this document, can mean a mammal that wants or needs to be immunized with the immunogenic compositions described herein. The mammal may be a human, chimpanzee, dog, cat, horse, cow, mouse or rat.

The term substantially complementary, when used in the context of this document, means that the first sequence has 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% identity to the complement of the second sequence in the region 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 the two sequences hybridize under stringent hybridization conditions.

The term substantially identical, when used in the context of this document, means that the first and second sequences have 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% identity in area 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,

- 9 043982

540 or more nucleotides or amino acids, or with respect to nucleic acids, if the first sequence is substantially complementary to the complement of the second sequence.

The term treat or treat, when used in the context of this document, can mean protecting an animal from a disease by means of preventing, suppressing, containing or completely eliminating the disease. Prevention of the disease involves administering an immunogenic composition according to this invention to the animal before the onset of the disease. Suppression of a disease involves administering an immunogenic composition according to this invention to an animal after the induction of the disease, but before its clinical manifestation. Containing the disease involves administering the immunogenic composition of the present invention to the animal after clinical manifestation of the disease.

The term variant, when used in the context of this document, in relation to a nucleic acid, means (i) a part or fragment of the specified nucleotide sequence; (ii) the complement of the specified nucleotide sequence or part thereof; (iii) a nucleic acid that is substantially identical to the specified nucleic acid or its complement; or (iv) a nucleic acid that hybridizes under stringent conditions with said nucleic acid, its complement, or sequences substantially identical thereto.

The term variant refers 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 that is substantially identical to the specified protein with an amino acid sequence that retains at least one biological activity. A conservative amino acid substitution, that is, the replacement of an amino acid with another amino acid with similar properties (eg, hydrophilicity, size and distribution of charged regions), is generally considered a minor change in the art. These minor changes can be identified, in particular, by taking into account the hydropathic amino acid index, as is known in the art. Kyte et al., J. Mol. Biol. 157:105-132 (1982). The hydropathic index of an amino acid is based on its hydrophobicity and charge. It is known in the art that amino acids with similar hydropathic indices can be substituted and still retain protein function. In one aspect, amino acids having hydropathic indices of ±2 are substituted.

Amino acid hydrophilicity can also be used to identify substitutions that result in proteins retaining biological function. Considering amino acid hydrophilicity in the context of a peptide allows one to calculate the highest local average hydrophilicity of that peptide, a useful value that has been reported to correlate well with antigenicity and immunogenicity. US Patent No. 4,554,101 is incorporated herein by reference in its entirety. Substitution of amino acids having similar hydrophilicity values can result in the peptides retaining biological activity, such as immunogenicity, as is known in the art. Substitutions can be made using amino acids having hydrophilicity values within ± 2 relative to each other. The hydrophobicity index and hydrophilicity value of an amino acid are influenced by the specific side chain of that amino acid. Consistent with this observation, amino acid substitutions that are compatible with biological function are thought to depend on the relative similarity of the amino acids and, in particular, on the side chains of those amino acids, as reflected in hydrophobicity, hydrophilicity, charge, size, and other properties.

A variant may be a nucleic acid sequence that is substantially identical over the entire length of the complete sequence of a gene or fragment thereof.

The nucleic acid sequence may have 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94%, 95, 96, 97, 98, 99, or 100% identity throughout the length of the gene sequence 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 a fragment thereof. The amino acid sequence may have 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity throughout its length amino acid sequence or fragment thereof.

The term vector, when used in the context of this document, means a nucleic acid 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 in one embodiment of the invention it is an expression plasmid. The vector may contain or have one or more heterologous nucleic acid sequences.

2. Synthetic consensus TERT.

The invention provides an optimized consensus sequence of the TERT antigen. In one embodiment, the antigen encoded by the optimized consensus sequence is capable of eliciting an immune response in a mammal. In one embodiment

- 10 043982 of the invention, the antigen encoded by the optimized consensus sequence may contain epitope(s) that make it particularly effective as an immunogen against which an immune response can be induced.

The optimized consensus sequence may be a consensus sequence derived from two or more native TERT antigens. The optimized consensus sequence may contain the consensus sequence and/or modification(s) for enhanced expression. Modification may include codon optimization, RNA optimization, addition of a Kozak sequence to enhance translation initiation, and/or addition of an immunoglobulin leader sequence to enhance immunogenicity. The TERT antigen encoded by the optimized consensus sequence may comprise a signal peptide, such as an immunoglobulin signal peptide, such as, but not limited to, an immunoglobulin E (IgE) or immunoglobulin (IgG) signal peptide. In some embodiments, the antigen encoded by the optimized consensus sequence may contain a hemagglutinin (HA) tag. The TERT antigen encoded by the optimized consensus sequence can be engineered to elicit stronger cellular and/or humoral immune responses than the corresponding native antigen. The TERT antigen, encoded by an optimized consensus sequence, can be developed to break tolerance and synergize with anticancer immune therapy.

In one embodiment of the invention, the optimized consensus TERT is designed to break tolerance to native human TERT. In one embodiment, the human optimized TERT consensus coding sequence is as set forth in SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 55. In one embodiment, the human optimized encoded consensus TERT antigen has the amino acid sequence set forth in SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO : 54 or SEQ ID NO: 56.

In one embodiment, the optimized consensus TERT is designed to break tolerance to native mouse TERT. In one embodiment, the mouse optimized consensus TERT coding sequence is as set forth in SEQ ID NO: 49 or SEQ ID NO: 51. In one embodiment, the mouse optimized consensus TERT encoded antigen has the amino acid sequence set forth in SEQ ID NO: 50 or SEQ ID NO: 52.

In one embodiment of the invention, the optimized consensus TERT is designed to break tolerance to native TERT in rhesus monkeys. In one embodiment, the rhesus macaque optimized consensus TERT coding sequence is as set forth in SEQ ID NO: 53 or SEQ ID NO: 55. In one embodiment, the mouse optimized encoded consensus TERT antigen has the amino acid sequence set forth in SEQ ID NO: 54 or SEQ ID NO: 56.

In one embodiment of the invention, the optimized encoded consensus TERT antigen is operably linked to one or more regulatory elements. In one embodiment of the invention, the regulatory element is a leader sequence. In one embodiment, the optimized consensus DNA sequence operably linked to the IgE leader coding sequence is provided in SEQ ID NO: 45, SEQ ID NO: 49, and SEQ ID NO: 53. In one embodiment, the optimized consensus TERT encoded antigen is operably associated with the IgE leader sequence is shown in SEQ ID NO: 46, SEQ ID NO: 50 and SEQ ID NO: 54.

In one embodiment of the invention, the regulatory element is a start codon. Therefore, in one embodiment, the invention provides a nucleic acid sequence set forth in SEQ ID NO: 47, SEQ ID NO: 51, SEQ ID NO: 55, or a fragment or homolog thereof, operably linked to a nucleotide sequence comprising a 5' start codon end. In one embodiment, the invention provides an amino acid sequence set forth in SEQ ID NO: 48, SEQ ID NO: 52, or SEQ ID NO: 56, or a fragment or homolog thereof, operably linked to the amino acid encoded by the start codon (e.g., methionine) at N-terminal

In one embodiment of the invention, the regulatory element is at least one stop codon. Therefore, in one embodiment, the invention relates to a nucleic acid sequence set forth in SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 55 or a fragment or homolog thereof operably linked to a nucleotide sequence containing at least one stop codon at the 3' end. In one embodiment of the invention, the nucleotide sequence is operably linked to two stop codons to enhance translation termination efficiency.

In one embodiment of the invention, the optimized consensus sequence encoding the TERT antigen may encode a peptide having the amino acid sequence

- 11 043982 presented in SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54 or SEQ ID NO: 56. In one embodiment of the invention, the optimized consensus sequence may have a nucleotide sequence represented by SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 55. In some embodiments, the sequence may represent is a nucleotide sequence having at least about 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity with respect to the entire length of the nucleotide sequence presented in SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SeQ ID NO: 55. In other embodiments, the sequence may be a nucleotide sequence that encodes an amino acid sequence having at least about 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with respect to the entire length of the amino acid sequence presented in SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO : 52, SEQ ID NO: 54 or SEQ ID NO: 56.

In some embodiments, the optimized TERT consensus antigen may be encoded by RNA, which is a transcript of a DNA sequence having at least about 96%, 97%, 98%, 99%, or 100% identity to the entire length of the nucleic acid sequence represented by the SEQ ID. NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 55. In some embodiments, the optimized TERT consensus antigen may be encoded by RNA, which encodes an amino acid sequence having at least about 96, 97, 98, 99, or 100% identity with respect to the entire length of the amino acid sequences represented by SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54 or SEQ ID NO: 56.

The optimized consensus encoded TERT antigen may be a peptide having the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8. In some embodiments, the antigen may have the amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with respect to the entire length of the amino acid sequence set forth in SEQ ID NO: 46, SEQ ID NO: 48 , SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54 or SEQ ID NO: 56.

Immunogenic protein fragments may be provided with amino acid sequences homologous to the immunogenic fragments set forth in SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, or SEQ ID NO: 56. Such 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%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of proteins that are 95% homologous to SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54 or SEQ ID NO: 56. Some embodiments of the invention provide immunogenic fragments that have 96% homology to immunogenic fragments of the consensus protein sequences presented herein. Some embodiments of the invention provide immunogenic fragments that have 97% homology to immunogenic fragments of the consensus protein sequences presented herein. Some embodiments of the invention provide immunogenic fragments that have 98% homology to immunogenic fragments of the consensus protein sequences presented herein. Some embodiments of the invention provide immunogenic fragments that have 99% homology to immunogenic fragments of the consensus protein sequences presented herein. In some embodiments, the immunogenic fragments comprise a leader sequence, such as, for example, an immunoglobulin leader sequence, such as an IgE leader sequence. In some embodiments, the immunogenic fragments do not contain a leader sequence.

In one embodiment, the immunogenic fragment of the optimized consensus TERT antigen encodes at least one immunodominant or subimmunodominant epitope of the optimized consensus full-length TERT antigen.

Some embodiments of the invention provide immunogenic fragments of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53 or SEQ ID NO: 55 containing 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 to at least 97%, at least 98%, or at least 99% of the total length of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53 or SEQ ID NO: 55. The immunogenic fragments may be at least 96%, at least 97%, at least 98%, or at least 99% homologous to the fragments of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 55. In some embodiments, the immunogenic fragments comprise sequences that encode a leader sequence

- 12 043982 ity, 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.

3. Immunogenic composition.

In one embodiment, the immunogenic composition of the present invention may comprise a synthetic TERT consensus antigen, a fragment thereof, or a variant thereof. In one embodiment, the TERT antigen is a human TERT antigen (hTERT). 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 expression of hTERT. Abnormal hTERT expression can also occur in hyperproliferative cells infected with HCV and HPV. Thus, immunotherapy for both 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. In one aspect, the hTERT cancer antigen can be further defined in US Patent Application No. 14/139,660, filed December 23, 2013, which is incorporated by reference in its entirety.

In one embodiment of the invention, the TERT antigen is a human TERT antigen. Non-human TERT antigens include, but are not limited to, mouse TERT (mTERT) and rhesus monkey TERT (rhTERT).

Expression of TERT in dendritic cells transfected with TERT genes can induce CD8+ cytotoxic T cells and induce CD4+ T cells in an antigen-specific manner. Therefore, the use of hTERT expression in antigen presenting cells (APCs) to delay aging and maintain their ability to present a selected antigen can be used in immunotherapeutic methods, such as the methods described herein.

The TERT antigen may be associated with or expressed in any number of cancers, including, but not limited to, melanoma, prostate cancer, liver cancer, cervical cancer, recurrent respiratory papillomatosis (RRP), anal cancer, head and neck cancer and cancers. blood diseases. Accordingly, the vaccine, if it contains the TERT antigen described herein, can be used to treat subjects suffering from any number of cancers, including, but not limited to, melanoma, prostate cancer, liver cancer, cervical cancer, recurrent respiratory papillomatosis (RRP), anal cancer, head and neck cancer and blood cancers.

The TERT antigen can induce an antigen-specific T cell response and/or high antibody titers, thereby eliciting or inducing an immune response that targets or responds to the cancer or tumor expressing the antigen. In some embodiments, the induced or evoked immune response may be a cellular, humoral, or both cellular and humoral immune response. 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 evoked immune response may reduce or inhibit one or more immunosuppressive factors that promote the growth of a tumor or cancer expressing an antigen, such as, but not limited to, factors that suppress MHC presentation, factors that increase levels of antigen-specific regulatory T cells (Treg), PD-L1, FasL, cytokines such as IL-10 and TFG-β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by immunosuppressive cells, CTLA-4, PD-1 , MDSCs, MCP-1 and immune checkpoint molecule, which are described in more detail below.

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

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

A nucleic acid encoding a TERT antigen or a TERT consensus antigen may also encode an immunoglobulin E (IgE) leader sequence. Nucleic acid encoding

- 13 043982 TERT antigen or TERT 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 TERT antigen or TERT consensus antigen via a peptide bond, respectively. The nucleic acid encoding a TERT antigen or a TERT consensus antigen may also contain a nucleotide sequence encoding an IgE leader sequence. In some embodiments, the nucleic acid encoding a TERT antigen or consensus TERT antigen does not contain a nucleotide sequence encoding an IgE leader sequence.

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

A nucleic acid encoding a TERT antigen or a TERT consensus antigen may be mutated relative to a wild-type TERT antigen such that one or more amino acids or residues in the amino acid sequence of the TERT antigen or the TERT consensus antigen, respectively, are replaced or replaced by another amino acid or residue. A nucleic acid encoding a TERT antigen or a TERT consensus antigen may be mutated relative to a wild-type TERT antigen such that one or more residues in the amino acid sequence of the TERT antigen or the TERT consensus antigen, respectively, are replaced or replaced by another residue, resulting in that the immune system is no longer tolerant to TERT in a mammal that is administered a nucleic acid encoding a TERT antigen or a TERT consensus antigen, a TERT antigen or a TERT consensus antigen, or a combination thereof. A nucleic acid encoding a TERT antigen or a TERT consensus antigen may be mutated relative to a wild-type TERT antigen such that arginine 589, aspartate 1005, or both arginine 589 and aspartate 1005 in the amino acid sequence of the TERT antigen or TERT consensus antigen is replaced or replaced by a residue tyrosine

In one aspect, the TERT antigen may be a nucleic acid sequence of SEQ ID NO: 23 or SEQ ID NO: 57, which encodes the amino acid sequence of SEQ ID NO: 24 and SEQ ID NO: 58, respectively. SEQ ID NO: 23 and SEQ ID NO: 57 encode a TERT antigen linked to an IgE leader sequence. In one embodiment, the TERT antigen may be coupled to an IgE leader sequence and an HA tag. In other embodiments, the TERT antigen may be free or not associated with an IgE leader sequence and/or an HA tag.

In some embodiments, the TERT antigen may be a nucleic acid sequence having at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95 , 96, 97, 98, 99, or 100% identity relative to the entire length of the nucleic acid sequence represented in SEQ ID NO: 23 or SEQ ID NO: 57. In other embodiments, the TERT antigen may be a nucleic acid sequence that encodes an amino acid a 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 relative to the entire length of the amino acid sequence set forth in SEQ ID NO: 24 or SEQ ID NO: 58. The TERT 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 with respect to the entire length of the amino acid sequence presented in SEQ ID NO: 24 or SEQ ID NO: 58.

Some embodiments of the invention relate to nucleic acid sequences encoding proteins homologous to the TERT antigen, an immunogenic fragment of the TERT antigen, and immunogenic fragments of homologous proteins. Nucleic acid molecules can be provided that encode immunogenic proteins that have up to 95% sequence homology, up to 96% sequence homology, up to 97% sequence homology, up to 98% sequence homology, and up to 99%. Likewise, nucleic acid sequences encoding immunogenic fragments presented herein and immunogenic fragments of proteins homologous to proteins presented herein are also provided.

Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 95% homology to the nucleic acid coding sequences provided herein. Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 96% homology to the nucleic acid coding sequences provided herein. Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 97% homology to the nucleic acid coding sequences provided herein. Some options

- 14 043982 embodiments of the invention relate to nucleic acid molecules that encode immunogenic proteins that have 98% homology to the nucleic acid coding sequences presented herein. Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 99% homology to the nucleic acid coding sequences provided herein. In some embodiments, nucleic acid molecules with coding sequences disclosed 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 nucleic acid sequences encoding proteins with a certain percentage of identity to the full-length TERT antigen, immunogenic fragments of the TERT antigen, and immunogenic protein fragments having identity to the TERT antigen. Nucleic acid molecules may also be provided that encode immunogenic proteins that have up to 80% identity to the full-length TERT sequence, up to 85% identity to the full-length sequence, up to 90% identity to the full-length TERT sequence, up to 91% identity to the full-length TERT sequence , up to 92% identity relative to the full-length TERT sequence, up to 93% identity relative to the full-length TERT sequence, up to 94% identity relative to the full-length TERT sequence, up to 95% identity relative to the full-length TERT sequence, up to 96% identity relative to the full-length TERT sequence, up to 97% identity relative to the full-length TERT sequence, up to 98% identity relative to the full-length TERT sequence, and up to 99% identity relative to the full-length TERT sequence. Likewise, nucleic acid sequences encoding the immunogenic fragments presented herein and immunogenic protein fragments with similar percentage identities as stated above to the TERT antigens presented herein are also provided.

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

Some embodiments of the invention relate to fragments of SEQ ID NO: 23 or SEQ ID NO: 57. The fragments may be at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70% , at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or according at least 99% of the sequence presented in SEQ ID NO: 23 or SEQ ID NO: 57. 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 or SEQ ID NO: 57. 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 the fragments of SEQ ID NO: 23 or SEQ ID NO: 57. In some embodiments, the fragments contain sequences that encode 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 TERT antigen is SEQ ID NO: 24 or SEQ ID NO: 58. The amino acid sequence of the TERT antigen linked to the IgE leader sequence is SEQ ID NO: 24 or SEQ ID NO: 58. The amino acid sequence of the TERT antigen , associated with the IgE leader sequence, can be connected to the HA tag.

Some embodiments of the invention relate to proteins that are homologous to SEQ ID NO: 24 or SEQ ID NO: 58. Some embodiments of the invention relate to immunogenic proteins that have 95% homology to the protein sequences presented in SEQ ID NO: 24 or SEQ ID NO: 58. Some embodiments of the invention relate to immunogenic proteins that have 96% homology to the protein sequences presented in SEQ ID

- 15 043982

NO: 24 or SEQ ID NO: 58. Some embodiments of the invention relate to immunogenic proteins that have 97% homology to the protein sequences presented in SEQ ID NO: 24 or SEQ ID NO: 58. Some embodiments of the invention relate to immunogenic proteins that have 98% homology to the protein sequences set forth in SEQ ID NO: 24 or SEQ ID NO: 58. Some embodiments of the invention provide immunogenic proteins that have 99% homology to the protein sequences set forth in SEQ ID NO: 24 or SEQ ID NO: 58.

Some embodiments of the invention provide proteins that are identical to SEQ ID NO: 24 or SEQ ID NO: 58. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 80% identical to the full-length amino acid sequence set forth in SEQ ID NO : 24 or SEQ ID NO: 58. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 85% identical to the full-length amino acid sequence presented in SEQ ID NO: 24 or SEQ ID NO: 58. Some embodiments of the invention refer to immunogenic proteins that have an amino acid sequence that is 90% identical to the full-length amino acid sequence set forth in SEQ ID NO: 24 or SEQ ID NO: 58. Some embodiments of the invention relate to immunogenic proteins that have an amino acid sequence that is 91 % identical to the full-length amino acid sequence set forth in SEQ ID NO: 24 or SEQ ID NO: 58. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 92% identical to the full-length amino acid sequence set forth in SEQ ID NO: 24 or SEQ ID NO: 58. Some embodiments of the invention relate to immunogenic proteins that have an amino acid sequence that is 93% identical to the full-length amino acid sequence presented in SEQ ID NO: 24 or SEQ ID NO: 58. Some embodiments of the invention relate to immunogenic proteins that have an amino acid sequence that is 94% identical to the full-length amino acid sequence set forth in SEQ ID NO: 24 or SEQ ID NO: 58. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 95% identical full-length amino acid sequence set forth in SEQ ID NO: 24 or SEQ ID NO: 58. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 96% identical to the full-length amino acid sequence set forth in SEQ ID NO: 24 or SEQ ID NO: 58. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 97% identical to the full-length amino acid sequence set forth in SEQ ID NO: 24 or SEQ ID NO: 58. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 98% identical to the full-length amino acid sequence set forth in SEQ ID NO: 24 or SEQ ID NO: 58. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 99% identical to the full-length amino acid the sequence presented in SEQ ID NO: 24 or SEQ ID NO: 58.

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 SEQ ID NO: 24 or SEQ ID NO: 58 may be provided. The 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%, according 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 presented in SEQ ID NO: 24 or SEQ ID NO: 58. 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 homologous to the immunogenic fragments of SEQ ID NO: 24 or SEQ ID NO: 58. Such immunogenic fragments may contain at least 10%, at least 15%, at least 20%,

- 16 043982 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 a protein that is 95% or more homologous to the sequence set forth in SEQ ID NO: 18. Some embodiments of the invention provide immunogenic fragments that have 96% homology to immunogenic fragments of the protein sequences presented in this document. Some embodiments of the invention provide immunogenic fragments that have 97% homology to the immunogenic fragments of the protein sequences presented herein. Some embodiments of the invention provide immunogenic fragments that have 98% homology to the immunogenic fragments of the protein sequences presented herein. Some embodiments of the invention provide immunogenic fragments that have 99% homology to the immunogenic fragments of the protein sequences presented 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: 24 or SEQ ID NO: 58. Such immunogenic fragments may contain at least 10%, at least 15%, at least 20%, at least 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: 24 or SEQ ID NO: 58. 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 stated herein with respect to the attachment of a signal peptide or leader sequence to the N-terminus of a protein, the signal peptide/leader sequence replaces the N-terminal methionine of the protein, which is encoded by the start codon of the nucleic acid sequence, as opposed to protein coding sequences without signal peptides.

Fragments of SEQ ID NO: 23 or SEQ ID NO: 57 may contain 30 or more nucleotides, including sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 or SEQ ID NO: 57 may contain 45 or more nucleotides, including sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 or SEQ ID NO: 57 may contain 60 or more nucleotides, including sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 or SEQ ID NO: 57 may contain 75 or more nucleotides, including sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 or SEQ ID NO: 57 may contain 90 or more nucleotides, including sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 or SEQ ID NO: 57 may contain 120 or more nucleotides, including sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 or SEQ ID NO: 57 may contain 150 or more nucleotides, including sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 or SEQ ID NO: 57 may contain 180 or more nucleotides, including sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 or SEQ ID NO: 57 may contain 210 or more nucleotides, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 or SEQ ID NO: 57 may contain 240 or more nucleotides, including sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 or SEQ ID NO: 57 may contain 270 or more nucleotides, including sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 or SEQ ID NO: 57 may contain 300 or more nucleotides, including sequences that encode an immunodominant epi

- 17 043982 top. In some embodiments, the fragments of SEQ ID NO: 23 or SEQ ID NO: 57 may contain 360 or more nucleotides, including sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 or SEQ ID NO: 57 may contain 420 or more nucleotides, including sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 or SEQ ID NO: 57 may contain 480 or more nucleotides, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 or SEQ ID NO: 57 may contain 540 or more nucleotides, including sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 or SEQ ID NO: 57 may contain 600 or more nucleotides, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 or SEQ ID NO: 57 may contain 300 or more nucleotides, including sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 or SEQ ID NO: 57 may contain 660 or more nucleotides, including sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 or SEQ ID NO: 57 may contain 720 or more nucleotides, including sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 or SEQ ID NO: 57 may contain 780 or more nucleotides, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 or SEQ ID NO: 57 may contain 840 or more nucleotides, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 or SEQ ID NO: 57 may contain 900 or more nucleotides, including sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 or SEQ ID NO: 57 may contain 960 or more nucleotides, including sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 or SEQ ID NO: 57 may contain 1020 or more nucleotides, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 or SEQ ID NO: 57 may contain 1080 or more nucleotides, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 or SEQ ID NO: 57 may contain 1140 or more nucleotides, including sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 or SEQ ID NO: 57 may contain 1200 or more nucleotides, including sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 or SEQ ID NO: 57 may contain 1260 or more nucleotides, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 or SEQ ID NO: 57 may contain 1320 or more nucleotides, including sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 or SEQ ID NO: 57 may contain 1380 or more nucleotides, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 or SEQ ID NO: 57 may contain 1440 or more nucleotides, including sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 or SEQ ID NO: 57 may contain 1500 or more nucleotides, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 or SEQ ID NO: 57 may contain 1560 or more nucleotides, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 or SEQ ID NO: 57 may contain 1620 or more nucleotides, including sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 or SEQ ID NO: 57 may contain 1680 or more nucleotides, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 or SEQ ID NO: 57 may contain 1740 or more nucleotides, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 or SEQ ID NO: 57 may contain 1800 or more nucleotides, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 or SEQ ID NO: 57 may contain 1860 or more nucleotides, including sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 or SEQ ID NO: 57 may contain 1920 or more nucleotides, including sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 or SEQ ID NO: 57 may contain 1980 or more nucleotides, including sequences that encode an immunodominant epitope. Somehow

- 18 043982 In some embodiments, fragments of SEQ ID NO: 23 or SEQ ID NO: 57 may contain 2040 or more nucleotides, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 or SEQ ID NO: 57 may contain 2100 or more nucleotides, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 or SEQ ID NO: 57 may contain 2160 or more nucleotides, including sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 or SEQ ID NO: 57 may contain 2220 or more nucleotides, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 or SEQ ID NO: 57 may contain 2280 or more nucleotides, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 or SEQ ID NO: 57 may contain 2340 or more nucleotides, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 or SEQ ID NO: 57 may contain 2400 or more nucleotides, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 or SEQ ID NO: 57 may contain 2460 or more nucleotides, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 or SEQ ID NO: 57 may contain 2520 or more nucleotides, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 or SEQ ID NO: 57 may contain 2580 or more nucleotides, including sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 or SEQ ID NO: 57 may contain 2640 or more nucleotides, including sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 or SEQ ID NO: 57 may contain 2,700 or more nucleotides, including sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ ID NO: 23 or SEQ ID NO: 57 may contain 2,760 or more nucleotides, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 or SEQ ID NO: 57 may contain 2820 or more nucleotides, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 or SEQ ID NO: 57 may contain 2880 or more nucleotides, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 or SEQ ID NO: 57 may contain 2940 or more nucleotides, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 or SEQ ID NO: 57 may contain 3000 or more nucleotides, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 or SEQ ID NO: 57 may contain 3060 or more nucleotides, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 or SEQ ID NO: 57 may contain 3120 or more nucleotides, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 or SEQ ID NO: 57 may contain 3180 or more nucleotides, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 or SEQ ID NO: 57 may contain 3240 or more nucleotides, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 or SEQ ID NO: 57 may contain 3300 or more nucleotides, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 or SEQ ID NO: 57 may contain 3360 or more nucleotides, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 or SEQ ID NO: 57 may contain 3420 or more nucleotides, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 or SEQ ID NO: 57 may contain 3480 or more nucleotides, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 23 or SEQ ID NO: 57 may contain coding sequences for IgE leader sequences. In some embodiments, the fragments of SEQ ID NO: 23 or SEQ ID NO: 57 do not contain IgE leader sequence coding 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 less than 180 nucleotides nucleotides, in some embodiments less than 210 nucleotides, in some embodiments

- 19 043982 lysis of the invention less than 240 nucleotides, in some embodiments less than 270 nucleotides, in some embodiments less than 300 nucleotides, in some embodiments less than 360 nucleotides, in some embodiments less than 420 nucleotides, in some embodiments less than 480 nucleotides, in some embodiments less than 540 nucleotides, in some embodiments less than 600 nucleotides, in some embodiments less than 660 nucleotides, in some embodiments less than 720 nucleotides, in some embodiments less than 780 nucleotides, in 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 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 in some embodiments, less than 1500 nucleotides, in some embodiments, less than 1560 nucleotides, in some embodiments, less than 1620 nucleotides, in some embodiments, less than 1680 nucleotides, in some embodiments, less than 1740 nucleotides, in some embodiments, less than 1800 nucleotides, in some embodiments less than 1860 nucleotides, in some embodiments less than 1920 nucleotides, in some embodiments less than 1980 nucleotides, in some embodiments less than 2040 nucleotides, in some embodiments less than 2100 nucleotides, in some embodiments implementations of the invention less than 2160 nucleotides, in some embodiments less than 2220 nucleotides, in some embodiments less than 2280 nucleotides, in some embodiments less than 2340 nucleotides, in some embodiments less than 2400 nucleotides, in some embodiments less than 2460 nucleotides , in some embodiments, less than 2520 nucleotides, in some embodiments, less than 2580 nucleotides, in some embodiments, less than 2640 nucleotides, in some embodiments, less than 2700 nucleotides, in some embodiments, less than 2760 nucleotides, in some embodiments of the invention is 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 of the invention less than 3060 nucleotides, in some embodiments of the invention 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 or SEQ ID NO: 58 may contain 15 or more amino acids, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 24 or SEQ ID NO: 58 may contain 18 or more amino acids, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 24 or SEQ ID NO: 58 may contain 21 or more amino acids, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 24 or SEQ ID NO: 58 may contain 24 or more amino acids, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 24 or SEQ ID NO: 58 may contain 30 or more amino acids, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 24 or SEQ ID NO: 58 may contain 36 or more amino acids, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 24 or SEQ ID NO: 58 may contain 42 or more amino acids, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 24 or SEQ ID NO: 58 may contain 48 or more amino acids, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 24 or SEQ ID NO: 58 may contain 54 or more amino acids, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 24 or SEQ ID NO: 58 may contain 60 or more amino acids, including sequences that encode an immunodominant epitope. In some variants

- 20 043982 implementations of the invention, fragments of SEQ ID NO: 24 or SEQ ID NO: 58 may contain 66 or more amino acids, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 24 or SEQ ID NO: 58 may contain 72 or more amino acids, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 24 or SEQ ID NO: 58 may contain 90 or more amino acids, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 24 or SEQ ID NO: 58 may contain 120 or more amino acids, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 24 or SEQ ID NO: 58 may contain 150 or more amino acids, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 24 or SEQ ID NO: 58 may contain 180 or more amino acids, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 24 or SEQ ID NO: 58 may contain 210 or more amino acids, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 24 or SEQ ID NO: 58 may contain 240 or more amino acids, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 24 or SEQ ID NO: 58 may contain 270 or more amino acids, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 24 or SEQ ID NO: 58 may contain 300 or more amino acids, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 24 or SEQ ID NO: 58 may contain 330 or more amino acids, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 24 or SEQ ID NO: 58 may contain 360 or more amino acids, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 24 or SEQ ID NO: 58 may contain 390 or more amino acids, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 24 or SEQ ID NO: 58 may contain 420 or more amino acids, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 24 or SEQ ID NO: 58 may contain 450 or more amino acids, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 24 or SEQ ID NO: 58 may contain 480 or more amino acids, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 24 or SEQ ID NO: 58 may contain 510 or more amino acids, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 24 or SEQ ID NO: 58 may contain 540 or more amino acids, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 24 or SEQ ID NO: 58 may contain 570 or more amino acids, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 24 or SEQ ID NO: 58 may contain 600 or more amino acids, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 24 or SEQ ID NO: 58 may contain 630 or more amino acids, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 24 or SEQ ID NO: 58 may contain 660 or more amino acids, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 24 or SEQ ID NO: 58 may contain 690 or more amino acids, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 24 or SEQ ID NO: 58 may contain 720 or more amino acids, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 24 or SEQ ID NO: 58 may contain 750 or more amino acids, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 24 or SEQ ID NO: 58 may contain 780 or more amino acids, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 24 or SEQ ID NO: 58 may contain 810 or more amino acids, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 24 or SEQ ID NO: 58 may contain 840 or more amino acids, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 24 or SEQ ID NO: 58 may contain 870 or more amino acids, including sequences that encode an immunodominant epitope. In some va

- 21 043982 In embodiments of the invention, the fragments of SEQ ID NO: 24 or SEQ ID NO: 58 may contain 900 or more amino acids, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 24 or SEQ ID NO: 58 may contain 930 or more amino acids, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 24 or SEQ ID NO: 58 may contain 960 or more amino acids, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 24 or SEQ ID NO: 58 may contain 990 or more amino acids, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 24 or SEQ ID NO: 58 may contain 1020 or more amino acids, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 24 or SEQ ID NO: 58 may contain 1050 or more amino acids, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 24 or SEQ ID NO: 58 may contain 1080 or more amino acids, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 24 or SEQ ID NO: 58 may contain 1110 or more amino acids, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 24 or SEQ ID NO: 58 may contain 1140 or more amino acids, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 24 or SEQ ID NO: 58 may contain 1170 or more amino acids, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 24 or SEQ ID NO: 58 may contain 1200 or more amino acids, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 24 or SEQ ID NO: 58 may contain 1230 or more amino acids, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 24 or SEQ ID NO: 58 may contain 1260 or more amino acids, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 24 or SEQ ID NO: 58 may contain 1290 or more amino acids, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 24 or SEQ ID NO: 58 may contain 1320 or more amino acids, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 24 or SEQ ID NO: 58 may contain 1350 or more amino acids, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 24 or SEQ ID NO: 58 may contain 1380 or more amino acids, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 24 or SEQ ID NO: 58 may contain 1410 or more amino acids, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 24 or SEQ ID NO: 58 may contain 1440 or more amino acids, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 24 or SEQ ID NO: 58 may contain 1470 or more amino acids, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 24 or SEQ ID NO: 58 may contain 1500 or more amino acids, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 24 or SEQ ID NO: 58 may contain coding sequences for IgE leader sequences. In some embodiments, the fragments of SEQ ID NO: 24 or SEQ ID NO: 58 do not contain IgE leader sequence coding 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 less than 54 amino acids amino acids, in some embodiments less than 60 amino acids, in some embodiments less than 72 amino acids, in some embodiments less than 90 amino acids, in some embodiments less than 120 amino acids, in some embodiments less than 150 amino acids, in some embodiments implementations of the invention less than 180 amino acids, in some embodiments less than 210 amino acids, in some embodiments less than 240 amino acids, in some embodiments less than 260 amino acids, in some embodiments less than 290 amino acids, in some embodiments less than 320 amino acids acid, in some embodiments less than 350 amino acids, in some embodiments

- 22 043982 less than 380 amino acids, in some embodiments less than 410 amino acids, in some embodiments less than 440 amino acids, in some embodiments less than 470 amino acids, in some embodiments less than 500 amino acids, in some embodiments less than 530 amino acids amino acids, in some embodiments less than 560 amino acids, in some embodiments less than 590 amino acids, in some embodiments less than 620 amino acids, in some embodiments less than 650 amino acids, in some embodiments less than 680 amino acids, in some in some embodiments, less than 710 amino acids, in some embodiments, less than 740 amino acids, in some embodiments, less than 770 amino acids, in some embodiments, less than 800 amino acids, in some embodiments, less than 830 amino acids, in some embodiments, less than 860 amino acids 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 implementations of the invention 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 in some embodiments, less than 1470 amino acids; and in some embodiments, less than 1500 amino acids.

In one embodiment, the TERT antigen is a synthetic TERT consensus antigen. In some embodiments, the synthetic consensus TERT contains 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 or more, 20 or more, 30 or more, or 50 or more amino acid mutations relative to the native TERT antigen.

In one embodiment, the hTERT consensus antigen may be the nucleic acid sequence of SEQ ID NO: 45, which encodes the amino acid sequence of SEQ ID NO: 46.

The sequence of SEQ ID NO: 45 encodes the hTERT consensus antigen linked to an IgE leader sequence. The hTERT consensus antigen can be coupled to an IgE leader sequence and an HA tag. In other embodiments, the hTERT consensus antigen may be free or not associated with an IgE leader sequence and/or an HA tag. In one embodiment, the hTERT consensus antigen may be a nucleic acid sequence of SEQ ID NO: 47 that encodes the amino acid sequence of SEQ ID NO: 48. The sequence of SEQ ID NO: 47 encodes an hTERT consensus antigen free or not associated with an IgE leader sequence and /or marked ON.

In one embodiment, the mTERT consensus antigen may be a nucleic acid sequence of SEQ ID NO: 49, which encodes the amino acid sequence of SEQ ID NO: 50.

The sequence of SEQ ID NO: 49 encodes the mTERT consensus antigen linked to an IgE leader sequence. The mTERT consensus antigen can be coupled to an IgE leader sequence and an HA tag. In other embodiments, the mTERT consensus antigen may be free or not associated with an IgE leader sequence and/or an HA tag. In one embodiment, the mTERT consensus antigen may be a nucleic acid sequence of SEQ ID NO: 51 that encodes the amino acid sequence of SEQ ID NO: 52. The sequence of SEQ ID NO: 51 encodes a mTERT consensus antigen free or not associated with an IgE leader sequence and /or marked ON.

In one embodiment, the rhTERT consensus antigen may be the nucleic acid sequence of SEQ ID NO: 53, which encodes the amino acid sequence of SEQ ID NO: 54.

The sequence of SEQ ID NO: 53 encodes the rhTERT consensus antigen linked to an IgE leader sequence. The rhTERT consensus antigen can be coupled to an IgE leader sequence and an HA tag. In other embodiments, the rhTERT consensus antigen may be free or not associated with an IgE leader sequence and/or an HA tag. In one embodiment, the rhTERT consensus antigen may be a nucleic acid sequence of SEQ ID NO: 55 that encodes the amino acid sequence of SEQ ID NO: 56. The sequence of SEQ ID NO: 55 encodes the consensus antigen

- 23 043982 rhTERT, free or not associated with an IgE leader sequence and/or an HA tag.

In some embodiments, the TERT consensus antigen may be a nucleic acid sequence having at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity relative to the entire length of the nucleic acid sequence presented in SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53 or SEQ ID NO: 55. In other embodiments, the TERT consensus antigen may be a nucleic acid 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 with respect to the entire length of the amino acid sequence presented in SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, or SEQ ID NO: 56. The TERT 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 with respect to the entire length of the amino acid sequence presented in SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO : 50, SEQ ID NO: 52, SEQ ID NO: 54 or SEQ ID NO: 56.

Some embodiments of the invention relate to nucleic acid sequences encoding proteins homologous to the TERT consensus antigen, an immunogenic fragment of the TERT consensus antigen, and immunogenic fragments of homologous proteins. Nucleic acid molecules can be provided that encode immunogenic proteins that have up to 95% sequence homology, up to 96% sequence homology, up to 97% sequence homology, up to 98% sequence homology, and up to 99%. Likewise, nucleic acid sequences encoding immunogenic fragments presented herein and immunogenic fragments of proteins homologous to proteins presented herein are also provided.

Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 95% homology to the nucleic acid coding sequences provided herein. Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 96% homology to the nucleic acid coding sequences provided herein. Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 97% homology to the nucleic acid coding sequences provided herein. Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 98% homology to the nucleic acid coding sequences provided herein. Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 99% homology to the nucleic acid coding sequences provided herein. In some embodiments, nucleic acid molecules with coding sequences disclosed 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 nucleic acid sequences encoding proteins with a certain percentage of identity to the full-length TERT consensus antigen, immunogenic fragments of the TERT consensus antigen, and immunogenic protein fragments having identity to the TERT consensus antigen. Nucleic acid molecules can also be proposed that encode immunogenic proteins that have up to 80% identity with respect to the full-length consensus TERT sequence, up to 85% identity with respect to the full-length sequence, up to 90% identity with respect to the full-length consensus TERT sequence, up to 91% identity with respect to the full-length consensus TERT sequence, up to 92% identity relative to the full-length consensus TERT sequence, up to 93% identity relative to the full-length consensus TERT sequence, up to 94% identity relative to the full-length consensus TERT sequence, up to 95% identity relative to the full-length consensus TERT sequence, up to 96% identity relative to the full length consensus TERT sequence, up to 97% identity with respect to the full-length consensus TERT sequence, up to 98% identity with respect to the full-length consensus TERT sequence, and up to 99% identity with respect to the full-length consensus TERT sequence. Likewise, nucleic acid sequences encoding the immunogenic fragments presented herein and immunogenic protein fragments with similar percentage identities as stated above to the TERT consensus antigens presented herein are also provided.

- 24 043982

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

Some embodiments of the invention are directed to portions of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 55. The portions may be at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% , at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the sequence presented in SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 55. 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: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53 or SEQ ID NO: 55. 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: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 55. In some embodiments, the fragments comprise sequences that encode 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.

Additionally, in one embodiment, the amino acid sequence of the TERT consensus antigen is SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, or SEQ ID NO: 56 The amino acid sequence of the TERT consensus antigen fused to the IgE leader sequence is SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, or SEQ ID NO: 56 The amino acid sequence of the TERT consensus antigen linked to an IgE leader sequence can be linked to an HA tag.

Some embodiments of the invention relate to proteins that are homologous to SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, or SEQ ID NO: 56. Some embodiments of the invention refer to immunogenic proteins that have 95% homology to the protein sequences presented in SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54 or SEQ ID NO: 56. Certain embodiments of the invention provide immunogenic proteins that have 96% homology to the protein sequences set forth in SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54 or SEQ ID NO: 56. Certain embodiments of the invention provide immunogenic proteins that have 97% homology to the protein sequences set forth in SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54 or SEQ ID NO: 56. Some embodiments of the invention provide immunogenic proteins that have 98% homology to the protein sequences presented in SEQ ID NO: 46. Some embodiments of the invention provide immunogenic proteins that have 99% homology to the protein sequences presented in SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54 or SEQ ID NO: 56.

Some embodiments of the invention provide proteins that are identical to SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, or SEQ ID NO: 56. Some embodiments of the invention refer to immunogenic proteins that have an amino acid sequence that is 80% identical to the full-length amino acid sequence set forth in SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54 or SEQ ID NO: 56. Certain embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 85% identical to the full-length amino acid sequence set forth in SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54 or SEQ ID NO: 56. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 90% identical to the full-length amino acid sequence set forth in SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, or SEQ ID NO: 56. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 91% identical to the full-length amino acid sequence presented in SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54 or SEQ ID NO: 56. Some

- 25 043982 Other embodiments of the invention relate to immunogenic proteins that have an amino acid sequence that is 92% identical to the full-length amino acid sequence set forth in SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54 or SEQ ID NO: 56. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 93% identical to the full-length amino acid sequence set forth in SEQ ID NO: 46, SEQ ID NO: 48 , SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, or SEQ ID NO: 56. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 94% identical to the full-length amino acid sequence presented in SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, or SEQ ID NO: 56. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence, which is 95% identical to the full-length amino acid sequence set forth in SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, or SEQ ID NO: 56. Some embodiments The inventions relate to immunogenic proteins that have an amino acid sequence that is 96% identical to the full-length amino acid sequence presented in SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54 or SEQ ID NO: 56. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 97% identical to the full-length amino acid sequence set forth in SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50 , SEQ ID NO: 52, SEQ ID NO: 54, or SEQ ID NO: 56. Certain embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 98% identical to the full-length amino acid sequence set forth in SEQ ID NO: 46. SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, or SEQ ID NO: 56. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 99% identical to the full-length amino acid sequence set forth in SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, or SEQ ID NO: 56.

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 SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54 or SEQ ID NO: 56 may be provided. The 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%, according to 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 presented in SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, or SEQ ID NO: 56. 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 homologous to the immunogenic fragments set forth in SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, or SEQ ID NO: 56. 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%, according to 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 shown in SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54 or SEQ ID NO: 56. Some embodiments of the invention provide immunogenic fragments that have 96% homologies to immunogenic fragments of the protein sequences presented herein. Some embodiments of the invention provide immunogenic fragments that have 97% homology to the immunogenic fragments of the protein sequences presented herein. Some embodiments of the invention from

- 26 043982 refers to immunogenic fragments that have 98% homology to the immunogenic fragments of the protein sequences presented in this document. Some embodiments of the invention provide immunogenic fragments that have 99% homology to the immunogenic fragments of the protein sequences presented 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 set forth in SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, or SEQ ID NO: 56. 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%, according to 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: 46, SEQ ID NO: 48, SEQ ID NO : 50, SEQ ID NO: 52, SEQ ID NO: 54, or SEQ ID NO: 56. 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 stated herein with respect to the attachment of a signal peptide or leader sequence to the N-terminus of a protein, the signal peptide/leader sequence replaces the N-terminal methionine of the protein, which is encoded by the start codon of the nucleic acid sequence, as opposed to protein coding sequences without signal peptides.

Fragments of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53 or SEQ ID NO: 55 may contain 30 or more nucleotides, including sequences that encode an immunodominant epitope . In some embodiments, the fragments of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 55 may contain 45 or more nucleotides, including sequences , which encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 55 may contain 60 or more nucleotides, including sequences , which encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 55 may contain 75 or more nucleotides, including sequences , which encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 55 may contain 90 or more nucleotides, including sequences , which encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 55 may contain 120 or more nucleotides, including sequences , which encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 55 may contain 150 or more nucleotides, including sequences , which encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 55 may contain 180 or more nucleotides, including sequences , which encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 55 may contain 210 or more nucleotides, including sequences , which encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 55 may contain 240 or more nucleotides, including sequences , which encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 55 may contain 270 or more nucleotides, including sequences , which encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 55 may contain 300 or more nucleotides, including sequences , which encode an immunodominant epitope. In some embodiments of the invention, the frag

- 27 043982 ments SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53 or SEQ ID NO: 55 may contain 360 or more nucleotides, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 55 may contain 420 or more nucleotides, including sequences , which encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 55 may contain 480 or more nucleotides, including sequences , which encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 55 may contain 540 or more nucleotides, including sequences , which encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 55 may contain 600 or more nucleotides, including sequences , which encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 55 may contain 300 or more nucleotides, including sequences , which encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SeQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 55 may contain 660 or more nucleotides, including sequences , which encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 55 may contain 720 or more nucleotides, including sequences , which encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 55 may contain 780 or more nucleotides, including sequences , which encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 55 may contain 840 or more nucleotides, including sequences , which encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SeQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 55 may contain 900 or more nucleotides, including sequences , which encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 55 may contain 960 or more nucleotides, including sequences , which encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 55 may contain 1020 or more nucleotides, including sequences , which encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 55 may contain 1080 or more nucleotides, including sequences , which encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 55 may contain 1140 or more nucleotides, including sequences , which encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 55 may contain 1200 or more nucleotides, including sequences , which encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 55 may contain 1260 or more nucleotides, including sequences , which encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 55 may contain 1320 or more nucleotides, including sequences , which encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 55 may contain 1380 or more nucleotides, including sequences , which encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 55 may contain 1440 or more nucleotides, including sequences , which encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 55 may contain 1500 or more nucleotides, including sequences , which encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 55 may contain 1560 or more nucleotides, including sequences , which encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 55 may contain 1620 or more nucleotides,

- 28 043982 including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 55 may contain 1680 or more nucleotides, including sequences , which encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 55 may contain 1740 or more nucleotides, including sequences , which encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 55 may contain 1800 or more nucleotides, including sequences , which encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 55 may contain 1860 or more nucleotides, including sequences , which encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 55 may contain 1920 or more nucleotides, including sequences , which encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 55 may contain 1980 or more nucleotides, including sequences , which encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 55 may contain 2040 or more nucleotides, including sequences , which encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 55 may contain 2100 or more nucleotides, including sequences , which encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 55 may contain 2160 or more nucleotides, including sequences , which encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 55 may contain 2220 or more nucleotides, including sequences , which encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 55 may contain 2280 or more nucleotides, including sequences , which encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 55 may contain 2340 or more nucleotides, including sequences , which encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 55 may contain 2400 or more nucleotides, including sequences , which encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 55 may contain 2460 or more nucleotides, including sequences , which encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 55 may contain 2520 or more nucleotides, including sequences , which encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 55 may contain 2580 or more nucleotides, including sequences , which encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 55 may contain 2640 or more nucleotides, including sequences , which encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 55 may contain 2700 or more nucleotides, including sequences , which encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 55 may contain 2760 or more nucleotides, including sequences , which encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 55 may contain 2820 or more nucleotides, including sequences , which encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 55 may contain 2880 or more nucleotides, including sequences , which encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 55 may contain 2940 or more nucleotides, including sequences , which encode an immunodominant epitope. In some embodiments, the SEQ ID NOs are:

- 29 043982

45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53 or SEQ ID NO: 55 may contain 3000 or more nucleotides, including sequences that encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 55 may contain 3060 or more nucleotides, including sequences , which encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 55 may contain 3120 or more nucleotides, including sequences , which encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 55 may contain 3180 or more nucleotides, including sequences , which encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 55 may contain 3240 or more nucleotides, including sequences , which encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID no: 55 may contain 3300 or more nucleotides, including sequences , which encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 55 may contain 3360 or more nucleotides, including sequences , which encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 55 may contain 3420 or more nucleotides, including sequences , which encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 55 may contain 3480 or more nucleotides, including sequences , which encode an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SeQ ID NO: 53, or SEQ ID NO: 55 may contain coding sequences for IgE leader sequences. In some embodiments, the fragments of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 55 do not contain IgE leader sequence coding 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 less than 180 nucleotides nucleotides, in some embodiments less than 210 nucleotides, in some embodiments less than 240 nucleotides, in some embodiments less than 270 nucleotides, in some embodiments less than 300 nucleotides, in some embodiments less than 360 nucleotides, in some embodiments implementations of the invention less than 420 nucleotides, in some embodiments of the invention less than 480 nucleotides, in some embodiments of the invention less than 540 nucleotides, in some embodiments of the invention less than 600 nucleotides, in some embodiments of the invention less than 660 nucleotides, in some embodiments of the invention 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 the invention is less than 1080 nucleotides, in some embodiments less than 1140 nucleotides, in some embodiments less than 1200 nucleotides, in some embodiments less than 1260 nucleotides, in some embodiments less than 1320 nucleotides, in some embodiments less than 1380 nucleotides, in some embodiments less than 1440 nucleotides, in some embodiments less than 1500 nucleotides, in some embodiments less than 1560 nucleotides, in some embodiments less than 1620 nucleotides, in some embodiments less than 1680 nucleotides, in some embodiments less than 1680 nucleotides less than 1740 nucleotides, in some embodiments less than 1800 nucleotides, in some embodiments less than 1860 nucleotides, in some embodiments less than 1920 nucleotides, in some embodiments less than 1980 nucleotides, in some embodiments less than 2040 nucleotides, in in some embodiments less than 2100 nucleotides, in some embodiments less than 2160 nucleotides, in some embodiments less than 2220 nucleotides, in some embodiments less than 2280 nucleotides, in some embodiments less than 2340 nucleotides, in some embodiments less 2400 nucleotides, in some embodiments less than 2460 nucleotides, in some embodiments less than 2520 nucleotides

- 30,043,982 nucleotides, in some embodiments less than 2580 nucleotides, in some embodiments less than 2640 nucleotides, in some embodiments less than 2700 nucleotides, in some embodiments less than 2760 nucleotides, in some embodiments less than 2820 nucleotides, in some embodiments, less than 2860 nucleotides, in some embodiments, less than 2940 nucleotides, in some embodiments, less than 3000 nucleotides, in some embodiments, less than 3060 nucleotides, in some embodiments, less than 3120 nucleotides, in some embodiments, less than 3060 nucleotides 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: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54 or SEQ ID NO: 56 may contain 15 or more amino acids, including sequences that contain an immunodominant epitope . In some embodiments, the fragments of SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, or SEQ ID NO: 56 may contain 18 or more amino acids, including sequences , which contain an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, or SEQ ID NO: 56 may contain 21 or more amino acids, including sequences , which contain an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SeQ ID nO: 52, SEQ ID NO: 54, or SEQ ID NO: 56 may contain 24 or more amino acids, including sequences , which contain an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, or SEQ ID NO: 56 may contain 30 or more amino acids, including sequences , which contain an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, or SEQ ID NO: 56 may contain 36 or more amino acids, including sequences , which contain an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, or SEQ ID NO: 56 may contain 42 or more amino acids, including sequences , which contain an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, or SEQ ID NO: 56 may contain 48 or more amino acids, including sequences , which contain an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, or SEQ ID NO: 56 may contain 54 or more amino acids, including sequences , which contain an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, or SEQ ID NO: 56 may contain 60 or more amino acids, including sequences , which contain an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, or SEQ ID NO: 56 may contain 66 or more amino acids, including sequences , which contain an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, or SEQ ID NO: 56 may contain 72 or more amino acids, including sequences , which contain an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, or SEQ ID NO: 56 may contain 90 or more amino acids, including sequences , which contain an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, or SEQ ID NO: 56 may contain 120 or more amino acids, including sequences , which contain an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, or SEQ ID NO: 56 may contain 150 or more amino acids, including sequences , which contain an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, or SEQ ID NO: 56 may contain 180 or more amino acids, including sequences , which contain an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, or SEQ ID NO: 56 may contain 210 or more amino acids, including sequences , which contain an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, or SEQ ID NO: 56 may contain 240 or more amino acids, including sequences , which contain an immunodominant epitope. In some variants

- 31 043982 each implementation of the invention, fragments of SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54 or SEQ ID NO: 56 may contain 270 or more amino acids, including sequences that contain an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, or SEQ ID NO: 56 may contain 300 or more amino acids, including sequences , which contain an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, or SEQ ID NO: 56 may contain 330 or more amino acids, including sequences , which contain an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, or SEQ ID NO: 56 may contain 360 or more amino acids, including sequences , which contain an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SeQ ID nO: 52, SEQ ID NO: 54, or SEQ ID NO: 56 may contain 390 or more amino acids, including sequences , which contain an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, or SEQ ID NO: 56 may contain 420 or more amino acids, including sequences , which contain an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, or SEQ ID NO: 56 may contain 450 or more amino acids, including sequences , which contain an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, or SEQ ID NO: 56 may contain 480 or more amino acids, including sequences , which contain an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SeQ ID nO: 52, SEQ ID NO: 54, or SEQ ID NO: 56 may contain 510 or more amino acids, including sequences , which contain an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, or SEQ ID NO: 56 may contain 540 or more amino acids, including sequences , which contain an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, or SEQ ID NO: 56 may contain 570 or more amino acids, including sequences , which contain an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, or SEQ ID NO: 56 may contain 600 or more amino acids, including sequences , which contain an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SeQ ID nO: 52, SEQ ID NO: 54, or SEQ ID NO: 56 may contain 630 or more amino acids, including sequences , which contain an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, or SEQ ID NO: 56 may contain 660 or more amino acids, including sequences , which contain an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, or SEQ ID NO: 56 may contain 690 or more amino acids, including sequences , which contain an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, or SEQ ID NO: 56 may contain 720 or more amino acids, including sequences , which contain an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SeQ ID nO: 52, SEQ ID NO: 54, or SEQ ID NO: 56 may contain 750 or more amino acids, including sequences , which contain an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, or SEQ ID NO: 56 may contain 780 or more amino acids, including sequences , which contain an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, or SEQ ID NO: 56 may contain 810 or more amino acids, including sequences , which contain an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, or SEQ ID NO: 56 may contain 840 or more amino acids, including sequences , which contain an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SeQ ID nO: 52, SEQ ID NO: 54, or SEQ ID NO: 56 may contain 870 or more amino acids, including sequences , which contain an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, or SEQ ID NO: 56 may contain 900 or more amino acids, including sequences , which contain an immunodominant epitope. In some embodiments, portions of SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, or SEQ ID NO: 56 may contain

- 32 043982 reap 930 or more amino acids, including sequences that contain an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, or SEQ ID no: 56 may contain 960 or more amino acids, including sequences , which contain an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SeQ ID nO: 52, SEQ ID NO: 54, or SEQ ID NO: 56 may contain 990 or more amino acids, including sequences , which contain an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, or SEQ ID NO: 56 may contain 1020 or more amino acids, including sequences , which contain an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, or SEQ ID NO: 56 may contain 1050 or more amino acids, including sequences , which contain an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, or SEQ ID NO: 56 may contain 1080 or more amino acids, including sequences , which contain an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, or SEQ ID NO: 56 may contain 1110 or more amino acids, including sequences , which contain an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, or SEQ ID NO: 56 may contain 1140 or more amino acids, including sequences , which contain an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, or SEQ ID NO: 56 may contain 1170 or more amino acids, including sequences , which contain an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, or SEQ ID NO: 56 may contain 1200 or more amino acids, including sequences , which contain an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, or SEQ ID NO: 56 may contain 1230 or more amino acids, including sequences , which contain an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, or SEQ ID NO: 56 may contain 1260 or more amino acids, including sequences , which contain an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, or SEQ ID NO: 56 may contain 1290 or more amino acids, including sequences , which contain an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, or SEQ ID NO: 56 may contain 1320 or more amino acids, including sequences , which contain an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, or SEQ ID NO: 56 may contain 1350 or more amino acids, including sequences , which contain an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SeQ ID nO: 52, SEQ ID NO: 54, or SEQ ID NO: 56 may contain 1380 or more amino acids, including sequences , which contain an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, or SEQ ID NO: 56 may contain 1410 or more amino acids, including sequences , which contain an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, or SEQ ID NO: 56 may contain 1440 or more amino acids, including sequences , which contain an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, or SEQ ID NO: 56 may contain 1470 or more amino acids, including sequences , which contain an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, or SEQ ID NO: 56 may contain 1500 or more amino acids, including sequences , which contain an immunodominant epitope. In some embodiments, the fragments of SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, or SEQ ID NO: 56 may contain coding sequences for IgE leader sequences. In some embodiments, the fragments of SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, or SEQ ID NO: 56 do not contain IgE leader sequence coding 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

- 33 043982 invention less than 48 amino acids, in some embodiments less than 54 amino acids, in some embodiments less than 60 amino acids, in some embodiments less than 72 amino acids, in some embodiments less than 90 amino acids, in some embodiments less 120 amino acids, in some embodiments less than 150 amino acids, in some embodiments less than 180 amino acids, in some embodiments less than 210 amino acids, in some embodiments less than 240 amino acids, in some embodiments less than 260 amino acids, in some in some embodiments, less than 290 amino acids, in some embodiments, less than 320 amino acids, in some embodiments, less than 350 amino acids, in some embodiments, less than 380 amino acids, in some embodiments, less than 410 amino acids, in some embodiments, less 440 amino acids, in some embodiments less than 470 amino acids, in some embodiments less than 500 amino acids, in some embodiments less than 530 amino acids, in some embodiments less than 560 amino acids, in some embodiments less than 590 amino acids, in some embodiments implementations of the invention less than 620 amino acids, in some embodiments less than 650 amino acids, in some embodiments less than 680 amino acids, in some embodiments less than 710 amino acids, in some embodiments less than 740 amino acids, in some embodiments less than 770 amino acids, in some embodiments less than 800 amino acids, in some embodiments less than 830 amino acids, in some embodiments less than 860 amino acids, in some embodiments less than 890 amino acids, in some embodiments less than 920 amino acids, in some embodiments implementations of the invention less than 950 amino acids, in some embodiments of the invention less than 980 amino acids, in some embodiments of the invention less than 1010 amino acids, in some embodiments of the invention less than 1040 amino acids, in some embodiments of the invention 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 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.

This invention relates to an anticancer immunogenic composition. The immunogenic composition may contain one or more cancer antigens. The immunogenic composition can prevent tumor growth. The immunogenic composition can reduce tumor growth. The immunogenic composition can prevent metastasis of tumor cells. Depending on the cancer antigen, the immunogenic composition may be targeted to treat cancers, including, but not limited to, liver cancer, prostate cancer, melanomas, blood cancers, head and neck cancer, glioblastoma, recurrent respiratory papillomatosis, anal cancer, cancer cervix, and brain cancer.

The first step in developing an immunogenic composition is to identify a cancer antigen that is not recognized by the immune system and is a self-antigen. An identified cancer antigen undergoes a change from self-antigen to foreign antigen for recognition by the immune system. Rearrangement of the nucleic acid and amino acid sequence of a recombinant cancer antigen from a self-antigen to a foreign antigen breaks the immune system's tolerance to the antigen. To break tolerance, several modification methods can be applied to the cancer antigen, as described below.

The recombinant cancer antigen of the immunogenic composition is not recognized as a self-antigen and therefore breaks tolerance. Breakdown of tolerance can induce an antigen-specific T-cell response and/or high titers of antibodies, thereby causing or inducing an immune response that targets or responds to the cancer or tumor expressing the antigen. In some embodiments, the induced or evoked immune response may be a cellular, humoral, or both cellular and humoral immune response. 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 evoked immune response may reduce or inhibit one or more immunosuppressive factors that promote the growth of a tumor or cancer expressing the antigen, such as, but not limited to, factors that

- 34 043982 which suppress MHC presentation, factors that increase the levels of antigen-specific regulatory T cells (Treg), PD-L1, FasL, cytokines such as IL-10 and TFG-β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by immunosuppressive cells, CTLA-4, PD-1, MDSCs, MCP-1 and immune checkpoint molecule.

In a specific embodiment, the immunogenic composition can mediate the clearance or prevent the growth of tumor cells by inducing (1) humoral immunity through B cell responses to generate antibodies that block the production of monocyte chemoattractant protein-1 (MCP-1), thereby slowing the development of suppressor tumors. myeloid-derived cells (MDSC) and suppressing tumor growth; (2) increasing levels of cytotoxic T lymphocytes such as CD8 ⁺ (CTL) to attack and kill tumor cells; (3) enhancing T-helper responses; (4) and enhancement of inflammatory responses by IFN-γ and TFN-α or a combination of the above. The immunogenic composition can increase tumor-free survival by 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 and 45%. The immunogenic composition can reduce tumor weight 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 immunogenic composition can prevent and block the increase in monocyte chemoattractant protein 1 (MCP-1), a cytokine secreted by myeloid-derived suppressor cells. The immunogenic composition 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 immunogenic composition may enhance the cellular immune response in a subject to which the immunogenic composition is administered by about 50-6000 times, about 50-5500 times, about 50 to 5000 times, about 50 to 4500 times, about 100 to 6000 times times, about 150 to 6000 times, about 200 to 6000 times, about 250 to 6000 times, or about 300 to 6000 times the cellular immune response in a subject not administered the immunogenic composition. In some embodiments, the immunogenic composition may enhance the cellular immune response in a subject to which the immunogenic composition is administered by about 50-fold, 100-fold, 150-fold, 200-fold, 250-fold, 300-fold, 350-fold, 400 times, 450 times, 500 times, 550 times, 600 times, 650 times, 700 times, 750 times, 800 times, 850 times, 900 times, 950 times, 1000 times , 1100 times, 1200 times, 1300 times, 1400 times, 1500 times, 1600 times, 1700 times, 1800 times, 1900 times, 2000 times, 2100 times, 2200 times, 2300 times, 2400 times, 2500 times, 2600 times, 2700 times, 2800 times, 2900 times, 3000 times, 3100 times, 3200 times, 3300 times, 3400 times, 3500 times , 3600 times, 3700 times, 3800 times, 3900 times, 4000 times, 4100 times, 4200 times, 4300 times, 4400 times, 4500 times, 4600 times, 4700 times, 4800 times, 4900 times, 5000 times, 5100 times, 5200 times, 5300 times, 5400 times, 5500 times, 5600 times, 5700 times, 5800 times, 5900 times or 6000 times compared to the cellular immune response in a subject not administered the immunogenic composition.

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

The immunogenic composition may be a nucleic acid vaccine. In one embodiment of the invention, the nucleic acid vaccine is a DNA vaccine. DNA vaccines are described in US Pat. Nos. 5,593,972, 5,739,118, 5817637, 5830876, 5962428, 5981505, 5580859, 5703055, and 5676594, which are incorporated herein by reference in their entirety. The DNA vaccine may additionally contain elements or reagents that prevent its integration into the chromosome.

The immunogenic composition may be an RNA vaccine. An RNA vaccine can be introduced into a cell. An RNA vaccine may additionally contain elements or reagents that prevent its integration into the chromosome.

The immunogenic composition 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 Patent Nos.

- 35 043982

4510245; 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, each of which is incorporated herein by reference.

The immunogenic composition of the present invention may have properties required for effective vaccines, such as safety, such that the vaccine itself does not cause disease or death; the ability to protect against diseases; induction of neutralizing antibody; induction of protective T cell responses; and ensuring ease of administration, few side effects, biological stability and low cost per dose. The immunogenic composition may achieve some or all of these properties by containing a cancer antigen, as discussed below.

As described in more detail below, the immunogenic composition 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 inhibition of any component in the immune system, such as MHC class 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 immunogenic composition can be further combined with antibodies to checkpoint inhibitors such as PD-1, PDL-1, CTLA4, TIM3 and LAG3 to enhance the stimulation of both cellular and humoral immune responses. The use of antibodies directed to immune checkpoint proteins prevents the T cell and/or B cell response from suppressing the immune checkpoint protein.

The immunogenic composition may also contain an antigen or a fragment or variant thereof. An antigen can be anything that triggers an immune response in a subject. The antigen may be a nucleic acid sequence, an amino acid sequence, or a combination thereof. The nucleic acid sequence may be DNA, RNA, cDNA, a variant thereof, a fragment thereof, or a combination thereof. The nucleic acid sequence may also contain additional sequences that encode linker or tag sequences that are linked to the antigen via a peptide bond. The amino acid sequence may be a protein, a peptide, a variant thereof, a fragment thereof, or a combination thereof.

The antigen may be contained in a protein, nucleic acid or fragment or variant thereof or combination thereof from any number of organisms, such as a virus, parasite, bacterium, fungus or mammal. The antigen may be associated with an autoimmune disease, allergy, or asthma. In other embodiments, the antigen may be associated with cancer, herpes, influenza, hepatitis B, hepatitis C, human papillomavirus (HPV) or human immunodeficiency virus (HIV).

Some antigens can cause a strong immune response. Other antigens may cause a weak immune response. The antigen may produce a higher immune response when combined with the TERT antigen.

Cancer antigens.

The immunogenic composition may contain one or more cancer antigens. The cancer antigen may be a nucleic acid sequence, an amino acid sequence, or a combination thereof. The nucleic acid sequence may be DNA, RNA, cDNA, a variant thereof, a fragment thereof, or a combination thereof. The nucleic acid sequence may also contain additional sequences that encode linker or tag sequences that are linked to the cancer antigen via 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.

In the context of the present invention, a tumor antigen or a hyperproliferative disorder antigen or an antigen associated with a hyperproliferative disorder refers to antigens that are common to specific hyperproliferative disorders such as cancer. The antigens discussed herein are included by way of example only. This list is not to be construed as exclusive, and additional examples will be apparent to those skilled in the art.

Cancer antigens or tumor antigens are proteins that are produced by tumor cells that trigger an immune response, particularly T cell-mediated immune responses. The choice of antigen binding fragment according to the invention will depend on the specific type of cancer being treated. Tumor antigens are well known in the art and include, for example, glioma-associated antigen, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX , human telomerase reverse transcriptase

- 36 043982 pelvis, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein, PSMA, Her2/neu, survivin and telomerase, prostate carcinoma antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor and mesothelin.

In one embodiment of the invention, the tumor antigen contains one or more cancer antigenic epitopes associated with a malignant tumor. Malignant tumors express a number of proteins that can serve as target antigens for immune attack. These molecules include, but are not limited to, tissue-specific antigens such as MART-1, tyrosinase and GP 100 in melanoma and prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to the group of transformation-related molecules, such as the HER-2/Neu/ErbB-2 oncogene. Another group of target antigens are oncofetal antigens, such as carcinoembryonic antigen (CEA). In B-cell lymphoma, tumor-specific immunoglobulin idiotype is a truly tumor-specific immunoglobulin antigen that is unique to an individual tumor. B cell differentiation antigens such as CD19, CD20, and CD37 are other candidates for target antigens in B cell lymphoma. Some of these antigens (CEA, HER-2, CD19, CD20, idiotype) have been used as targets for passive monoclonal antibody immunotherapy with limited success.

The type of tumor antigen mentioned in the invention may also be a tumor specific antigen (TSA) or a tumor associated antigen (TAA). TSA is unique to tumor cells and is not found in other cells in the body. The TAA-associated antigen is not unique to the tumor cell and is instead also expressed on the normal cell under conditions that cannot induce a state of immunological tolerance to the antigen. Expression of an antigen in a tumor can occur under conditions that allow the immune system to respond to the antigen. TAAs may be antigens that are expressed on normal cells during fetal development when the immune system is immature and unable to respond, or they may be antigens that are normally present at extremely low levels in normal cells but that are expressed at much higher levels. levels on tumor cells.

Non-limiting examples of TSA or TAA antigens include the following: Differentiation antigens such as MART-1/Melan-A (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2 and tumor-specific multilineage antigens such as MAGE -1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor suppressor genes such as p53, Ras, HER-2/neu; unique tumor antigens resulting from chromosomal translocations; such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens such as Epstein-Barr virus EBVA antigens and human papillomavirus (HPV) E6 and E7 antigens. Other large protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175 , M344, MA-50, MG7Ag, MOV18, NB/70K, NY-CO-1, RCAS1, SDCCAG16, TA-90\Mac-2-binding protein\cyclophilin C-associated protein, TAAL6, TAG72, TLP, and TPS .

The TERT antigen and, optionally, one or more antibodies targeting one or more immune checkpoint proteins may be associated or combined with a tumor antigen or a fragment or variant thereof. Cancer markers are known proteins that are present or activated in certain cancer cells. Using a methodology for producing antigens that present such markers to break self-tolerance, a cancer vaccine can be created. Such cancer vaccines may contain a TERT antigen and, optionally, one or more antibodies targeting one or more immune checkpoint proteins to enhance the immune response, and optionally one or more additional tumor antigens. The following are some typical tumor antigens:

(1) Tyrosinase (Tyr).

The immunogenic composition of the present invention may contain a tyrosinase (Tyr) antigen, a fragment thereof, or a variant thereof. Tyrosinase is a copper-containing enzyme with catalytic activity against tyrosine hydroxylase and dopa oxidase, which can be found in microorganisms and tissues of plants and animals. Specifically, tyrosinase catalyzes the production of melanin and other pigments by oxidizing 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 may become dysregulated, resulting in increased melanin synthesis. Accordingly, tyrosinase may be a cancer antigen

- 37 043982 nom related to melanoma. In subjects suffering from melanoma, tyrosinase may be a target for recognition by cytotoxic T cells. However, in some cases, the immune response to cancer or tumor (including melanoma) may be suppressed, resulting in a microenvironment that supports tumor formation and/or growth and thus disease progression.

Immune suppression 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 can be a heterogeneous population of myeloid progenitor cells and immature myeloid cells (IMC). Markers of MDSC may include expression of Gr-1 and CD11b (ie, Gr-1 ⁺ and CD11b ⁺ cells).

MDSC circulation may increase due to chronic infection, and increased MDSC populations may be associated with autoimmunity and inflammation. In particular, an increase in MDSCs (or presence in a tumor or cancerous tissue) may promote tumor growth and evade immune detection and/or regulation, and thus MDSCs may influence immune responses to tumor 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 tumor-free mice. One such difference may be the activation of the production of the chemokine MCP-1, which is secreted by MDSCs. MCP-1 can stimulate cell migration by signaling through CCR2, a G protein-coupled receptor (GPCR) found on monocytes, endothelial cells and T cells. Accordingly, MCP-1 may induce endothelial cell migration, thereby promoting vascularization. Blocking MCP-1 using neutralizing antibodies can inhibit angiogenesis and therefore may lead to reduced tumor metastasis and increased survival. As such, MCP-1 can be considered an angiogenic factor. In addition to secreting MCP-1, MDSCs can secrete growth factors, thereby further promoting tumor growth.

The Tyr antigen can induce an antigen-specific T cell response and/or high antibody titers, thereby eliciting or inducing an immune response that targets or responds to the cancer or tumor expressing the antigen. In some embodiments, the induced or evoked immune response may be a cellular, humoral, or both cellular and humoral immune response. 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 evoked immune response may reduce or inhibit one or more immunosuppressive factors that promote the growth of a tumor or cancer expressing an antigen, such as, but not limited to, factors that suppress MHC presentation, factors that increase levels of antigen-specific regulatory T cells (Treg), PD-L1, FasL, cytokines such as IL-10 and TFG-β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by immunosuppressive cells, CTLA-4, PD-1 , MDSCs, MCP-1 and immune checkpoint molecule, which are described in more detail below.

As described herein, the Tyr antigen induces antigen-specific T cell responses and high titer antibody responses against cancer or tumor cells (eg, melanoma cells). In particular, Tyr antigen is an important target for immune-mediated clearance by inducing (1) humoral immunity through B cell responses to generate antibodies that block the production of monocyte chemoattractant protein-1 (MCP-1), thereby slowing the development of myeloid-derived suppressor cells ( MDSC) and suppressing tumor growth; (2) increasing levels of cytotoxic T lymphocytes such as CD8 ⁺ (CTL) to attack and kill tumor cells; (3) enhancing T-helper responses; and (4) enhancing inflammatory responses with IFN-γ and TFN-α or a combination of the above. As such, a protective immune response against tumor formation and tumor growth is provided by immunogenic compositions containing Tyr antigen (e.g., the Tyr consensus antigen, which is described in more detail below), since these immunogenic compositions prevent immune suppression by reducing the population of MDSCs found in the cancer or tumor tissue. tissue and block the vascularization of cancerous or tumor tissue by reducing the production or secretion of MCP-1. Accordingly, any user can formulate an immunogenic composition according to this invention to include the Tyr antigen to provide broad immunity against tumor formation, tumor metastasis and tumor growth.

Tyr antigen may contain protein epitopes that make them particularly effective as immunogens against which anti-Tyr immune responses 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.

- 38 043982

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

The Tyr consensus antigen may be a nucleic acid sequence of SEQ ID NO: 1 that encodes the amino acid sequence of SEQ ID NO: 2. The sequence of SEQ ID NO: 1 encodes a Tyr consensus protein linked to an IgE leader sequence. The Tyr consensus protein can be coupled to an IgE leader sequence and an HA tag. In other embodiments, the Tyr consensus protein may be free or not associated with an IgE leader sequence and/or an HA tag.

In some embodiments, the Tyr consensus antigen may be a nucleic acid sequence having at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity relative to the entire length of the nucleic acid sequence set forth in SEQ ID NO: 1. In other embodiments, the Tyr consensus antigen may be a nucleic acid 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 relative to the entire amino acid length sequence set forth in SEQ ID NO: 2. The 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 with respect to the entire length of the amino acid sequence presented in SEQ ID NO: 2.

Some embodiments of the invention relate to nucleic acid sequences encoding proteins homologous to the Tyr consensus protein, an immunogenic fragment of the Tyr consensus protein, and immunogenic fragments of homologous proteins. Nucleic acid molecules may be provided that encode immunogenic proteins that have up to 95% homology to a consensus sequence, up to 96% homology to a consensus sequence, up to 97% homology to a consensus sequence, up to 98% homology to a consensus sequence, and up to 99% homology to a consensus sequence. . Likewise, nucleic acid sequences encoding immunogenic fragments presented herein and immunogenic fragments of proteins homologous to proteins presented herein are also provided.

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

- 39 043982 cabbage soup homologous protein sequences described in this document.

Some embodiments of the invention relate to nucleic acid sequences encoding proteins with a certain percentage of identity to full-length Tyr consensus proteins, immunogenic fragments of the Tyr consensus protein, and immunogenic protein fragments having identity to the Tyr consensus protein. Nucleic acid molecules can also be proposed that encode immunogenic proteins that have up to 80% identity with respect to the full-length Tyr consensus sequence, up to 85% identity with respect to the full-length 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 Tyr consensus sequence, up to 92% identity relative to the full-length Tyr consensus sequence, up to 93% identity relative to the full-length Tyr consensus sequence, up to 94% identity relative to the full-length Tyr consensus sequence, up to 95% identity relative to the full-length Tyr consensus sequence, up to 96% identity relative to the full-length Tyr Tyr consensus sequence, up to 97% identity with respect to the full-length Tyr consensus sequence, up to 98% identity with respect to the full-length Tyr consensus sequence, and up to 99% identity with respect to the full-length Tyr consensus sequence. Likewise, nucleic acid sequences encoding the immunogenic fragments presented herein and immunogenic protein fragments with similar percentage identities as stated above to the Tyr proteins presented herein are also provided.

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

Some embodiments of the invention relate to fragments of SEQ ID NO: 1. The fragments may be at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% , at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the sequence, set forth in SEQ ID NO: 1. The fragments may be at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous to the fragments of SEQ ID NO: 1. Fragments may be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, according to 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 In embodiments of the invention, the fragments contain sequences that encode 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 ID NO: 2. The amino acid sequence of the Tyr consensus protein associated with the IgE leader sequence is SEQ ID NO: 2. The amino acid sequence of the Tyr consensus protein associated with the IgE leader sequence may be connected to the HA mark.

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 presented in SEQ ID NO: 2. Some embodiments of the invention relate to immunogenic proteins that have 96% homology to the consensus protein sequences presented in SEQ ID NO: 2. Some embodiments of the invention relate to immunogenic proteins that have 97% homology to the consensus protein sequences presented in SEQ ID NO: 2. Some embodiments The inventions relate to immunogenic proteins that have 98% homology to the consensus protein sequences presented in SEQ ID NO: 2. Some embodiments of the invention relate to immunogenic proteins that have 99% homology to the consensus protein sequences presented in SEQ ID NO: 2 .

Some embodiments of the invention provide proteins that are identical to SEQ ID NO: 2. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 80% identical to the full-length consensus amino

- 40 043982 acid sequence presented in SEQ ID NO: 2. Some embodiments of the invention relate to immunogenic proteins that have an amino acid sequence that is 85% identical to the full-length consensus amino acid sequence presented in SEQ ID NO: 2. Some embodiments of the invention refer to immunogenic proteins that have an amino acid sequence that is 90% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 2. Some embodiments of the invention relate to immunogenic proteins that have an amino acid sequence that is 91% identical to the full-length consensus amino acid sequence sequence set forth in SEQ ID NO: 2. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 92% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 2. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 93% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 2. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 94% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 2. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 95% identical to the full-length consensus amino acid sequence presented in SEQ ID NO: 2. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence a sequence that is 96% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 2. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 97% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 2. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 98% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 2. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 99% identical to the full-length consensus amino acid sequence shown in SEQ ID NO: 2.

In some embodiments, the protein does not contain a leader sequence. In some embodiments, the protein does not contain an IgE leader sequence. 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 may be provided SEQ ID NO: 2. 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%, according 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 presented 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.

Immunogenic protein fragments may be provided with amino acid sequences homologous 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 at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70 %, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of a protein that is 95% or more homologous to the sequence set forth in SEQ ID NO: 2. Some embodiments of the invention provide immunogenic fragments that have 96% homology to immunogenic fragments of the consensus protein sequences provided herein. Some embodiments of the invention provide immunogenic fragments that have 97% homology to immunogenic fragments of the consensus protein sequences presented herein. Neko

- 41 043982 Other embodiments of the invention provide immunogenic fragments that have 98% homology to the immunogenic fragments of the consensus protein sequences presented herein. Some embodiments of the invention provide immunogenic fragments that have 99% homology to immunogenic fragments of the consensus protein sequences presented 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 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: 2. In some embodiments The fragments of the invention 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 stated herein with respect to the attachment of a signal peptide or leader sequence to the N-terminus of a protein, the signal peptide/leader sequence replaces the N-terminal methionine of the protein, which is encoded by the start codon of the nucleic acid sequence, as opposed to protein coding sequences without signal peptides.

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

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

As described above for tyrosinase, tyrosinase-dependent protein 1 (TYRP-1) may also be involved in melanin synthesis and the pigment mechanisms of melanocytes and may be recognized by the immune system in subjects suffering from melanoma.

Accordingly, TYRP-1 may be an antigen associated with melanoma.

The TRYP-1 antigen can induce an antigen-specific T-cell response and/or high antibody titers, thereby eliciting or inducing an immune response that targets or responds to the cancer or tumor expressing the antigen. In some embodiments, the induced or evoked immune response may be a cellular, humoral, or both cellular and humoral immune response. 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 evoked immune response may reduce or inhibit one or more immunosuppressive factors that promote the growth of a tumor or cancer expressing an antigen, such as, but not limited to, factors that suppress MHC presentation, factors that increase levels of antigen-specific regulatory T cells (Treg), PD-L1, FasL, cytokines such as IL-10 and TFG-β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by immunosuppressive cells, CTLA-4, PD-1 , MDSCs, MCP-1 and immune checkpoint molecule, which are described in more detail below.

TYRP-1 antigen may contain protein epitopes that make them particularly effective as immunogens against which anti-TYRP-1 immune responses 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 nucleic acid sequence encoding the TYRP-1 consensus antigen may

- 42 043982 be optimized in terms of the use of codons and corresponding RNA transcripts. The nucleic acid encoding the TYRP-1 consensus antigen can be codon-optimized and RNA-optimized for expression. In some embodiments, the nucleic acid sequence encoding the TYRP-1 consensus antigen may contain a Kozak sequence (eg, GCC ACC) to improve translation efficiency. The nucleic acid encoding the TYRP-1 consensus antigen may contain multiple stop codons (eg, TGA TGA) to enhance 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 via 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 contain a nucleotide sequence encoding an IgE leader sequence.

The TYRP-1 consensus antigen may be a nucleic acid sequence of SEQ ID NO: 3 that encodes the amino acid sequence of SEQ ID NO: 4. The sequence of SEQ ID NO: 3 encodes a TYRP-1 consensus protein fused to an IgE leader sequence. The TYRP-1 consensus protein can be coupled to an IgE leader sequence and an HA tag. In other embodiments, the TYRP-1 consensus protein may be free or not associated with an IgE leader sequence and/or an HA tag.

In some embodiments, the TYRP-1 consensus antigen may be a nucleic acid sequence having at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity relative to the entire length of the nucleic acid sequence set forth in SEQ ID NO: 3. In other embodiments, the TYRP-1 consensus antigen may be a nucleic acid sequence that encodes an amino acid a 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 relative to the entire length of the amino acid sequence set forth 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%. Identity of 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with respect to the entire length of the amino acid sequence presented in SEQ ID NO: 4.

Some embodiments of the invention relate to nucleic acid 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. Nucleic acid molecules may be provided that encode immunogenic proteins that have up to 95% homology to a consensus sequence, up to 96% homology to a consensus sequence, up to 97% homology to a consensus sequence, up to 98% homology to a consensus sequence, and up to 99% homology to a consensus sequence. . Likewise, nucleic acid sequences encoding immunogenic fragments presented herein and immunogenic fragments of proteins homologous to proteins presented herein are also provided.

Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 95% homology to the nucleic acid coding sequences provided herein. Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 96% homology to the nucleic acid coding sequences provided herein. Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 97% homology to the nucleic acid coding sequences provided herein. Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 98% homology to the nucleic acid coding sequences provided herein. Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 99% homology to the nucleic acid coding sequences provided herein. In some embodiments, nucleic acid molecules with coding sequences disclosed 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.

- 43 043982

Some embodiments of the invention relate to nucleic acid sequences encoding proteins with a certain percentage of identity to full-length TYRP-1 consensus proteins, immunogenic fragments of the TYRP-1 consensus protein, and immunogenic protein fragments having identity to the TYRP-1 consensus protein. Nucleic acid molecules can also be proposed 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 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 to the full-length TYRP-1 consensus sequence, up to 96% identity to the full-length TYRP-1 consensus sequence, up to 97% identity to the full-length TYRP-1 consensus sequence, up to 98% identity to the full-length TYRP-1 consensus sequence and up to 99% identity relative to the full-length TYRP-1 consensus sequence. Likewise, nucleic acid sequences encoding the immunogenic fragments presented herein and immunogenic protein fragments with similar percentage identities as stated above to the TYRP-1 proteins presented herein are also provided.

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

Some embodiments of the invention relate to fragments of SEQ ID NO: 3. The fragments may be at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% , at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the sequence, set forth in SEQ ID NO: 3. The fragments may be at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous to the fragments of SEQ ID NO: 3. Fragments may be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, according to 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 In embodiments of the invention, the fragments contain sequences that encode 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 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 can be connected to 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 presented in SEQ ID NO: 4. Some embodiments of the invention relate to immunogenic proteins that have 96% homology to the consensus protein sequences presented in SEQ ID NO: 4. Some embodiments of the invention relate to immunogenic proteins that have 97% homology to the consensus protein sequences presented in SEQ ID NO: 4. Some embodiments The inventions relate to immunogenic proteins that have 98% homology to the consensus protein sequences presented in SEQ ID NO: 4. Some embodiments of the invention relate to immunogenic proteins that have 99% homology to the consensus protein sequences presented in SEQ ID NO: 4 .

Some embodiments of the invention provide proteins that are identical to SEQ ID NO: 4. Some embodiments of the invention provide immunogenic proteins that have

- 44 043982 an amino acid sequence that is 80% identical to the full-length consensus amino acid sequence presented in SEQ ID NO: 4. Some embodiments of the invention relate to immunogenic proteins that have an amino acid sequence that is 85% identical to the full-length consensus amino acid sequence presented in SEQ ID NO: 4. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 90% identical to the full-length consensus amino acid sequence presented in SEQ ID NO: 4. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence a sequence that is 91% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 4. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 92% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 4. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 93% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 4. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 94% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 4. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 95% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 4. Some embodiments embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 96% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 4. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 97% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 4. Some embodiments of the invention relate to immunogenic proteins that have an amino acid sequence that is 98% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 4. Some embodiments of the invention relate to immunogenic proteins that have an amino acid sequence that is 99% identical to the full-length consensus amino acid sequence 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 may be provided SEQ ID NO: 4. 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%, according 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 presented in SEQ ID NO: 4. 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 homologous to the immunogenic fragments of SEQ ID NO: 4. Such immunogenic fragments may contain at least 10%, at least 15%, at least 20%, at least 25%, at least at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70 %, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of a protein that is 95% or more homologous to the sequence set forth in SEQ ID NO: 4. Some embodiments of the invention provide immunogenic fragments that have 96% homology to immunogenic fragments of the consensus protein sequences provided herein.

Some embodiments of the invention relate to immunogenic fragments that

- 45 043982 have 97% homology to the immunogenic fragments of the consensus protein sequences presented in this document. Some embodiments of the invention provide immunogenic fragments that have 98% homology to immunogenic fragments of the consensus protein sequences presented herein. Some embodiments of the invention provide immunogenic fragments that have 99% homology to immunogenic fragments of the consensus protein sequences presented 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: 4. Such immunogenic fragments may contain at least 10%, at least 15%, at least 20%, at least 25%, at least 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 of the invention 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 stated herein with respect to the attachment of a signal peptide or leader sequence to the N-terminus of a protein, the signal peptide/leader sequence replaces the N-terminal methionine of the protein, which is encoded by the start codon of the nucleic acid sequence, as opposed to protein coding sequences without signal peptides.

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

The immunogenic composition of the present invention may comprise 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-characterized melanocyte-specific enzyme that, in combination with tyrosinase and TYRP1, plays a role in the conversion of L-tyrosine to melanin in melanocytes. DCT specifically catalyzes the tautomerization of chromium L-DOPA melanin precursors to 5,6-dihydroindole-2-carboxylic acid (DHICA), which is subsequently oxidized by TYRP1 (as discussed above) to form eumelanin. Studies have shown that TYRP2/DCT may be a mediator of drug resistance in melanoma cells with specificity for DNA-damaging agents. Since TYRP2/DCT has been frequently reported to be highly expressed in melanomas, this melanocyte-specific enzyme plays an important role in promoting the intrinsic resistant phenotype of melanomas to various DNA-damaging antitumor drugs.

As described above for tyrosinase, tyrosinase-dependent protein 2 (TYRP-2) may also be involved in melanin synthesis and 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 an antigen associated with melanoma.

The TRYP-2 antigen can induce an antigen-specific T-cell response and/or high antibody titers, thereby eliciting or inducing an immune response that targets or responds to the cancer or tumor expressing the antigen. In some embodiments, the induced or evoked immune response may be a cellular, humoral, or both cellular and humoral immune response. 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 evoked immune response may reduce or inhibit one or more immunosuppressive factors that promote the growth of a tumor or cancer expressing an antigen, such as, but not limited to, factors that suppress MHC presentation, factors that increase levels of antigen-specific regulatory T cells (Treg), PD-L1, FasL, cytokines such as IL-10 and TFG-β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by immunosuppressive cells, CTLA-4, PD-1 , MDSCs, MCP-1 and immune checkpoint molecule, which are described in more detail below.

- 46 043982

TYRP2 antigen may contain protein epitopes that make them particularly effective as immunogens against which anti-TYRP2 immune responses 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 nucleic acid sequence encoding the TYRP2 consensus antigen can be optimized for codon usage and corresponding RNA transcripts. The nucleic acid encoding the TYRP2 consensus antigen can be codon optimized and RNA optimized for expression. In some embodiments, the nucleic acid sequence encoding the TYRP2 consensus antigen may contain a Kozak sequence (eg, GCC ACC) to improve translation efficiency. The nucleic acid encoding the TYRP2 consensus antigen may contain multiple stop codons (eg, TGA TGA) to enhance 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 via 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 contain a nucleotide sequence encoding an IgE leader sequence.

The TYRP2 consensus antigen may be a nucleic acid sequence of SEQ ID NO: 5 that encodes the amino acid sequence of SEQ ID NO: b. SEQ ID NO: 5 encodes the TYRP2 consensus protein fused to an IgE leader sequence. The TYRP2 consensus protein can be coupled to an IgE leader sequence and an HA tag. In other embodiments, the TYRP2 consensus protein may be free or not associated with an IgE leader sequence and/or an HA tag.

In some embodiments, the TYRP2 consensus antigen may be a nucleic acid sequence having at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity relative to the entire length of the nucleic acid sequence set forth in SEQ ID NO: 5. In other embodiments, the TYRP2 consensus antigen may be a nucleic acid 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 relative to the entire amino acid length sequence set forth in SEQ ID NO: 6. The 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% identity with respect to the entire length of the amino acid sequence presented in SEQ ID NO: 6.

Some embodiments of the invention relate to nucleic acid sequences encoding proteins homologous to the TYRP2 consensus protein, an immunogenic fragment of the TYRP2 consensus protein, and immunogenic fragments of homologous proteins. Nucleic acid molecules may be provided that encode immunogenic proteins that have up to 95% homology to a consensus sequence, up to 96% homology to a consensus sequence, up to 97% homology to a consensus sequence, up to 98% homology to a consensus sequence, and up to 99% homology to a consensus sequence. . Likewise, nucleic acid sequences encoding immunogenic fragments presented herein and immunogenic fragments of proteins homologous to proteins presented herein are also provided.

Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 95% homology to the nucleic acid coding sequences provided herein. Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 96% homology to the nucleic acid coding sequences provided herein. Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 97% homology to the nucleic acid coding sequences provided herein. Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 98% homology to the nucleic acid coding sequences provided herein. Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 99% homology to the nucleic acid coding sequences provided herein. In no

- 47 043982 in which embodiments of the invention, nucleic acid molecules with coding sequences disclosed herein that are homologous to the 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 herein.

Some embodiments of the invention relate to nucleic acid sequences encoding proteins with a certain percentage of identity to full-length TYRP2 consensus proteins, immunogenic fragments of the TYRP2 consensus protein, and immunogenic protein fragments having identity to the TYRP2 consensus protein. Nucleic acid molecules that encode immunogenic proteins that have up to 80% identity with respect to the full-length TYRP2 consensus sequence, up to 85% identity with respect to the full-length TYRP2 sequence, up to 90% identity with respect to the full-length TYRP2 consensus sequence, up to 91% identity with respect to 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 full-length TYRP2 consensus sequence, up to 97% identity to the full-length TYRP2 consensus sequence, up to 98% identity to the full-length TYRP2 consensus sequence, and up to 99% identity to the full-length TYRP2 consensus sequence. Likewise, nucleic acid sequences encoding the immunogenic fragments presented herein and immunogenic protein fragments with similar percentage identities as stated above to the TYRP2 proteins presented herein are also provided.

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

Some embodiments of the invention relate to fragments of SEQ ID NO: 5. The fragments may be at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% , at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the sequence, set forth in SEQ ID NO: 5. The fragments may be at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous to the fragments of SEQ ID NO: 5. Fragments may be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, according to 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: 5. In some In embodiments of the invention, the fragments contain sequences that encode 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 connected to the HA mark.

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

- 48 043982

NO: 6. Certain embodiments of the invention provide 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 SEQ ID NO: 6. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 80% identical to the full-length consensus amino acid sequence presented in SEQ ID NO: 6. Some embodiments embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 85% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 6. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 90% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 6. Some embodiments of the invention relate to immunogenic proteins that have an amino acid sequence that is 91% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 6. Some embodiments of the invention relate to immunogenic proteins that have an amino acid sequence that is 92% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 6. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 93% identical to the full-length consensus amino acid sequence, set forth in SEQ ID NO: 6. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 94% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 6. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 95% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 6. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 96% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 6. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 97% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 6. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that which is 98% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 6. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 99% identical to the full-length consensus amino acid sequence 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. 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 may be provided SEQ ID NO: 6. 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%, according 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 presented in SEQ ID NO: 6. 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 homologous to the immunogenic fragments of SEQ ID NO: 6. Such immunogenic fragments may contain at least 10%, at least 15%, at least 20%, at least 25%, at least 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 By

- 49 043982 at least 99% of a protein that is 95% or more homologous to the sequence presented in SEQ ID NO: 6. Some embodiments of the invention provide immunogenic fragments that have 96% homology to the immunogenic fragments of the consensus protein sequences presented herein document. Some embodiments of the invention provide immunogenic fragments that have 97% homology to immunogenic fragments of the consensus protein sequences presented herein. Some embodiments of the invention provide immunogenic fragments that have 98% homology to immunogenic fragments of the consensus protein sequences presented herein. Some embodiments of the invention provide immunogenic fragments that have 99% homology to immunogenic fragments of the consensus protein sequences presented 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: 6. Such immunogenic fragments may contain at least 10%, at least 15%, at least 20%, at least 25%, at least 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 of the invention 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 stated herein with respect to the attachment of a signal peptide or leader sequence to the N-terminus of a protein, the signal peptide/leader sequence replaces the N-terminal methionine of the protein, which is encoded by the start codon of the nucleic acid sequence, as opposed to protein coding sequences without signal peptides.

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

The immunogenic composition of the present invention may contain Melanoma-associated Antigen 4 (MAGEA4), a fragment thereof, or a variant thereof. MAGE-A4, encoded by the MAGE-A4 gene, is a 317 amino acid protein expressed in male germ cells and tumor cells of various histological types, such as gastrointestinal, esophageal and lung carcinomas. MAGE-A4 binds to the oncoprotein, gankyrin. This specific binding of MAGE-A4 is mediated by its C-terminus. Studies have shown that exogenous MAGE-A4 can partially inhibit the 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, indicating that interactions between gankyrin and MAGE-A4 inhibit gankyrin-mediated carcinogenesis. It is likely that MAGE expression in tumor tissue is not a cause but a result of tumorigenesis, and MAGE genes participate in the immune process by targeting early tumor cells for destruction.

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

The MAGEA4 antigen can induce an antigen-specific T cell response and/or high antibody titers, thereby eliciting or inducing an immune response that targets or responds to the cancer or tumor expressing the antigen. In some embodiments, the induced or evoked immune response may be a cellular, humoral, or both cellular and humoral immune response. 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 immunosuppressive factors that promote tumor or cancer growth, expression

- 50 043982 antigen, such as, but not limited to, factors that suppress MHC presentation, factors that increase levels of antigen-specific regulatory T cells (Treg), PD-L1, FasL, cytokines such as IL-10 and TFG- β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by immunosuppressive cells, CTLA-4, PD-1, MDSCs, MCP-1 and immune checkpoint molecule, which are described in more detail below.

MAGEA4 antigen may contain protein epitopes that make them particularly effective as immunogens against which anti-MAGEA4 immune responses 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 nucleic acid sequence encoding the MAGEA4 consensus antigen can be optimized for codon usage and corresponding RNA transcripts. The nucleic acid encoding the MAGEA4 consensus antigen can be codon optimized and RNA optimized for expression. In some embodiments, the nucleic acid sequence encoding the MAGEA4 consensus antigen may contain a Kozak sequence (eg, GCC ACC) to improve translation efficiency.

The nucleic acid encoding the MAGEA4 consensus antigen may contain multiple stop codons (eg, TGA TGA) to enhance 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 via 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 contain a nucleotide sequence encoding an IgE leader sequence.

The MAGEA4 consensus antigen may be the nucleic acid 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 fused to an IgE leader sequence. The MAGEA4 consensus protein can be coupled to an IgE leader sequence and an HA tag. In other embodiments, the MAGEA4 consensus protein may be free or not associated with an IgE leader sequence and/or an HA tag.

In some embodiments, the MAGEA4 consensus antigen may be a nucleic acid sequence having at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity relative to the entire length of the nucleic acid sequence set forth in SEQ ID NO: 7. In other embodiments, the MAGEA4 consensus antigen may be a nucleic acid 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 relative to the entire amino acid length sequence set forth 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%. Identity of 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with respect to the entire length of the amino acid sequence presented in SEQ ID NO: 8.

Some embodiments of the invention relate to nucleic acid sequences encoding proteins homologous to the MAGEA4 consensus protein, an immunogenic fragment of the MAGEA4 consensus protein, and immunogenic fragments of homologous proteins. Nucleic acid molecules may be provided that encode immunogenic proteins that have up to 95% homology to a consensus sequence, up to 96% homology to a consensus sequence, up to 97% homology to a consensus sequence, up to 98% homology to a consensus sequence, and up to 99% homology to a consensus sequence. . Likewise, nucleic acid sequences encoding immunogenic fragments presented herein and immunogenic fragments of proteins homologous to proteins presented herein are also provided.

Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 95% homology to the nucleic acid coding sequences provided herein. Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 96% homology to the nucleic acid coding sequences provided herein. Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 97% homology to the nucleic acid coding sequences provided herein. Some options

- 51 043982 embodiments of the invention relate to nucleic acid molecules that encode immunogenic proteins that have 98% homology to the nucleic acid coding sequences presented herein. Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 99% homology to the nucleic acid coding sequences provided herein. In some embodiments, nucleic acid molecules with coding sequences disclosed 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 nucleic acid sequences encoding proteins with a certain percentage of identity to full-length MAGEA4 consensus proteins, immunogenic fragments of the MAGEA4 consensus protein, and immunogenic protein fragments having identity to the MAGEA4 consensus protein. Nucleic acid molecules can also be proposed that encode immunogenic proteins that have up to 80% identity with respect to the full-length MAGEA4 consensus sequence, up to 85% identity with respect to the full-length sequence, up to 90% identity with respect to the full-length MAGEA4 consensus sequence, up to 91% identity with respect 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. Likewise, nucleic acid sequences encoding the immunogenic fragments presented herein and immunogenic protein fragments with similar percentage identities as stated above to the MAGEA4 proteins presented herein are also provided.

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

Some embodiments of the invention relate to fragments of SEQ ID NO: 7. The fragments may be at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% , at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the sequence, set forth in SEQ ID NO: 7. The fragments may be at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous to the fragments of SEQ ID NO: 7. Fragments may be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, according to 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: 7. In some In embodiments of the invention, the fragments contain sequences that encode 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 may be connected to the HA mark.

Some embodiments of the invention relate to proteins that are homologous to SEQ ID NO: 8. Some embodiments of the invention relate to immunogenic proteins that have 95% homology to the consensus protein sequences presented in SEQ ID NO: 8. Some embodiments of the invention relate to immunogenic proteins that have

- 52 043982

96% homology to the consensus protein sequences presented in SEQ ID NO: 8. Some embodiments of the invention relate to immunogenic proteins that have 97% homology to the consensus protein sequences presented in SEQ ID NO: 8. Some embodiments of the invention relate to immunogenic proteins proteins that have 98% homology to the consensus protein sequences presented in SEQ ID NO: 8. Some embodiments of the invention provide immunogenic proteins that have 99% homology to the consensus protein sequences presented in SEQ ID NO: 8.

Some embodiments of the invention provide proteins that are identical to SEQ ID NO: 8. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 80% identical to the full-length consensus amino acid sequence presented in SEQ ID NO: 8. Some embodiments Embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 85% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 8. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 90% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 8. Some embodiments of the invention relate to immunogenic proteins that have an amino acid sequence that is 91% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 8. Some embodiments of the invention relate to immunogenic proteins that have an amino acid sequence that is 92% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 8. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 93% identical to the full-length consensus amino acid sequence, set forth in SEQ ID NO: 8. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 94% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 8. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 95% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 8. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 96% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 8. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 97% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 8. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that which is 98% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 8. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 99% identical to the full-length consensus amino acid sequence set forth 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 may be provided SEQ ID NO: 8. 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%, according 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 presented in SEQ ID NO: 8. 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 homologous to the immunogenic fragments of SEQ ID NO: 8 may be provided. Such immunogenic fragments may

- 53 043982 must 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 a protein that is 95% or more homologous to the sequence presented in SEQ ID NO: 8. Some embodiments of the invention provide immunogenic fragments that have 96% homology to immunogenic fragments of the consensus protein sequences presented herein. Some embodiments of the invention provide immunogenic fragments that have 97% homology to immunogenic fragments of the consensus protein sequences presented herein. Some embodiments of the invention provide immunogenic fragments that have 98% homology to immunogenic fragments of the consensus protein sequences presented herein.

Some embodiments of the invention provide immunogenic fragments that have 99% homology to immunogenic fragments of the consensus protein sequences presented 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: 8. Such immunogenic fragments may contain at least 10%, at least 15%, at least 20%, at least 25%, at least 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 of the invention 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 stated herein with respect to the attachment of a signal peptide or leader sequence to the N-terminus of a protein, the signal peptide/leader sequence replaces the N-terminal methionine of the protein, which is encoded by the start codon of the nucleic acid sequence, as opposed to protein coding sequences without signal peptides.

(5) Growth hormone releasing hormone (GHRH).

The immunogenic composition of the present invention may contain the cancer antigen somatotropin-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, regulator of metabolism and body structure, from the pituitary gland. GHRH also stimulates the production of growth hormone. GHRH antagonists can inhibit the growth of various cancers, such as osteosarcoma, glioblastoma, prostate cancer, kidney cancer, pancreatic cancer, colorectal cancer and breast cancer. Accordingly, GHRH may be an antigen associated with various tumors.

The GHRH antigen can induce an antigen-specific T cell response and/or high antibody titers, thereby eliciting or inducing an immune response that targets or responds to the cancer or tumor expressing the antigen. In some embodiments, the induced or evoked immune response may be a cellular, humoral, or both cellular and humoral immune response. 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 evoked immune response may reduce or inhibit one or more immunosuppressive factors that promote the growth of a tumor or cancer expressing an antigen, such as, but not limited to, factors that suppress MHC presentation, factors that increase levels of antigen-specific regulatory T cells (Treg), PD-L1, FasL,

- 54 043982 cytokines such as IL-10 and TFG-β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by immunosuppressive cells, CTLA-4, PD-1, MDSCs, MCP-1 and immune checkpoint molecule , which are described in more detail below.

GHRH antigen may contain protein epitopes that make them particularly effective as immunogens against which anti-GHRH immune responses 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 nucleic acid sequence encoding the GHRH consensus antigen can be optimized for codon usage and corresponding RNA transcripts. The nucleic acid encoding the GHRH consensus antigen can be codon optimized and RNA optimized for expression. In some embodiments, the nucleic acid sequence encoding the GHRH consensus antigen may contain a Kozak sequence (eg, GCC ACC) to improve translation efficiency. The nucleic acid encoding the GHRH consensus antigen may contain multiple stop codons (eg, TGA TGA) to enhance 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 via 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 contain a nucleotide sequence encoding an IgE leader sequence.

The GHRH consensus antigen may be a nucleic acid sequence of SEQ ID NO: 9 that encodes the amino acid sequence of SEQ ID NO: 10. The sequence of SEQ ID NO: 9 encodes a GHRH consensus protein fused to an IgE leader sequence. The GHRH consensus protein can be coupled to an IgE leader sequence and an HA tag. In other embodiments, the GHRH consensus protein may be free or not associated with an IgE leader sequence and/or an HA tag.

In some embodiments, the GHRH consensus antigen may be a nucleic acid sequence having at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity relative to the entire length of the nucleic acid sequence set forth in SEQ ID NO: 9. In other embodiments, the GHRH consensus antigen may be a nucleic acid 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 relative to the entire amino acid length sequence set forth 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%. Identity of 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with respect to the entire length of the amino acid sequence presented in SEQ ID NO: 10.

Some embodiments of the invention relate to nucleic acid sequences encoding proteins homologous to the GHRH consensus protein, an immunogenic fragment of the GHRH consensus protein, and immunogenic fragments of homologous proteins. Nucleic acid molecules may be provided that encode immunogenic proteins that have up to 95% homology to a consensus sequence, up to 96% homology to a consensus sequence, up to 97% homology to a consensus sequence, up to 98% homology to a consensus sequence, and up to 99% homology to a consensus sequence. . Likewise, nucleic acid sequences encoding immunogenic fragments presented herein and immunogenic fragments of proteins homologous to proteins presented herein are also provided.

Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 95% homology to the nucleic acid coding sequences provided herein. Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 96% homology to the nucleic acid coding sequences provided herein. Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 97% homology to the nucleic acid coding sequences provided herein. Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 98% homology to nucleic acid coding sequences,

- 55 043982 presented in this document. Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 99% homology to the nucleic acid coding sequences provided herein. In some embodiments, nucleic acid molecules with coding sequences disclosed 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 nucleic acid sequences encoding proteins with a certain percentage of identity to full-length GHRH consensus proteins, immunogenic fragments of the GHRH consensus protein, and immunogenic protein fragments having identity to the GHRH consensus protein. Nucleic acid molecules can also be proposed that encode immunogenic proteins that have up to 80% identity with respect to the full-length GHRH consensus sequence, up to 85% identity with respect to the full-length sequence, up to 90% identity with respect to the full-length GHRH consensus sequence, up to 91% identity with respect to the full-length GHRH consensus sequence, up to 92% identity relative to the full-length GHRH consensus sequence, up to 93% identity relative to the full-length GHRH consensus sequence, up to 94% identity relative to the full-length GHRH consensus sequence, up to 95% identity relative to the full-length GHRH consensus sequence, up to 96% identity relative to the full length GHRH consensus sequence, up to 97% identity with respect to the full-length GHRH consensus sequence, up to 98% identity with respect to the full-length GHRH consensus sequence, and up to 99% identity with respect to the full-length GHRH consensus sequence. Likewise, nucleic acid sequences encoding the immunogenic fragments presented herein and immunogenic protein fragments with similar percentage identities as stated above to the GHRH proteins presented herein are also provided.

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

Some embodiments of the invention relate to fragments of SEQ ID NO: 9. The fragments may be at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% , at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the sequence, set forth in SEQ ID NO: 9. The fragments may be at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous to the fragments of SEQ ID NO: 9. Fragments may be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, according to 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: 9. In some In embodiments of the invention, the fragments contain sequences that encode 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 connected to the HA mark.

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 presented in SEQ ID NO: 10. Some embodiments of the invention relate to immunogenic proteins that have 96% homology with respect to the consensus protein sequences presented in SEQ ID NO: 10. Some embodiments of the invention relate to immunogenic proteins that

- 56 043982 which have 97% homology to the consensus protein sequences presented in SEQ ID NO: 10. Some embodiments of the invention relate to immunogenic proteins that have 98% homology to the consensus protein sequences presented in SEQ ID NO: 10. Some embodiments implementations of the invention relate to immunogenic proteins that have 99% homology with respect to the consensus protein sequences presented in SEQ ID NO: 10.

Some embodiments of the invention provide proteins that are identical to SEQ ID NO: 10. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 80% identical to the full-length consensus amino acid sequence presented in SEQ ID NO: 10. Some embodiments embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 85% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 10. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 90% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 10. Some embodiments of the invention relate to immunogenic proteins that have an amino acid sequence that is 91% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 10. Some embodiments of the invention relate to immunogenic proteins that have an amino acid sequence that is 92% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 10. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 93% identical to the full-length consensus amino acid sequence, set forth in SEQ ID NO: 10. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 94% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 10. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 95% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 10. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 96% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 10. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 97% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 10. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that which is 98% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 10. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 99% identical to the full-length consensus amino acid sequence set forth 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 may be provided SEQ ID NO: 10. 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%, according 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 presented in SEQ ID NO: 10. 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 homologous to the immunogenic fragments of SEQ ID NO: 10. Such immunogenic fragments may contain at least 10%, at least 15%, at least 20%, at least 25%, at least at least 30%, at least 35%, at least 40%, at least 45%, according to

- 57 043982 at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of a protein that is 95% or more homologous to the sequence shown in SEQ ID NO: 10. Some embodiments of the invention provide immunogenic fragments that have 96% homology to immunogenic fragments of the consensus protein sequences presented herein. Some embodiments of the invention provide immunogenic fragments that have 97% homology to immunogenic fragments of the consensus protein sequences presented herein. Some embodiments of the invention provide immunogenic fragments that have 98% homology to immunogenic fragments of the consensus protein sequences presented herein. Some embodiments of the invention provide immunogenic fragments that have 99% homology to immunogenic fragments of the consensus protein sequences presented 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: 10. Such immunogenic fragments may contain at least 10%, at least 15%, at least 20%, at least 25%, at least 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 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.

As stated herein with respect to the attachment of a signal peptide or leader sequence to the N-terminus of a protein, the signal peptide/leader sequence replaces the N-terminal methionine of the protein, which is encoded by the start codon of the nucleic acid sequence, as opposed to protein coding sequences without signal peptides.

(6) MART-1/Melan-A.

The immunogenic composition of the present invention may comprise 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, trafficking and processing, which is required for melanosome structure and maturation. Accordingly, MART-1 is required for the regulation of mammalian pigmentation. Defects in melanosome maturation are associated with cancer susceptibility. MART-1 can be expressed in numerous cancers, 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 be associated with the endoplasmic reticulum and melanosomes. MelanA can be recognized by cytotoxic T cells as an antigen on melanoma cells, but can also be associated with other tumors that are of melanocytic origin or differentiation (ie, cells that have melanosomes), such as clear cell sarcoma and melanotic neurofibroma. Accordingly, Melan-A may be an antigen associated with various tumors derived from cells having melanosomes.

The Melan-A antigen can induce an antigen-specific T-cell response and/or high antibody titers, thereby eliciting or inducing an immune response that targets or responds to the cancer or tumor expressing the antigen. In some embodiments, the induced or evoked immune response may be a cellular, humoral, or both cellular and humoral immune response. 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 implementations

- 58 043982 tions of the invention, the induced or evoked immune response may reduce or inhibit one or more immunosuppressive factors that promote the growth of a tumor or cancer expressing an antigen, for example, but not limited to, factors that suppress MHC presentation, factors that increase levels of antigen-specific regulatory T cells (Treg), PD-L1, FasL, cytokines such as IL-10 and TFG-β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by immunosuppressive cells, CTLA-4, PD-1 , MDSCs, MCP-1 and immune checkpoint molecule, which are described in more detail below.

Melan-A antigen may contain protein epitopes that make them particularly effective as immunogens against which anti-Melan-A immune responses 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 nucleic acid sequence encoding the Melan-A consensus antigen can be optimized in terms of codon usage and corresponding RNA transcripts. The nucleic acid encoding the Melan-A consensus antigen can be codon-optimized and RNA-optimized for expression. In some embodiments, the nucleic acid sequence encoding the Melan-A consensus antigen may contain a Kozak sequence (eg, GCC ACC) to improve 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 via 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 contain a nucleotide sequence encoding an IgE leader sequence.

The Melan-A consensus antigen may be the nucleic acid 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 fused to an IgE leader sequence. The Melan-A consensus protein can be coupled to an IgE leader sequence and an HA tag. In other embodiments, the Melan-A consensus protein may be free or not associated with an IgE leader sequence and/or an HA tag.

In some embodiments, the Melan-A consensus antigen may be a nucleic acid sequence having at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity relative to the entire length of the nucleic acid sequence set forth in SEQ ID NO: 11. In other embodiments, the Melan-A consensus antigen may be a nucleic acid sequence that encodes an amino acid a 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 relative to the entire length of the amino acid sequence set forth 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%. Identity of 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with respect to the entire length of the amino acid sequence presented in SEQ ID NO: 12.

Some embodiments of the invention relate to nucleic acid 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. Nucleic acid molecules may be provided that encode immunogenic proteins that have up to 95% homology to a consensus sequence, up to 96% homology to a consensus sequence, up to 97% homology to a consensus sequence, up to 98% homology to a consensus sequence, and up to 99% homology to a consensus sequence. . Likewise, nucleic acid sequences encoding immunogenic fragments presented herein and immunogenic fragments of proteins homologous to proteins presented herein are also provided.

Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 95% homology to the nucleic acid coding sequences provided herein. Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 96% homology to the nucleic acid coding sequences provided herein. Some embodiments of the invention relate to nucleic acid molecules

- 59 043982 acids that encode immunogenic proteins that have 97% homology with the nucleic acid coding sequences presented in this document. Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 98% homology to the nucleic acid coding sequences provided herein. Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 99% homology to the nucleic acid coding sequences provided herein. In some embodiments, nucleic acid molecules with coding sequences disclosed 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 nucleic acid sequences encoding proteins with a certain percentage of identity to full-length Melan-A consensus proteins, immunogenic fragments of the Melan-A consensus protein, and immunogenic protein fragments having identity to the Melan-A consensus protein. Nucleic acid molecules can also be proposed 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 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 to the full-length Melan-A consensus sequence, up to 96% identity to the full-length Melan-A consensus sequence, up to 97% identity to the full-length Melan-A consensus sequence, up to 98% identity to the full-length Melan-A consensus sequence, up to 99% identity relative to the full-length Melan-A consensus sequence. Likewise, nucleic acid sequences encoding the immunogenic fragments presented herein and immunogenic protein fragments with similar percentage identities as stated above to the Melan-A proteins presented herein are also provided.

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

Some embodiments of the invention relate to fragments of SEQ ID NO: 11. The fragments may be at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% , at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the sequence, set forth in 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 the fragments of SEQ ID NO: 11. Fragments may be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, according to 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: 11. In some In embodiments of the invention, the fragments contain sequences that encode 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 can be connected to 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 presented in SEQ ID NO: 12. Some embodiments The inventions relate to immunogenic proteins that have 96% homology to the consensus protein sequences presented in SEQ ID NO: 12. Some embodiments of the invention relate to immunogenic proteins that have 97% homology to the consensus protein sequences presented in SEQ ID NO: 12 Some embodiments of the invention provide immunogenic proteins that have 98% homology to the consensus protein sequences set forth in SEQ ID NO: 12. Some embodiments of the invention provide 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 SEQ ID NO: 12. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 80% identical to the full-length consensus amino acid sequence presented in SEQ ID NO: 12. Some embodiments embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 85% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 12. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 90% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 12. Some embodiments of the invention relate to immunogenic proteins that have an amino acid sequence that is 91% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 12. Some embodiments of the invention relate to immunogenic proteins that have an amino acid sequence that is 92% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 12. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 93% identical to the full-length consensus amino acid sequence, set forth in SEQ ID NO: 12. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 94% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 12. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 95% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 12. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 96% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 12. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 97% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 12. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that which is 98% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 12. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 99% identical to the full-length consensus amino acid sequence 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 may be provided SEQ ID NO: 12. 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%, according 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 presented in SEQ ID NO: 12. 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.

- 61 043982

Immunogenic protein fragments may be provided with amino acid sequences homologous 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 at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70 %, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of a protein that is 95% or more homologous to the sequence set forth in SEQ ID NO: 12. Some embodiments of the invention provide immunogenic fragments that have 96% homology to the immunogenic fragments of the consensus protein sequences provided herein. Some embodiments of the invention provide immunogenic fragments that have 97% homology to immunogenic fragments of the consensus protein sequences presented herein. Some embodiments of the invention provide immunogenic fragments that have 98% homology to immunogenic fragments of the consensus protein sequences presented herein. Some embodiments of the invention provide immunogenic fragments that have 99% homology to immunogenic fragments of the consensus protein sequences presented 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, according to 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: 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 stated herein with respect to the attachment of a signal peptide or leader sequence to the N-terminus of a protein, the signal peptide/leader sequence replaces the N-terminal methionine of the protein, which is encoded by the start codon of the nucleic acid sequence, as opposed to protein coding sequences without signal peptides.

(7) NY-ESO-1.

The immunogenic composition of the present invention may comprise New York esophageal cancer antigen-1 (NY-ESO-1; also referred to as CTAG1), a fragment thereof, or a variant thereof. NYESO-1, encoded by the CTAG1B gene, is a 180 amino acid long protein with a glycine-rich N-terminal region and an extremely hydrophobic C-terminal region. NY-ESO-1 has limited expression in normal tissues, but is often found in cancer. NY-ESO-1 can be expressed in numerous cancers, including, but not limited to, bladder cancer, colorectal cancer, esophageal cancer, gastric cancer, hepatocarcinoma, head and neck cancer, melanoma, non-small cell lung cancer, ovarian cancer, pancreatic cancer glands, synovial carcinoma and prostate cancer.

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

The NY-ESO-1 antigen can induce an antigen-specific T cell response and/or high antibody titers, thereby eliciting or inducing an immune response that targets or responds to cancer or tumor expressing the antigen. In some embodiments, the induced or evoked immune response may be a cellular, humoral, or both cellular and humoral immune response. 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

- 62 043982 one or more immunosuppressive factors that promote the growth of a tumor or cancer expressing an antigen, for example, but not limited to, factors that suppress MHC presentation, factors that increase levels of antigen-specific regulatory T cells (Treg), PD-L1 , FasL, cytokines such as IL-10 and TFG-β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by immunosuppressive cells, CTLA-4, PD-1, MDSCs, MCP-1 and immune checkpoint molecule , which are described in more detail below.

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

The NY-ESO-1 antigen may contain protein epitopes that make them particularly effective as immunogens against which anti-NYESO-1 immune responses can be induced. The NY-ESO-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 nucleic acid sequence encoding the NY-ESO-1 consensus antigen can be optimized in terms of codon usage and corresponding RNA transcripts. The nucleic acid encoding the NY-ESO-1 consensus antigen can be codon-optimized and RNA-optimized for expression. In some embodiments, the nucleic acid sequence encoding the NY-ESO-1 consensus antigen may contain a Kozak sequence (eg, GCC ACC) to improve translation efficiency. The nucleic acid encoding the NY-ESO-1 consensus antigen may contain multiple stop codons (eg, TGA TGA) to enhance 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 via 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 contain a nucleotide sequence encoding an IgE leader sequence.

The NY-ESO-1 consensus antigen may be the nucleic acid 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 fused to an IgE leader sequence. The NY-ESO-1 consensus protein can be coupled to an IgE leader sequence and an HA tag. In other embodiments, the NY-ESO-1 consensus protein may

- 63 043982 may be free or not associated with an IgE leader sequence and/or an HA tag.

In some embodiments, the NY-ESO-1 consensus antigen may be a nucleic acid sequence having at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity relative to the entire length of the nucleic acid sequence set forth in SEQ ID NO: 13. In other embodiments, the NY-ESO-1 consensus antigen may be a nucleic acid sequence which 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 with respect to the entire length of the amino acid sequence presented 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 with respect to the entire length of the amino acid sequence presented in SEQ ID NO: 14.

Some embodiments of the invention relate to nucleic acid 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. Nucleic acid molecules may be provided that encode immunogenic proteins that have up to 95% homology to a consensus sequence, up to 96% homology to a consensus sequence, up to 97% homology to a consensus sequence, up to 98% homology to a consensus sequence, and up to 99% homology to a consensus sequence. . Likewise, nucleic acid sequences encoding immunogenic fragments presented herein and immunogenic fragments of proteins homologous to proteins presented herein are also provided.

Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 95% homology to the nucleic acid coding sequences provided herein. Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 96% homology to the nucleic acid coding sequences provided herein. Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 97% homology to the nucleic acid coding sequences provided herein. Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 98% homology to the nucleic acid coding sequences provided herein. Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 99% homology to the nucleic acid coding sequences provided herein. In some embodiments, nucleic acid molecules with coding sequences disclosed 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 nucleic acid sequences encoding proteins with a certain percentage of identity to the full-length NY-ESO-1 consensus proteins, immunogenic fragments of the NY-ESO-1 consensus protein, and immunogenic protein fragments having identity to the NY-ESO-1 consensus protein . Nucleic acid molecules may also be provided 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 sequence, up to identity with respect to identity identity with respect to identity identity with respect to relatively relatively relatively relatively relatively relatively relatively full-size full-size full-size full-size full-size full-size full-size full-size consensus consensus consensus consensus consensus consensus consensus consensus sequence sequence sequence sequence sequence sequence sequence

NY-ESO-1, NY-ESO-1, NY-ESO-1, NY-ESO-1, NY-ESO-1, NY-ESO-1, NY-ESO-1, NY-ESO-1, full-length consensus sequences NY-ESO-1 and before before before before before before before

90%

91%

92%

93%

94%

95%

96%

97%

98%

99% full-length consensus sequence of NY-ESO-1. Likewise, nucleic acid sequences encoding the immunogenic fragments presented herein and immunogenic protein fragments with similar percentage identities as stated above to the NY-ESO-1 proteins presented herein are also provided.

- 64 043982

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

Some embodiments of the invention relate to fragments of SEQ ID NO: 13. The fragments may be at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% , at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the sequence, set forth in 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 the fragments of SEQ ID NO: 13. Fragments may be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, according to 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: 13. In some In embodiments of the invention, the fragments contain sequences that encode 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 an IgE leader sequence can be coupled to 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 presented in SEQ ID NO: 14. Some embodiments of the invention relate to immunogenic proteins that have 96% homology to the consensus protein sequences presented in SEQ ID NO: 14. Some embodiments of the invention relate to immunogenic proteins that have 97% homology to the consensus protein sequences presented in SEQ ID NO: 14. Some embodiments The inventions relate to immunogenic proteins that have 98% homology to the consensus protein sequences presented in SEQ ID NO: 14. Some embodiments of the invention relate to immunogenic proteins that have 99% homology to the consensus protein sequences presented in SEQ ID NO: 14 .

Some embodiments of the invention provide proteins that are identical to SEQ ID NO: 14. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 80% identical to the full-length consensus amino acid sequence presented in SEQ ID NO: 14. Some embodiments embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 85% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 14. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 90% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 14. Some embodiments of the invention relate to immunogenic proteins that have an amino acid sequence that is 91% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 14. Some embodiments of the invention relate to immunogenic proteins that have an amino acid sequence that is 92% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 14. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 93% identical to the full-length consensus amino acid sequence, set forth in SEQ ID NO: 14. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 94% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 14. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 95% identical to the full-length consensus amino acid sequence presented in SEQ ID NO: 14. Some variants

- 65 043982 embodiments of the invention relate to immunogenic proteins that have an amino acid sequence that is 96% identical to the full-length consensus amino acid sequence presented in SEQ ID NO: 14. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 97% identical to % identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 14. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 98% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 14. Some embodiments The inventions relate to immunogenic proteins that have an amino acid sequence that is 99% identical to the full-length consensus amino acid sequence presented 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 may be provided SEQ ID NO: 14. 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%, according 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 presented in SEQ ID NO: 14. 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 homologous to the immunogenic fragments of SEQ ID NO: 14. Such immunogenic fragments may contain at least 10%, at least 15%, at least 20%, at least 25%, at least at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70 %, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of a protein that is 95% or more homologous to the sequence set forth in SEQ ID NO: 14. Some embodiments of the invention provide immunogenic fragments that have 96% homology to the immunogenic fragments of the consensus protein sequences presented herein. Some embodiments of the invention provide immunogenic fragments that have 97% homology to immunogenic fragments of the consensus protein sequences presented herein. Some embodiments of the invention provide immunogenic fragments that have 98% homology to immunogenic fragments of the consensus protein sequences presented herein. Some embodiments of the invention provide immunogenic fragments that have 99% homology to immunogenic fragments of the consensus protein sequences presented 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: 14. Such immunogenic fragments may contain at least 10%, at least 15%, at least 20%, at least 25%, at least 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 invention

- 66 043982 tion 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 stated herein with respect to the attachment of a signal peptide or leader sequence to the N-terminus of a protein, the signal peptide/leader sequence replaces the N-terminal methionine of the protein, which is encoded by the start codon of the nucleic acid sequence, as opposed to protein coding sequences without signal peptides.

(8) NY-ESO-2.

The immunogenic composition of the present invention may comprise New York esophageal cancer antigen-2 (NY-ESO-2; also known as testicular cancer 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 testicular cancer 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. Additionally, NY-ESO-2 can be detected in 25-50% of tumor samples from melanomas, non-small cell lung cancer, bladder cancer, prostate cancer, and head and neck cancer. The gene encoding NY-ESO-2 also contains an alternative open reading frame that encodes a protein called CAMEL, a tumor antigen that is recognized by melanoma-specific cytotoxic T lymphocytes.

Like NY-ESO-1, NY-ESO-2 can be expressed in the testis and ovary. NY-ESO-2 may also be associated with various types of cancer and is immunogenic in subjects suffering from cancer or tumors. Accordingly, NY-ESO-2 may be an antigen associated with numerous tumors.

The NY-ESO-2 antigen can induce an antigen-specific T-cell response and/or high antibody titers, thereby eliciting or inducing an immune response that targets or responds to the cancer or tumor expressing the antigen. In some embodiments, the induced or evoked immune response may be a cellular, humoral, or both cellular and humoral immune response. 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 evoked immune response may reduce or inhibit one or more immunosuppressive factors that promote the growth of a tumor or cancer expressing an antigen, such as, but not limited to, factors that suppress MHC presentation, factors that increase levels of antigen-specific regulatory T cells (Treg), PD-L1, FasL, cytokines such as IL-10 and TFG-β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by immunosuppressive cells, CTLA-4, PD-1 , MDSCs, MCP-1 and immune checkpoint molecule, which are described in more detail below.

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

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

- 67 043982 times, 1400 times, 1500 times, 1600 times, 1700 times, 1800 times, 1900 times, 2000 times, 2100 times, 2200 times, 2300 times, 2400 times, 2500 times, 2600 times, 2700 times, 2800 times, 2900 times, 3000 times, 3100 times, 3200 times, 3300 times, 3400 times, 3500 times, 3600 times, 3700 times , 3800 times, 3900 times, 4000 times, 4100 times, 4200 times, 4300 times, 4400 times, 4500 times, 4600 times, 4700 times, 4800 times, 4900 times, 5000-fold, 5100-fold, 5200-fold, 5300-fold, 5400-fold, 5500-fold, 5600-fold, 5700-fold, 5800-fold, 5900-fold, or 6000-fold compared to IFN-γ levels in subject who was not injected with NY-ESO-2 antigen.

The NY-ESO-2 antigen may contain protein epitopes that make them particularly effective as immunogens against which anti-NYESO-2 immune responses can be induced. The NY-ESO-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 nucleic acid sequence encoding the NY-ESO-2 consensus antigen can be optimized in terms of codon usage and corresponding RNA transcripts. The nucleic acid encoding the NY-ESO-2 consensus antigen can be codon-optimized and RNA-optimized for expression. In some embodiments, the nucleic acid sequence encoding the NY-ESO-2 consensus antigen may contain a Kozak sequence (eg, GCC ACC) to improve translation efficiency. The nucleic acid encoding the NY-ESO-2 consensus antigen may contain multiple stop codons (eg, TGA TGA) to enhance 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 via 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 contain a nucleotide sequence encoding an IgE leader sequence.

The NY-ESO-2 consensus antigen may be the nucleic acid sequence of SEQ ID NO: 15, which encodes the amino acid sequence of SEQ ID NO: 16. The sequence of SEQ ID NO: 1 encodes the NY-ESO-2 consensus protein fused to an IgE leader sequence. The NY-ESO-2 consensus protein can be coupled to an IgE leader sequence and an HA tag. In other embodiments, the NY-ESO-2 consensus protein may be free or not associated with an IgE leader sequence and/or an HA tag.

In some embodiments, the NY-ESO-2 consensus antigen may be a nucleic acid sequence having at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity relative to the entire length of the nucleic acid sequence set forth in SEQ ID NO: 15. In other embodiments, the NY-ESO-2 consensus antigen may be a nucleic acid sequence which 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 with respect to the entire length of the amino acid sequence presented 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 with respect to the entire length of the amino acid sequence presented in SEQ ID NO: 16.

Some embodiments of the invention relate to nucleic acid 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. Nucleic acid molecules may be provided that encode immunogenic proteins that have up to 95% homology to a consensus sequence, up to 96% homology to a consensus sequence, up to 97% homology to a consensus sequence, up to 98% homology to a consensus sequence, and up to 99% homology to a consensus sequence. . Likewise, nucleic acid sequences encoding immunogenic fragments presented herein and immunogenic fragments of proteins homologous to proteins presented herein are also provided.

Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 95% homology to the nucleic acid coding sequences provided herein. Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 96% homology to the nucleic acid coding sequences provided herein. Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 97% homology to the nucleic acid coding sequences provided herein. Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 98% homology to the nucleic acid coding sequences provided herein. Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 99% homology to the nucleic acid coding sequences provided herein. In some embodiments, nucleic acid molecules with coding sequences disclosed 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 nucleic acid sequences encoding proteins with a certain percentage of identity to full-length NY-ESO-2 consensus proteins, immunogenic fragments of the NY-ESO-2 consensus protein, and immunogenic protein fragments having identity to the NY-ESO-2 consensus protein . Nucleic acid molecules may also be provided 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 sequence, up to identity with respect to identity identity with respect to identity identity with respect to relatively relatively relatively relatively relatively relatively relatively full-size full-size full-size full-size full-size full-size full-size full-size consensus consensus consensus consensus consensus consensus consensus consensus sequence sequence sequence sequence sequence sequence sequence

NY-ESO-2, NY-ESO-2, NY-ESO-2, NY-ESO-2, NY-ESO-2, NY-ESO-2, NY-ESO-2, NY-ESO-2, full-length consensus sequences NY-ESO-2 and before before before before before before before before

90%

91%

92%

93%

94%

95%

96%

97%

98%

99% full-length consensus sequence of NY-ESO-2. Likewise, nucleic acid sequences encoding the immunogenic fragments presented herein and immunogenic protein fragments with similar percentage identities as stated above to the NY-ESO-2 proteins presented herein are also provided.

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

Some embodiments of the invention relate to fragments of SEQ ID NO: 15. The fragments may be at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% , at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the sequence, set forth in 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 the fragments of SEQ ID NO: 15. Fragments may be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, according to 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: 15. In some In embodiments of the invention, the fragments contain sequences that encode 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 an IgE leader sequence can be coupled to an HA tag.

Some embodiments of the invention relate to proteins that are homologous to SEQ ID NO: 16. Some embodiments of the invention relate to immunogenic proteins that have

- 69 043982 have 95% homology to the consensus protein sequences presented in SEQ ID NO: 16. Some embodiments of the invention relate to immunogenic proteins that have 96% homology to the consensus protein sequences presented in SEQ ID NO: 16. Some embodiments The inventions relate to immunogenic proteins that have 97% homology to the consensus protein sequences presented in SEQ ID NO: 16. Some embodiments of the invention relate to immunogenic proteins that have 98% homology to the consensus protein sequences presented in SEQ ID NO: 16 Some embodiments of the invention provide 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 to SEQ ID NO: 16. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 80% identical to the full-length consensus amino acid sequence presented in SEQ ID NO: 16. Some embodiments embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 85% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 16. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 90% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 16. Some embodiments of the invention relate to immunogenic proteins that have an amino acid sequence that is 91% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 16. Some embodiments of the invention relate to immunogenic proteins that have an amino acid sequence that is 92% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 16. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 93% identical to the full-length consensus amino acid sequence, set forth in SEQ ID NO: 16. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 94% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 16. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 95% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 16. Some embodiments of the invention relate to immunogenic proteins that have an amino acid sequence that is 96% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 16. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 97% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 16. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that which is 98% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 16. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 99% identical to the full-length consensus amino acid sequence set forth 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 may be provided SEQ ID NO: 16. 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%, according 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 presented in SEQ ID NO: 16. 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.

- 70 043982

Immunogenic protein fragments may be provided with amino acid sequences homologous to the immunogenic fragments of SEQ ID NO: 16. Such immunogenic fragments may contain at least 10%, at least 15%, at least 20%, at least 25%, at least at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70 %, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of a protein that is 95% or more homologous to the sequence set forth in SEQ ID NO: 16. Some embodiments of the invention provide immunogenic fragments that have 96% homology to the immunogenic fragments of the consensus protein sequences presented herein. Some embodiments of the invention provide immunogenic fragments that have 97% homology to immunogenic fragments of the consensus protein sequences presented herein. Some embodiments of the invention provide immunogenic fragments that have 98% homology to immunogenic fragments of the consensus protein sequences presented herein. Some embodiments of the invention provide immunogenic fragments that have 99% homology to immunogenic fragments of the consensus protein sequences presented 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: 16. Such immunogenic fragments may contain at least 10%, at least 15%, at least 20%, at least 25%, at least 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 in amino acid sequence, set forth in SEQ ID NO: 16. 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 stated herein with respect to the attachment of a signal peptide or leader sequence to the N-terminus of a protein, the signal peptide/leader sequence replaces the N-terminal methionine of the protein, which is encoded by the start codon of the nucleic acid sequence, as opposed to protein coding sequences without signal peptides.

(9)PRAME.

The immunogenic composition of this invention may contain a PRAME antigen, a fragment thereof, or a variant thereof. PRAME, encoded by the PRAME gene, is a 509 amino acid protein expressed in the testis, placenta, endometrium, ovary, adrenal gland, and tissues derived from melanoma, lung carcinoma, renal carcinoma, and head and neck carcinoma. PRAME is also expressed in adult and pediatric acute leukemia and multiple myeloma. PRAME contains an immunogenic nonapeptide capable of inducing a cytotoxic response upon presentation of HLA-A24. Studies show that overexpression of PRAME in cultured cells causes caspase-independent cell death, leading to lower proliferation rates. Other studies demonstrate that overexpression of PRAME also provides growth or survival benefits by antagonizing retinoic acid receptor (RAR) signaling and is involved in the oncogenic process as a contributory cause. Interference of RAR signaling results in loss of regulation of cell proliferation, development, and differentiation.

PRAME may have a similar expression pattern to testicular cancer antigens MAG, BAGE and GAGE, namely expression in the testis. 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 can induce an antigen-specific T-cell response and/or high antibody titers, thereby eliciting or inducing an immune response that targets or responds to the cancer or tumor expressing the antigen. In some embodiments, the induced or elicited immune response may be cellular, humoral, or

- 71 043982 both cellular and humoral immune response. 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 evoked immune response may reduce or inhibit one or more immunosuppressive factors that promote the growth of a tumor or cancer expressing an antigen, such as, but not limited to, factors that suppress MHC presentation, factors that increase levels of antigen-specific regulatory T cells (Treg), PD-L1, FasL, cytokines such as IL-10 and TFG-β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by immunosuppressive cells, CTLA-4, PD-1 , MDSCs, MCP-1 and immune checkpoint molecule, which are described in more detail below.

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

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

PRAME antigen may contain protein epitopes that make them particularly effective as immunogens against which anti-PRAME immune responses 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 nucleic acid sequence encoding the PRAME consensus antigen can be optimized for codon usage and corresponding RNA transcripts. The nucleic acid encoding the PRAME consensus antigen can be codon optimized and RNA optimized for expression. In some embodiments, the nucleic acid sequence encoding the PRAME consensus antigen may contain a Kozak sequence (eg, GCC ACC) to improve translation efficiency. The nucleic acid encoding the PRAME consensus antigen may contain multiple stop codons (eg, TGA TGA) to enhance 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 via 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 contain a nucleotide sequence encoding an IgE leader sequence.

The PRAME consensus antigen may be a nucleic acid sequence

- 72 043982 you SEQ ID NO: 17, which encodes the amino acid sequence SEQ ID NO: 18. The sequence SEQ ID NO: 17 encodes the PRAME consensus protein connected to the IgE leader sequence. The PRAME consensus protein can be coupled to an IgE leader sequence and an HA tag. In other embodiments, the PRAME consensus protein may be free or not associated with an IgE leader sequence and/or an HA tag.

In some embodiments, the PRAME consensus antigen may be a nucleic acid sequence having at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity relative to the entire length of the nucleic acid sequence set forth in SEQ ID NO: 17. In other embodiments, the PRAME consensus antigen may be a nucleic acid 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 relative to the entire amino acid length sequence set forth in SEQ ID NO: 18. The PRAME 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 with respect to the entire length of the amino acid sequence presented in SEQ ID NO: 18.

Some embodiments of the invention relate to nucleic acid sequences encoding proteins homologous to the PRAME consensus protein, an immunogenic fragment of the PRAME consensus protein, and immunogenic fragments of homologous proteins. Nucleic acid molecules may be provided that encode immunogenic proteins that have up to 95% homology to a consensus sequence, up to 96% homology to a consensus sequence, up to 97% homology to a consensus sequence, up to 98% homology to a consensus sequence, and up to 99% homology to a consensus sequence. . Likewise, nucleic acid sequences encoding immunogenic fragments presented herein and immunogenic fragments of proteins homologous to proteins presented herein are also provided.

Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 95% homology to the nucleic acid coding sequences provided herein. Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 96% homology to the nucleic acid coding sequences provided herein. Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 97% homology to the nucleic acid coding sequences provided herein. Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 98% homology to the nucleic acid coding sequences provided herein. Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 99% homology to the nucleic acid coding sequences provided herein. In some embodiments, nucleic acid molecules with coding sequences disclosed 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 nucleic acid sequences encoding proteins with a certain percentage of identity to full-length PRAME consensus proteins, immunogenic fragments of the PRAME consensus protein, and immunogenic protein fragments having identity to the PRAME consensus protein. Nucleic acid molecules can also be proposed 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 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 with respect to the full-length PRAME consensus sequence, up to 98% identity with respect to the full-length PRAME consensus sequence, and up to 99% identity with respect to the full-length PRAME consensus sequence. Similarly, nucleic acid sequences have also been proposed

- 73 043982 encoding immunogenic fragments presented herein, and immunogenic protein fragments with similar percentage identities as indicated above to the PRAME proteins presented herein.

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

Some embodiments of the invention relate to fragments of SEQ ID NO: 17. The fragments may be at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% , at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the sequence, set forth in 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 the fragments of SEQ ID NO: 17. Fragments may be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, according to 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: 17. In some In embodiments of the invention, the fragments contain sequences that encode 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 connected to the HA mark.

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 presented in SEQ ID NO: 18. Some embodiments of the invention relate to immunogenic proteins that have 96% homology to the consensus protein sequences presented in SEQ ID NO: 18. Some embodiments of the invention relate to immunogenic proteins that have 97% homology to the consensus protein sequences presented in SEQ ID NO: 18. Some embodiments The inventions relate to immunogenic proteins that have 98% homology to the consensus protein sequences presented in SEQ ID NO: 18. Some embodiments of the invention relate to immunogenic proteins that have 99% homology to the consensus protein sequences presented in SEQ ID NO: 18 .

Some embodiments of the invention provide proteins that are identical to SEQ ID NO: 18. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 80% identical to the full-length consensus amino acid sequence presented in SEQ ID NO: 18. Some embodiments embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 85% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 18. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 90% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 18. Some embodiments of the invention relate to immunogenic proteins that have an amino acid sequence that is 91% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 18. Some embodiments of the invention relate to immunogenic proteins that have an amino acid sequence that is 92% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 18. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 93% identical to the full-length consensus amino acid sequence, presented in SEQ ID NO: 18. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 94% identical to the full-length consensus amino acid sequence presented

- 74 043982 in SEQ ID NO: 18. Some embodiments of the invention relate to immunogenic proteins that have an amino acid sequence that is 95% identical to the full-length consensus amino acid sequence presented in SEQ ID NO: 18. Some embodiments of the invention relate to immunogenic proteins that have an amino acid sequence that is 96% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 18. Some embodiments of the invention relate to immunogenic proteins that have an amino acid sequence that is 97% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 18. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 98% identical to the full-length consensus amino acid sequence presented in SEQ ID NO: 18. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence a sequence that is 99% identical to the full-length consensus amino acid sequence presented 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 may be provided SEQ ID NO: 18. 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%, according 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 presented in SEQ ID NO: 18. 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 homologous to the immunogenic fragments of SEQ ID NO: 18. Such immunogenic fragments may contain at least 10%, at least 15%, at least 20%, at least 25%, at least at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70 %, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of a protein that is 95% or more homologous to the sequence set forth in SEQ ID NO: 18. Some embodiments of the invention provide immunogenic fragments that have 96% homology to the immunogenic fragments of the consensus protein sequences provided herein. Some embodiments of the invention provide immunogenic fragments that have 97% homology to immunogenic fragments of the consensus protein sequences presented herein. Some embodiments of the invention provide immunogenic fragments that have 98% homology to immunogenic fragments of the consensus protein sequences presented herein. Some embodiments of the invention provide immunogenic fragments that have 99% homology to immunogenic fragments of the consensus protein sequences presented 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: 18. Such immunogenic fragments may contain at least 10%, at least 15%, at least 20%, at least 25%, at least 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

- 75 043982 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 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 stated herein with respect to the attachment of a signal peptide or leader sequence to the N-terminus of a protein, the signal peptide/leader sequence replaces the N-terminal methionine of the protein, which is encoded by the start codon of the nucleic acid sequence, as opposed to protein coding sequences without signal peptides.

(10) PSA.

The immunogenic composition of the present invention may contain prostate-specific antigen (PSA; also known as seminoprotein gamma or kallikrein-3 (KLK3)), a fragment thereof, or a variant thereof. PSA is an androgen-regulated serine protease produced by prostate epithelial cells and prostate cancer cells and is encoded by the KLK3 gene. PSA is often used as a serum marker in prostate cancer. PSA is a member of the tissue kallikrein family and cleaves the semenogelins in the seminal coagulum after proenzyme cleavage to release the active enzyme, thereby liquefying the sperm so that the sperm can have fluidity. In addition, the enzymatic activity of PSA is regulated by zinc concentration, and high concentrations of zinc inhibit the enzymatic activity of PSA.

The PSA antigen can induce an antigen-specific T-cell response and/or high antibody titers, thereby eliciting or inducing an immune response that targets or responds to the cancer or tumor expressing the antigen. In some embodiments, the induced or evoked immune response may be a cellular, humoral, or both cellular and humoral immune response. 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 evoked immune response may reduce or inhibit one or more immunosuppressive factors that promote the growth of a tumor or cancer expressing an antigen, such as, but not limited to, factors that suppress MHC presentation, factors that increase levels of antigen-specific regulatory T cells (Treg), PD-L1, FasL, cytokines such as IL-10 and TFG-β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by immunosuppressive cells, CTLA-4, PD-1 , MDSCs, MCP-1 and immune checkpoint molecule, which are described in more detail below.

PSA antigen may contain protein epitopes that make them particularly effective as immunogens against which anti-PSA immune responses can be induced. The PSA 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 PSA consensus antigen may be a nucleic acid sequence of SEQ ID NO: 63, which encodes the amino acid sequence of SEQ ID NO: 64. The PSA consensus protein may be linked to an IgE leader sequence and an HA tag. In other embodiments, the PSA consensus protein may be free or not associated with an IgE leader sequence and/or an HA tag. The PSA consensus sequence may be operably linked to a regulatory sequence, including, but not limited to, a start codon and at least one stop codon.

In some embodiments, the PSA consensus antigen may be a nucleic acid sequence having at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity relative to the entire length of the nucleic acid sequence set forth in SEQ ID NO: 63. In other embodiments, the PSA consensus antigen may be a nucleic acid 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 relative to the entire amino acid length sequence set forth in SEQ ID NO: 64. The PSA 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: 64.

Some embodiments of the invention relate to nucleic acid sequences

- 76 043982 slot encoding proteins homologous to the PSA consensus protein, an immunogenic fragment of the PSA consensus protein and immunogenic fragments of homologous proteins. Nucleic acid molecules may be provided that encode immunogenic proteins that have up to 95% homology to a consensus sequence, up to 96% homology to a consensus sequence, up to 97% homology to a consensus sequence, up to 98% homology to a consensus sequence, and up to 99% homology to a consensus sequence. . Likewise, nucleic acid sequences encoding immunogenic fragments presented herein and immunogenic fragments of proteins homologous to proteins presented herein are also provided.

Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 95% homology to the nucleic acid coding sequences provided herein. Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 96% homology to the nucleic acid coding sequences provided herein. Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 97% homology to the nucleic acid coding sequences provided herein. Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 98% homology to the nucleic acid coding sequences provided herein. Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 99% homology to the nucleic acid coding sequences provided herein. In some embodiments, nucleic acid molecules with coding sequences disclosed 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 nucleic acid sequences encoding proteins with a certain percentage of identity to full-length PSA consensus proteins, immunogenic fragments of the PSA consensus protein, and immunogenic protein fragments having identity to the PSA consensus protein. Nucleic acid molecules may also be provided that encode immunogenic proteins that have up to 80% identity to the full-length PSA consensus sequence, up to 85% identity to the full-length sequence, up to 90% identity to the full-length PSA consensus sequence, up to 91% identity to the full-length PSA consensus sequence, up to 92% identity with respect to the full-length PSA consensus sequence, up to 93% identity with respect to the full-length PSA consensus sequence, up to 94% identity with respect to the full-length PSA consensus sequence, up to 95% identity with respect to the full-length PSA consensus sequence, up to 96% identity with respect to the full-length PSA consensus sequence, up to 97% identity to the full-length PSA consensus sequence, up to 98% identity to the full-length PSA consensus sequence, and up to 99% identity to the full-length PSA consensus sequence. Likewise, nucleic acid sequences encoding the immunogenic fragments presented herein and immunogenic protein fragments with similar percentage identities as stated above to the PSA proteins presented herein are also provided.

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

Some embodiments of the invention relate to fragments of SEQ ID NO: 63. The fragments may be at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% , at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the sequence, set forth in SEQ ID NO: 63. The fragments may be at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous to the fragments of SEQ ID NO: 63. Fragments may be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, according to at least 94%, at least 95%, according to

- 77 043982 are at least 96%, at least 97%, at least 98%, or at least 99% identical to the fragments of SEQ ID NO: 63. In some embodiments, the fragments contain sequences that encode 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 PSA consensus protein is SEQ ID NO: 64. The amino acid sequence of the PSA consensus protein associated with the IgE leader sequence is SEQ ID NO: 64. The amino acid sequence of the PSA consensus protein associated with the IgE leader sequence may be connected to the HA mark.

Some embodiments of the invention relate to proteins that are homologous to SEQ ID NO: 64. Some embodiments of the invention relate to immunogenic proteins that have 95% homology to the consensus protein sequences presented in SEQ ID NO: 64. Some embodiments of the invention relate to immunogenic proteins that have 96% homology to the consensus protein sequences presented in SEQ ID NO: 64. Some embodiments of the invention relate to immunogenic proteins that have 97% homology to the consensus protein sequences presented in SEQ ID NO: 64. Some embodiments The inventions relate to immunogenic proteins that have 98% homology to the consensus protein sequences presented in SEQ ID NO: 64. Some embodiments of the invention relate to immunogenic proteins that have 99% homology to the consensus protein sequences presented in SEQ ID NO: 64 .

Some embodiments of the invention provide proteins that are identical to SEQ ID NO: 64. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 80% identical to the full-length consensus amino acid sequence presented in SEQ ID NO: 64. Some embodiments embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 85% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 64. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 90% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 64. Some embodiments of the invention relate to immunogenic proteins that have an amino acid sequence that is 91% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 64. Some embodiments of the invention relate to immunogenic proteins that have an amino acid sequence that is 92% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 64. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 93% identical to the full-length consensus amino acid sequence, set forth in SEQ ID NO: 64. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 94% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 64. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 95% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 64. Some embodiments of the invention relate to immunogenic proteins that have an amino acid sequence that is 96% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 64. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 97% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 64. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that which is 98% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 64. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 99% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 64.

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

- 78 043982 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 may be provided SEQ ID NO: 64. 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%, according 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 presented in SEQ ID NO: 64. 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 homologous to the immunogenic fragments of SEQ ID NO: 64. Such immunogenic fragments may contain at least 10%, at least 15%, at least 20%, at least 25%, at least at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70 %, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of a protein that is 95% or more homologous to the sequence set forth in SEQ ID NO: 64. Some embodiments of the invention provide immunogenic fragments that have 96% homology to the immunogenic fragments of the consensus protein sequences provided herein. Some embodiments of the invention provide immunogenic fragments that have 97% homology to immunogenic fragments of the consensus protein sequences presented herein. Some embodiments of the invention provide immunogenic fragments that have 98% homology to immunogenic fragments of the consensus protein sequences presented herein. Some embodiments of the invention provide immunogenic fragments that have 99% homology to immunogenic fragments of the consensus protein sequences presented 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: 64. 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, according to 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: 64. 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 stated herein with respect to the attachment of a signal peptide or leader sequence to the N-terminus of a protein, the signal peptide/leader sequence replaces the N-terminal methionine of the protein, which is encoded by the start codon of the nucleic acid sequence, as opposed to protein coding sequences without signal peptides.

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

- 79 043982 to increase the efficiency of translation termination.

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 via 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 contain a nucleotide sequence encoding an IgE leader sequence.

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

(11) PSMA.

The immunogenic composition of the present invention may contain cancer prostate-specific membrane antigen (PSMA; also known as glutamate carboxypeptidase II (GCPII), N-acetyl-L-aspartyl-L-glutamate peptidase I (NAALADase I) and NAAG peptidase), a fragment thereof, or option. PSMA is encoded by the folate hydrolase 1 (FOLH1) gene. PSMA is a zinc metalloenzyme found in membranes and extracellular spaces. PSMA is highly expressed in the human prostate and has increased expression levels in prostate cancer. PSMA has also been found to be overexpressed in other forms of cancer, such as solid tumors of the kidney, breast and colon.

PSMA antigen can induce an antigen-specific T-cell response and/or high antibody titers, thereby eliciting or inducing an immune response that targets or responds to cancer or tumor expressing the antigen. In some embodiments, the induced or evoked immune response may be a cellular, humoral, or both cellular and humoral immune response. 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 evoked immune response may reduce or inhibit one or more immunosuppressive factors that promote the growth of a tumor or cancer expressing an antigen, such as, but not limited to, factors that suppress MHC presentation, factors that increase levels of antigen-specific regulatory T cells (Treg), PD-L1, FasL, cytokines such as IL-10 and TFG-β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by immunosuppressive cells, CTLA-4, PD-1 , MDSCs, MCP-1 and immune checkpoint molecule, which are described in more detail below.

PSMA antigen may contain protein epitopes that make them particularly effective as immunogens against which anti-PSMA immune responses 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 PSMA consensus antigen may be a nucleic acid sequence of SEQ ID NO: 65 that encodes the amino acid sequence of SEQ ID NO: 66. The PSMA consensus protein may be linked to an IgE leader sequence and an HA tag. In other embodiments, the PSMA consensus protein may be free or not associated with an IgE leader sequence and/or an HA tag. The PSMA consensus sequence may be operably linked to a regulatory sequence, including, but not limited to, a start codon and at least one stop codon.

In some embodiments, the PSMA consensus antigen may be a nucleic acid sequence having at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity relative to the entire length of the nucleic acid sequence set forth in SEQ ID No: 65. In other embodiments, the PSMA consensus antigen may be a nucleic acid 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 relative to the entire amino acid length sequence set forth in SEQ ID NO: 66. The PSMA 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 with respect to the entire length of the amino acid sequence presented in SEQ ID NO: 66.

Some embodiments of the invention relate to nucleic acid sequences encoding proteins homologous to the PSMA consensus protein, an immunogenic fragment of con

- 80 043982 PSMA sensus protein and immunogenic fragments of homologous proteins. Nucleic acid molecules may be provided that encode immunogenic proteins that have up to 95% homology to a consensus sequence, up to 96% homology to a consensus sequence, up to 97% homology to a consensus sequence, up to 98% homology to a consensus sequence, and up to 99% homology to a consensus sequence. . Likewise, nucleic acid sequences encoding immunogenic fragments presented herein and immunogenic fragments of proteins homologous to proteins presented herein are also provided.

Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 95% homology to the nucleic acid coding sequences provided herein. Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 96% homology to the nucleic acid coding sequences provided herein. Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 97% homology to the nucleic acid coding sequences provided herein. Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 98% homology to the nucleic acid coding sequences provided herein. Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 99% homology to the nucleic acid coding sequences provided herein. In some embodiments, nucleic acid molecules with coding sequences disclosed 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 nucleic acid sequences encoding proteins with a certain percentage of identity to full-length PSMA consensus proteins, immunogenic fragments of the PSMA consensus protein, and immunogenic protein fragments having identity to the PSMA consensus protein. Nucleic acid molecules can also be proposed that encode immunogenic proteins that have up to 80% identity with respect to the full-length PSMA consensus sequence, up to 85% identity with respect to the full-length sequence, up to 90% identity with respect to the full-length PSMA consensus sequence, up to 91% identity with respect to the full-length PSMA consensus sequence, up to 92% identity relative to the full-length PSMA consensus sequence, up to 93% identity relative to the full-length PSMA consensus sequence, up to 94% identity relative to the full-length PSMA consensus sequence, up to 95% identity relative to the full-length PSMA consensus sequence, up to 96% identity relative to the full length PSMA consensus sequence, up to 97% identity with respect to the full-length PSMA consensus sequence, up to 98% identity with respect to the full-length PSMA consensus sequence, and up to 99% identity with respect to the full-length PSMA consensus sequence. Likewise, nucleic acid sequences encoding the immunogenic fragments presented herein and immunogenic protein fragments with similar percentage identities as stated above to the PSMA proteins presented herein are also provided.

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

Some embodiments of the invention are directed to fragments of SEQ ID NO: 65. The fragments may be at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% , at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the sequence, set forth in SEQ ID NO: 65. The fragments may be at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous to the fragments of SEQ ID NO: 65. Fragments may be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, according to at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%

- 81 043982 are identical to the fragments of SEQ ID NO: 65. In some embodiments, the fragments contain sequences that encode 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 PSMA consensus protein is SEQ ID NO: 66. The amino acid sequence of the PSMA consensus protein associated with the IgE leader sequence is SEQ ID NO: 66. The amino acid sequence of the PSMA consensus protein associated with the IgE leader sequence may be connected to the HA mark.

Some embodiments of the invention relate to proteins that are homologous to SEQ ID NO: 66. Some embodiments of the invention relate to immunogenic proteins that have 95% homology to the consensus protein sequences presented in SEQ ID NO: 66. Some embodiments of the invention relate to immunogenic proteins that have 96% homology to the consensus protein sequences presented in SEQ ID NO: 66. Some embodiments of the invention relate to immunogenic proteins that have 97% homology to the consensus protein sequences presented in SEQ ID NO: 66. Some embodiments The inventions relate to immunogenic proteins that have 98% homology to the consensus protein sequences presented in SEQ ID NO: 66. Some embodiments of the invention relate to immunogenic proteins that have 99% homology to the consensus protein sequences presented in SEQ ID NO: 66 .

Some embodiments of the invention provide proteins that are identical to SEQ ID NO: 66. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 80% identical to the full-length consensus amino acid sequence presented in SEQ ID NO: 66. Some embodiments Embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 85% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 66. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 90% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 66. Some embodiments of the invention relate to immunogenic proteins that have an amino acid sequence that is 91% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 66. Some embodiments of the invention relate to immunogenic proteins that have an amino acid sequence that is 92% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 66. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 93% identical to the full-length consensus amino acid sequence, set forth in SEQ ID NO: 66. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 94% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 66. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 95% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 66. Some embodiments of the invention relate to immunogenic proteins that have an amino acid sequence that is 96% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 66. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 97% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 66. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that which is 98% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 66. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 99% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 66.

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%,

- 82 043982 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 may be provided SEQ ID NO: 66. 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%, according 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 presented in SEQ ID NO: 66. 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 homologous to the immunogenic fragments of SEQ ID NO: 66. Such immunogenic fragments may contain at least 10%, at least 15%, at least 20%, at least 25%, at least at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70 %, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of a protein that is 95% or more homologous to the sequence set forth in SEQ ID NO: 66. Some embodiments of the invention provide immunogenic fragments that have 96% homology to the immunogenic fragments of the consensus protein sequences provided herein.

Some embodiments of the invention provide immunogenic fragments that have 97% homology to immunogenic fragments of the consensus protein sequences presented herein. Some embodiments of the invention provide immunogenic fragments that have 98% homology to immunogenic fragments of the consensus protein sequences presented herein. Some embodiments of the invention provide immunogenic fragments that have 99% homology to immunogenic fragments of the consensus protein sequences presented 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: 66. Such immunogenic fragments may contain at least 10%, at least 15%, at least 20%, at least 25%, at least 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 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: 66. 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 stated herein with respect to the attachment of a signal peptide or leader sequence to the N-terminus of a protein, the signal peptide/leader sequence replaces the N-terminal methionine of the protein, which is encoded by the start codon of the nucleic acid sequence, as opposed to protein coding sequences without signal peptides.

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

- 83 043982 to increase the efficiency of translation termination.

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 via 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 contain a nucleotide sequence encoding an IgE leader sequence.

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

(12) STEAP.

The immunogenic composition of the present invention may contain the cancer antigen six 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 tissues and has increased expression levels in cancer cells. STEAP is proposed to be a six-transmembrane protein and is a cell surface antigen found at cell junctions.

The STEAP antigen can induce an antigen-specific T-cell response and/or high antibody titers, thereby eliciting or inducing an immune response that targets or responds to the cancer or tumor expressing the antigen. In some embodiments, the induced or evoked immune response may be a cellular, humoral, or both cellular and humoral immune response. 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 evoked immune response may reduce or inhibit one or more immunosuppressive factors that promote the growth of a tumor or cancer expressing an antigen, such as, but not limited to, factors that suppress MHC presentation, factors that increase levels of antigen-specific regulatory T cells (Treg), PD-L1, FasL, cytokines such as IL-10 and TFG-β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by immunosuppressive cells, CTLA-4, PD-1 , MDSCs, MCP-1 and immune checkpoint molecule, which are described in more detail below.

STEAP antigen may contain protein epitopes that make them particularly effective as immunogens against which anti-STEAP immune responses 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 STEAP consensus antigen may be a nucleic acid sequence of SEQ ID NO: 67 that encodes the amino acid sequence of SEQ ID NO: 68. The STEAP consensus protein may be linked to an IgE leader sequence and an HA tag. In other embodiments, the STEAP consensus protein may be free or not associated with an IgE leader sequence and/or an HA tag. The STEAP consensus sequence may be operably linked to a regulatory sequence, including, but not limited to, a start codon and at least one stop codon.

In some embodiments, the STEAP consensus antigen may be a nucleic acid sequence having at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity relative to the entire length of the nucleic acid sequence set forth in SEQ ID NO: 67. In other embodiments, the STEAP consensus antigen may be a nucleic acid 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 relative to the entire amino acid length sequence set forth in SEQ ID NO: 68. The STEAP 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: 68.

Some embodiments of the invention relate to nucleic acid sequences encoding proteins homologous to the STEAP consensus protein, an immunogenic fragment of the STEAP consensus protein, and immunogenic fragments of homologous proteins. Nucleic acid molecules can be proposed that encode immunogenic proteins that have up to 95%

- 84 043982 homology relative to the consensus sequence, up to 96% homology relative to the consensus sequence, up to 97% homology relative to the consensus sequence, up to 98% homology relative to the consensus sequence and up to 99%. Likewise, nucleic acid sequences encoding immunogenic fragments presented herein and immunogenic fragments of proteins homologous to proteins presented herein are also provided.

Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 95% homology to the nucleic acid coding sequences provided herein. Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 96% homology to the nucleic acid coding sequences provided herein. Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 97% homology to the nucleic acid coding sequences provided herein. Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 98% homology to the nucleic acid coding sequences provided herein. Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 99% homology to the nucleic acid coding sequences provided herein. In some embodiments, nucleic acid molecules with coding sequences disclosed 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 nucleic acid sequences encoding proteins with a certain percentage of identity to full-length STEAP consensus proteins, immunogenic fragments of the STEAP consensus protein, and immunogenic protein fragments having identity to the STEAP consensus protein. Nucleic acid molecules can also be proposed that encode immunogenic proteins that have up to 80% identity with respect to the full-length STEAP consensus sequence, up to 85% identity with respect to the full-length STEAP sequence, up to 90% identity with respect to the full-length STEAP consensus sequence, up to 91% identity with respect to full-length STEAP consensus sequence, up to 92% identity relative to the full-length STEAP consensus sequence, up to 93% identity relative to the full-length STEAP consensus sequence, up to 94% identity relative to the full-length STEAP consensus sequence, up to 95% identity relative to the full-length STEAP consensus sequence, up to 96% identity relative to full-length STEAP consensus sequence, up to 97% identity relative to the full-length STEAP consensus sequence, up to 98% identity relative to the full-length STEAP consensus sequence, and up to 99% identity relative to the full-length STEAP consensus sequence. Likewise, nucleic acid sequences encoding the immunogenic fragments presented herein and immunogenic protein fragments with similar percentage identities as stated above to the STEAP proteins presented herein are also provided.

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

Some embodiments of the invention relate to fragments of SEQ ID NO: 67. The fragments may be at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% , at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the sequence, set forth in SEQ ID NO: 67. The fragments may be at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous to the fragments of SEQ ID NO: 67. Fragments may be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, according to 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: 67. In some In embodiments of the invention, the fragments contain sequences that encode a leader sequence, such as, for example,

- 85 043982 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 STEAP consensus protein is SEQ ID NO: 68. The amino acid sequence of the STEAP consensus protein associated with the IgE leader sequence is SEQ ID NO: 68. The amino acid sequence of the STEAP consensus protein associated with the IgE leader sequence may be connected to the HA mark.

Some embodiments of the invention relate to proteins that are homologous to SEQ ID NO: 68. Some embodiments of the invention relate to immunogenic proteins that have 95% homology to the consensus protein sequences presented in SEQ ID NO: 68. Some embodiments of the invention relate to immunogenic proteins that have 96% homology to the consensus protein sequences presented in SEQ ID NO: 68. Some embodiments of the invention relate to immunogenic proteins that have 97% homology to the consensus protein sequences presented in SEQ ID NO: 68. Some embodiments The inventions relate to immunogenic proteins that have 98% homology to the consensus protein sequences presented in SEQ ID NO: 68. Some embodiments of the invention relate to immunogenic proteins that have 99% homology to the consensus protein sequences presented in SEQ ID NO: 68 .

Some embodiments of the invention provide proteins that are identical to SEQ ID NO: 68. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 80% identical to the full-length consensus amino acid sequence presented in SEQ ID NO: 68. Some embodiments embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 85% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 68. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 90% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 68. Some embodiments of the invention relate to immunogenic proteins that have an amino acid sequence that is 91% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 68. Some embodiments of the invention relate to immunogenic proteins that have an amino acid sequence that is 92% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 68. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 93% identical to the full-length consensus amino acid sequence, set forth in SEQ ID NO: 68. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 94% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 68. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 95% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 68. Some embodiments of the invention relate to immunogenic proteins that have an amino acid sequence that is 96% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 68. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 97% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 68. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that which is 98% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 68. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 99% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 68.

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%,

- 86 043982 at least 98% or at least 99% of the consensus protein. Immunogenic fragments may be provided SEQ ID NO: 68. 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%, according 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 presented in SEQ ID NO: 68. 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 homologous to the immunogenic fragments of SEQ ID NO: 68. Such immunogenic fragments may contain at least 10%, at least 15%, at least 20%, at least 25%, at least at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70 %, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of a protein that is 95% or more homologous to the sequence set forth in SEQ ID NO: 68. Some embodiments of the invention provide immunogenic fragments that have 96% homology to the immunogenic fragments of the consensus protein sequences provided herein. Some embodiments of the invention provide immunogenic fragments that have 97% homology to immunogenic fragments of the consensus protein sequences presented herein. Some embodiments of the invention provide immunogenic fragments that have 98% homology to immunogenic fragments of the consensus protein sequences presented herein. Some embodiments of the invention provide immunogenic fragments that have 99% homology to immunogenic fragments of the consensus protein sequences presented 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: 68. Such immunogenic fragments may contain at least 10%, at least 15%, at least 20%, at least 25%, at least 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: 68. In some embodiments The fragments of the invention 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 stated herein with respect to the attachment of a signal peptide or leader sequence to the N-terminus of a protein, the signal peptide/leader sequence replaces the N-terminal methionine of the protein, which is encoded by the start codon of the nucleic acid sequence, as opposed to protein coding sequences without signal peptides.

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

The nucleic acid encoding the STEAP consensus antigen may also encode a leader

- 87 043982 sequence of immunoglobulin E (IgE). 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 via 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 contain a nucleotide sequence encoding an IgE leader sequence.

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

(13) PSCA.

The immunogenic composition 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 activated in many cancers, including prostate, bladder, and pancreatic cancer.

The PSCA antigen can induce an antigen-specific T-cell response and/or high antibody titers, thereby eliciting or inducing an immune response that targets or responds to the cancer or tumor expressing the antigen. In some embodiments, the induced or evoked immune response may be a cellular, humoral, or both cellular and humoral immune response. 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 evoked immune response may reduce or inhibit one or more immunosuppressive factors that promote the growth of a tumor or cancer expressing an antigen, such as, but not limited to, factors that suppress MHC presentation, factors that increase levels of antigen-specific regulatory T cells (Treg), PD-L1, FasL, cytokines such as IL-10 and TFG-β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by immunosuppressive cells, CTLA-4, PD-1 , MDSCs, MCP-1 and immune checkpoint molecule, which are described in more detail below.

PSCA antigen may contain protein epitopes that make them particularly effective as immunogens against which anti-PSCA immune responses 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 PSCA consensus antigen may be a nucleic acid sequence of SEQ ID NO: 69, which encodes the amino acid sequence of SEQ ID NO: 70. The PSCA consensus protein may be linked to an IgE leader sequence and an HA tag. In other embodiments, the PSCA consensus protein may be free or not associated with an IgE leader sequence and/or an HA tag. The PSCA consensus sequence may be operably linked to a regulatory sequence, including, but not limited to, a start codon and at least one stop codon.

In some embodiments, the PSCA consensus antigen may be a nucleic acid sequence having at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity relative to the entire length of the nucleic acid sequence set forth in SEQ ID No: 69. In other embodiments, the PSCA consensus antigen may be a nucleic acid 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 relative to the entire amino acid length sequence set forth in SEQ ID NO: 70. The PSCA 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 with respect to the entire length of the amino acid sequence presented in SEQ ID NO: 70.

Some embodiments of the invention relate to nucleic acid sequences encoding proteins homologous to the PSCA consensus protein, an immunogenic fragment of the PSCA consensus protein, and immunogenic fragments of homologous proteins. Nucleic acid molecules can be proposed that encode immunogenic proteins that have up to 95% homology with respect to the consensus sequence, up to 96% homology with respect to the consensus sequence, up to 97% homology with respect to the consensus sequence, up to 98%

- 88 043982 homology relative to the consensus sequence and up to 99%. Likewise, nucleic acid sequences encoding immunogenic fragments presented herein and immunogenic fragments of proteins homologous to proteins presented herein are also provided.

Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 95% homology to the nucleic acid coding sequences provided herein. Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 96% homology to the nucleic acid coding sequences provided herein. Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 97% homology to the nucleic acid coding sequences provided herein. Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 98% homology to the nucleic acid coding sequences provided herein. Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 99% homology to the nucleic acid coding sequences provided herein. In some embodiments, nucleic acid molecules with coding sequences disclosed 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 nucleic acid sequences encoding proteins with a certain percentage of identity to full-length PSCA consensus proteins, immunogenic fragments of the PSCA consensus protein, and immunogenic protein fragments having identity to the PSCA consensus protein. Nucleic acid molecules that encode immunogenic proteins that have up to 80% identity with respect to the full-length PSCA consensus sequence, up to 85% identity with respect to the full-length PSCA sequence, up to 90% identity with respect to the full-length PSCA consensus sequence, up to 91% identity with respect to full-length PSCA consensus sequence, up to 92% identity relative to the full-length PSCA consensus sequence, up to 93% identity relative to the full-length PSCA consensus sequence, up to 94% identity relative to the full-length PSCA consensus sequence, up to 95% identity relative to the full-length PSCA consensus sequence, up to 96% identity relative to full-length PSCA consensus sequence, up to 97% identity to the full-length PSCA consensus sequence, up to 98% identity to the full-length PSCA consensus sequence, and up to 99% identity to the full-length PSCA consensus sequence. Likewise, nucleic acid sequences encoding the immunogenic fragments presented herein and immunogenic protein fragments with similar percentage identities as stated above to the PSCA proteins presented herein are also provided.

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

Some embodiments of the invention relate to fragments of SEQ ID NO: 69. The fragments may be at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% , at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the sequence, set forth in SEQ ID NO: 69. The fragments may be at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous to the fragments of SEQ ID NO: 69. Fragments may be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, according to 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: 69. In some In embodiments of the invention, the fragments contain sequences that encode 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,

- 89 043982 which 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 PSCA consensus protein is SEQ ID NO: 70. The amino acid sequence of the PSCA consensus protein associated with the IgE leader sequence is SEQ ID NO: 70. The amino acid sequence of the PSCA consensus protein associated with the IgE leader sequence may be connected to the HA mark.

Some embodiments of the invention relate to proteins that are homologous to SEQ ID NO: 70. Some embodiments of the invention relate to immunogenic proteins that have 95% homology to the consensus protein sequences presented in SEQ ID NO: 70. Some embodiments of the invention relate to immunogenic proteins that have 96% homology to the consensus protein sequences presented in SEQ ID NO: 70. Some embodiments of the invention relate to immunogenic proteins that have 97% homology to the consensus protein sequences presented in SEQ ID NO: 70. Some embodiments The inventions relate to immunogenic proteins that have 98% homology to the consensus protein sequences presented in SEQ ID NO: 70. Some embodiments of the invention relate to immunogenic proteins that have 99% homology to the consensus protein sequences presented in SEQ ID NO: 70 .

Some embodiments of the invention provide proteins that are identical to SEQ ID NO: 70. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 80% identical to the full-length consensus amino acid sequence presented in SEQ ID NO: 70. Some embodiments Embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 85% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 70. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 90% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 70. Some embodiments of the invention relate to immunogenic proteins that have an amino acid sequence that is 91% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 70. Some embodiments of the invention relate to immunogenic proteins that have an amino acid sequence that is 92% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 70. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 93% identical to the full-length consensus amino acid sequence, set forth in SEQ ID NO: 70. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 94% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 70. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 95% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 70. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 96% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 70. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 97% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 70. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that which is 98% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 70. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 99% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 70.

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 may be provided SEQ ID NO: 70. Immunogenic fragments may contain at least 10%,

- 90 043982 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%, according to at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the sequence set forth in SEQ ID NO: 70. 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 homologous to the immunogenic fragments of SEQ ID NO: 70. Such immunogenic fragments may contain at least 10%, at least 15%, at least 20%, at least 25%, at least at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70 %, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of a protein that is 95% or more homologous to the sequence set forth in SEQ ID NO: 70. Some embodiments of the invention provide immunogenic fragments that have 96% homology to the immunogenic fragments of the consensus protein sequences provided herein. Some embodiments of the invention provide immunogenic fragments that have 97% homology to immunogenic fragments of the consensus protein sequences presented herein. Some embodiments of the invention provide immunogenic fragments that have 98% homology to immunogenic fragments of the consensus protein sequences presented herein. Some embodiments of the invention provide immunogenic fragments that have 99% homology to immunogenic fragments of the consensus protein sequences presented 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: 70. Such immunogenic fragments may contain at least 10%, at least 15%, at least 20%, at least 25%, at least 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: 70. In some embodiments The fragments of the invention 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 stated herein with respect to the attachment of a signal peptide or leader sequence to the N-terminus of a protein, the signal peptide/leader sequence replaces the N-terminal methionine of the protein, which is encoded by the start codon of the nucleic acid sequence, as opposed to protein coding sequences without signal peptides.

The nucleic acid sequence encoding the PSCA consensus antigen can be optimized in terms of codon usage and corresponding RNA transcripts. The nucleic acid encoding the PSCA consensus antigen can be codon-optimized and RNA-optimized for expression. In some embodiments, the nucleic acid sequence encoding the PSCA consensus antigen may contain a Kozak sequence (eg, GCC ACC) to improve translation efficiency. The nucleic acid encoding the PSCA consensus antigen may contain multiple stop codons (eg, TGA TGA) to enhance 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 additionally encode an IgE leader sequence such that the amino acid

- 91 043982 lot sequence of the IgE leader sequence is linked to the amino acid sequence of the PSCA consensus antigen via 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 contain a nucleotide sequence encoding an IgE leader sequence.

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

(14) MAGE A1.

The immunogenic composition of the present invention may contain 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 is dependent on DNA methylation for its repression in normal somatic tissues. These genes are activated in many tumor types through the genome-wide demethylation process that often accompanies tumorigenesis. Particularly during neoplastic transformation, these genes are activated, expressed, and can become antigenic targets that are recognized and attacked by the immune system. Therefore, MAGE genes participate in the immune process by targeting some early tumor cells for immune destruction. MAGE A1 can be expressed in numerous cancers, including, but not limited to, melanomas, lung carcinomas and squamous cell carcinomas of the esophagus.

The MAGE A1 antigen can induce an antigen-specific T-cell response and/or high antibody titers, thereby eliciting or inducing an immune response that targets or responds to the cancer or tumor expressing the antigen. In some embodiments, the induced or evoked immune response may be a cellular, humoral, or both cellular and humoral immune response. 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 evoked immune response may reduce or inhibit one or more immunosuppressive factors that promote the growth of a tumor or cancer expressing an antigen, such as, but not limited to, factors that suppress MHC presentation, factors that increase levels of antigen-specific regulatory T cells (Treg), PD-L1, FasL, cytokines such as IL-10 and TFG-β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by immunosuppressive cells, CTLA-4, PD-1 , MDSCs, MCP-1 and immune checkpoint molecule, which are described in more detail below.

(15) WT1.

The immunogenic composition of the present invention may comprise 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 plays a role in normal development of the genitourinary system and interacts with numerous factors, such as p53, a known tumor suppressor, and the serine protease HtrA2, which cleaves WT1 at several sites after cytotoxic drug treatment.

Mutation of WT1 can lead to tumor or carcinogenesis, 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 tract tissue, lymphatic tissue, and lung tissue. Accordingly, Wilms tumor can be considered a metastatic tumor. Wilms tumor usually occurs in young children (eg, under 5 years of age) in both sporadic and hereditary forms. 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.

The WT-1 antigen can induce an antigen-specific T cell response and/or high antibody titers, thereby eliciting or inducing an immune response that targets or responds to the cancer or tumor expressing the antigen. In some embodiments, the induced or evoked immune response may be a cellular, humoral, or both cellular and humoral immune response. 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 immunosuppressive factors that promote the growth of a tumor or cancer expressing an antigen, such as, but not limited to, factors that inhibit MHC presentation, factors, which increase levels of antigen-specific regulatory T cells (Treg), PD-L1, FasL, cytokines such as IL-10 and TFG-β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by immunosuppressive cells, CTLA-4 , PD-1, MDSCs, MCP-1 and immune checkpoint molecule, which are described in more detail below.

Accordingly, the immunogenic composition can be used to treat subjects suffering from Wilms tumor. The immunogenic composition can be used to treat subjects suffering from any number of cancers, including, but not limited to, melanoma, prostate cancer, liver cancer, cervical cancer, recurrent respiratory papillomatosis (RRP), anal cancer, head cancer and neck and blood cancer. The immunogenic composition can also be used to treat subjects with 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 (ie, mutated WT1 genes or sequences) are provided herein.

The native WT1 gene transcripts are processed into multiple mRNAs, and the resulting proteins are not all of equal value in inducing an immune response. The WT1 mutant genes described herein escape alternative processing by producing a single full-length transcript and resulting in stronger induction of effector T and B cell responses. The first mutated WT1 sequence is referred to as CON WT1 modified zinc fingers or ConWT 1 L. SEQ ID NO: 19 is a nucleic acid sequence encoding the WT1 antigen CON WT1 modified zinc fingers. SEQ ID NO: 20 is the amino acid sequence of the WT1 CON WT1 antigen with modified zinc fingers. The second mutated WT1 sequence is called CON WT1 without zinc fingers or ConWT1-S. SEQ ID NO: 21 is a nucleic acid 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) to enhance expression. Modification may include codon optimization, RNA optimization, addition of a Kozak sequence (eg, GCC ACC) to enhance translation initiation, and/or addition of an immunoglobulin leader sequence to enhance the immunogenicity of the WT1 antigen. The WT1 antigen may 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 comprise a hemagglutinin (HA) tag. The WT1 consensus antigen can be engineered 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 a stronger and broader cellular and/or humoral immune response 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 represents any residue other than histidine (H). Specifically, the one or more mutations are that residues 312, 317, 330, 334, 342, 347, 36o 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 nucleic acid of SEQ ID NO: 19, which encodes SEQ ID NO: 20. In some embodiments, the WT1 consensus antigen may be a nucleic acid sequence having at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity relative to the entire length of the nucleic acid sequence represented by SEQ ID NO: 19. In others In embodiments of the invention, the WT1 consensus antigen may be a nucleic acid sequence that encodes an amino acid sequence having

- 93 043982 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 relative to the entire the length of the amino acid sequence shown in SEQ ID NO: 20.

In other embodiments, the WT1 consensus antigen may be a nucleic acid 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 with respect to the entire length of the amino acid sequence presented in SEQ ID NO: 20, provided that residues 312, 317, 342 and 347 of SEQ ID NO: 20 are any residues other than cysteine (C), and residues 330, 334, 360 and 364 of SEQ ID NO: 20 are any residues other than histidine (H). In other embodiments, the WT1 consensus antigen may be a nucleic acid 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 with respect to the entire length of the amino acid sequence presented in SEQ ID NO: 20, provided that residues 312, 317, 330, 334, 342, 347, 360 and 364 SEQ ID NO: 20 is 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 with respect to the entire length of the amino acid sequence set forth in SEQ ID NO: 20. The WT1 consensus antigen may be an amino acid sequence having at least 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 with respect to the entire length of the amino acid sequence represented in SEQ ID NO: 20, provided that residues 312, 317, 342 and 347 of SEQ ID NO: 20 are residues other than cysteine (C), and residues 330, 334, 360 and 364 of SEQ ID NO: 20 represent any residues other than histidine (H). In some embodiments, the WT1 consensus antigen may be an amino acid sequence having at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95 , 96, 97, 98 or 99% identity with respect to the entire length of the amino acid sequence presented 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 a nucleic acid of SEQ ID NO: 21 that encodes SEQ ID NO: 22. In some embodiments, the WT1 consensus antigen may be a nucleic acid sequence having at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity relative to the entire length of the nucleic acid sequence represented by SEQ ID NO: 21. In others In embodiments of the invention, the WT1 consensus antigen may be a nucleic acid 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 with respect to the entire length of the amino acid sequence presented 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 with respect to the entire length of the amino acid sequence presented in SEQ ID NO: 22.

Immunogenic fragments of SEQ ID NO: 20 and SEQ ID NO: 22 may be provided. 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% 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 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of SEQ ID NO: 20, provided that if residues 312, 317, 342 and 347 of SEQ ID NO: 20 are present in the immunogenic fragment, then these residues are any residues other than cysteine (C), and provided that if residues 330, 334, 360 and 364 of SEQ ID NO: 20 are present in immunogenic fragment, then these residues are any residues 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% SEQ ID NO: 20, provided that

- 94 043982 if residues 312, 317, 330, 334, 342, 347, 360 and 364 of SEQ ID NO: 20 are present in the immunogenic fragment, then these residues are glycine (G).

In some embodiments, the immunogenic 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 may be provided with amino acid sequences identical to the immunogenic fragments of SEQ ID NOs: 20 and 22. 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% proteins having 95% or more identity to SEQ ID NO: 20 and/or SEQ ID NO: 22. Some embodiments of the invention provide immunogenic fragments that have 96% or more identity to immunogenic fragments of the WT1 protein sequences presented herein document. Some embodiments of the invention provide immunogenic fragments that have 97% or more identity with the immunogenic fragments of the WT1 protein sequences provided herein. Some embodiments of the invention provide immunogenic fragments that have 98% or more identity with the immunogenic fragments of the WT1 protein sequences provided herein. Some embodiments of the invention provide immunogenic fragments that have 99% or more identity with the immunogenic fragments of the WT1 protein sequences provided 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 the immunogenic fragments of 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 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% 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 may be provided with nucleotide sequences identical to the immunogenic fragments of SEQ ID NO: 19 and SEQ ID NO: 21. 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 more identity to SEQ ID NO: 19 and/or SEQ ID NO: 21. Some embodiments of the invention provide immunogenic fragments that have 96% or more identity to the immunogenic fragments WT1 nucleic acid sequences presented herein. Some embodiments of the invention provide immunogenic fragments that have 97% or more identity to the immunogenic fragments of the WT1 nucleic acid sequences provided herein. Some embodiments of the invention provide immunogenic fragments that have 98% or more identity to the immunogenic fragments of the WT1 nucleic acid sequences provided herein. Some embodiments of the invention provide immunogenic fragments that have 99% or more identity to the immunogenic fragments of the WT1 nucleic acid sequences provided 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.

(16) FAP.

The immunogenic composition of the present invention may contain the cancer antigen fibroblast activating protein (FAP), a fragment thereof, or a variant thereof. The FAP antigen can induce an antigen-specific T-cell response and/or high antibody titers, thereby eliciting or inducing an immune response that targets or responds to the cancer or tumor expressing the antigen. In some embodiments, the induced or elicited immune response may be a cellular, humoral, or both cellular and humoral immune response.

- 95 043982 answer. 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 evoked immune response may reduce or inhibit one or more immunosuppressive factors that promote the growth of a tumor or cancer expressing an antigen, such as, but not limited to, factors that suppress MHC presentation, factors that increase levels of antigen-specific regulatory T cells (Treg), PD-L1, FasL, cytokines such as IL-10 and TFG-β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by immunosuppressive cells, CTLA-4, PD-1 , MDSCs, MCP-1 and immune checkpoint molecule, which are described in more detail below.

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

The FAP consensus antigen may be a nucleic acid sequence of SEQ ID NO: 59, which encodes the amino acid sequence of SEQ ID NO: 60. The FAP consensus protein may be linked to an IgE leader sequence and an HA tag. In other embodiments, the FAP consensus protein may be free or not associated with an IgE leader sequence and/or an HA tag. The FAP consensus sequence may be operably linked to a regulatory sequence, including, but not limited to, a start codon and at least one stop codon.

In some embodiments, the FAP consensus antigen may be a nucleic acid sequence having at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity relative to the entire length of the nucleic acid sequence set forth in SEQ ID NO: 59. In other embodiments, the FAP consensus antigen may be a nucleic acid 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 relative to the entire amino acid length sequence set forth in SEQ ID NO: 60. The FAP 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 with respect to the entire length of the amino acid sequence presented in SEQ ID NO: 60.

Some embodiments of the invention relate to nucleic acid sequences encoding proteins homologous to the FAP consensus protein, an immunogenic fragment of the FAP consensus protein, and immunogenic fragments of homologous proteins. Nucleic acid molecules may be provided that encode immunogenic proteins that have up to 95% homology to a consensus sequence, up to 96% homology to a consensus sequence, up to 97% homology to a consensus sequence, up to 98% homology to a consensus sequence, and up to 99% homology to a consensus sequence. . Likewise, nucleic acid sequences encoding immunogenic fragments presented herein and immunogenic fragments of proteins homologous to proteins presented herein are also provided.

Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 95% homology to the nucleic acid coding sequences provided herein. Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 96% homology to the nucleic acid coding sequences provided herein. Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 97% homology to the nucleic acid coding sequences provided herein. Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 98% homology to the nucleic acid coding sequences provided herein. Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 99% homology to the nucleic acid coding sequences provided herein. In some embodiments, nucleic acid molecules with coding sequences disclosed 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.

- 96 043982

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

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

Some embodiments of the invention relate to fragments of SEQ ID NO: 59. The fragments may be at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% , at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the sequence, set forth in SEQ ID NO: 59. The fragments may be at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous to the fragments of SEQ ID NO: 59. Fragments may be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, according to 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: 59. In some In embodiments of the invention, the fragments contain sequences that encode 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 FAP consensus protein is SEQ ID NO: 60. The amino acid sequence of the FAP consensus protein associated with the IgE leader sequence is SEQ ID NO: 60. The amino acid sequence of the FAP consensus protein associated with the IgE leader sequence may be connected to the HA mark.

Some embodiments of the invention relate to proteins that are homologous to SEQ ID NO: 60. Some embodiments of the invention relate to immunogenic proteins that have 95% homology to the consensus protein sequences presented in SEQ ID NO: 60. Some embodiments of the invention relate to immunogenic proteins that have 96% homology to the consensus protein sequences presented in SEQ ID NO: 60. Some embodiments of the invention relate to immunogenic proteins that have 97% homology to the consensus protein sequences presented in SEQ ID NO: 60. Some embodiments The inventions relate to immunogenic proteins that have 98% homology to the consensus protein sequences presented in SEQ ID NO: 60. Some embodiments of the invention relate to immunogenic proteins that have 99% homology to the consensus protein sequences presented in SEQ ID NO: 60 .

Some embodiments of the invention provide proteins that are identical to SEQ ID NO: 60. Some embodiments of the invention provide immunogenic proteins that have

- 97 043982 an amino acid sequence that is 80% identical to the full-length consensus amino acid sequence presented in SEQ ID NO: 60. Some embodiments of the invention relate to immunogenic proteins that have an amino acid sequence that is 85% identical to the full-length consensus amino acid sequence presented in SEQ ID NO: 60. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 90% identical to the full-length consensus amino acid sequence presented in SEQ ID NO: 60. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence a sequence that is 91% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 60. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 92% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 60. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 93% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 60. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 94% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 60. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 95% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 60. Some embodiments Embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 96% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 60. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 97% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 60. Some embodiments of the invention relate to immunogenic proteins that have an amino acid sequence that is 98% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 60. Some embodiments of the invention relate to immunogenic proteins that have an amino acid sequence that is 99% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 60.

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 may be provided SEQ ID NO: 60. 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%, according 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 presented in SEQ ID NO: 60. 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 homologous to the immunogenic fragments of SEQ ID NO: 60. Such immunogenic fragments may contain at least 10%, at least 15%, at least 20%, at least 25%, at least at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70 %, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of a protein that is 95% or more homologous to the sequence set forth in SEQ ID NO: 60. Some embodiments of the invention provide immunogenic fragments that have 96% homology to the immunogenic fragments of the consensus protein sequences provided herein. Some embodiments of the invention provide immunogenic fragments that have 97% homology to the immunogenic

- 98 043982 fragments of consensus protein sequences presented in this document. Some embodiments of the invention provide immunogenic fragments that have 98% homology to immunogenic fragments of the consensus protein sequences presented herein. Some embodiments of the invention provide immunogenic fragments that have 99% homology to immunogenic fragments of the consensus protein sequences presented 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: 60. Such immunogenic fragments may contain at least 10%, at least 15%, at least 20%, at least 25%, at least 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: 60. In some embodiments The fragments of the invention 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 stated herein with respect to the attachment of a signal peptide or leader sequence to the N-terminus of a protein, the signal peptide/leader sequence replaces the N-terminal methionine of the protein, which is encoded by the start codon of the nucleic acid sequence, as opposed to protein coding sequences without signal peptides.

The nucleic acid sequence encoding the FAP consensus antigen can be optimized in terms of codon usage and corresponding RNA transcripts.

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

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

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

(17) FSHR.

The immunogenic composition of the present invention may contain the cancer antigen follicle-stimulating hormone receptor (FSHR), a fragment thereof, or a variant thereof. The FSHR antigen can induce an antigen-specific T-cell response and/or high antibody titers, thereby eliciting or inducing an immune response that targets or responds to the cancer or tumor expressing the antigen. In some embodiments, the induced or evoked immune response may be a cellular, humoral, or both cellular and humoral immune response. In some embodiments, the induced or evoked cellular immune response may include the induction or secretion of interferon-gamma (IFNγ) and/or tumor necrosis factor alpha (TNF-α). In other embodiments, the induced or elicited immune response may reduce or inhibit one or more immunosuppressive factors that promote the growth of a tumor or cancer expressing an antigen, such as, but not limited to, factors that inhibit MHC presentation, factors that

- 99 043982 increases levels of antigen-specific regulatory T cells (Treg), PD-L1, FasL, cytokines such as IL-10 and TFG-β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by immunosuppressive cells, CTLA -4, PD-1, MDSCs, MCP-1 and immune checkpoint molecule, which are described in more detail below.

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

The FSHR consensus antigen may be a nucleic acid sequence of SEQ ID NO: 61 that encodes the amino acid sequence of SEQ ID NO: 62. The FSHR consensus protein may be linked to an IgE leader sequence and an HA tag. In other embodiments, the FSHR consensus protein may be free or not associated with an IgE leader sequence and/or an HA tag. The FSHR consensus sequence may be operably linked to a regulatory sequence, including, but not limited to, a start codon and at least one stop codon.

In some embodiments, the FSHR consensus antigen may be a nucleic acid sequence having at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity relative to the entire length of the nucleic acid sequence set forth in SEQ ID NO: 61. In other embodiments, the FSHR consensus antigen may be a nucleic acid 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 relative to the entire amino acid length sequence set forth in SEQ ID NO: 62. The FSHR 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 with respect to the entire length of the amino acid sequence presented in SEQ ID NO: 62.

Some embodiments of the invention relate to nucleic acid sequences encoding proteins homologous to the FSHR consensus protein, an immunogenic fragment of the FSHR consensus protein, and immunogenic fragments of homologous proteins. Nucleic acid molecules may be provided that encode immunogenic proteins that have up to 95% homology to a consensus sequence, up to 96% homology to a consensus sequence, up to 97% homology to a consensus sequence, up to 98% homology to a consensus sequence, and up to 99% homology to a consensus sequence. . Likewise, nucleic acid sequences encoding immunogenic fragments presented herein and immunogenic fragments of proteins homologous to proteins presented herein are also provided.

Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 95% homology to the nucleic acid coding sequences provided herein. Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 96% homology to the nucleic acid coding sequences provided herein. Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 97% homology to the nucleic acid coding sequences provided herein. Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 98% homology to the nucleic acid coding sequences provided herein. Some embodiments of the invention provide nucleic acid molecules that encode immunogenic proteins that have 99% homology to the nucleic acid coding sequences provided herein. In some embodiments, nucleic acid molecules with coding sequences disclosed 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 nucleic acid sequences encoding proteins with a certain percentage of identity to full-length FSHR consensus proteins, immunogenic fragments of the FSHR consensus protein, and immunogenic protein fragments having identity to the FSHR consensus protein. Nucleic acid molecules can also be proposed that encode immunogenic proteins that have up to 80% identity with respect to the full-length FSHR consensus sequence, up to 85% identity with respect to the full-length FSHR sequence, up to 90% identity with

- 100 043982 relative to the full-length FSHR consensus sequence, up to 91% identity relative to the full-length FSHR consensus sequence, up to 92% identity relative to the full-length FSHR consensus sequence, up to 93% identity relative to the full-length FSHR consensus sequence, up to 94% identity relative to the full-length FSHR consensus sequence, up to 95% identity to the full-length FSHR consensus sequence, up to 96% identity to the full-length FSHR consensus sequence, up to 97% identity to the full-length FSHR consensus sequence, up to 98% identity to the full-length FSHR consensus sequence, and up to 99% identity to the full-length FSHR consensus sequence. Likewise, nucleic acid sequences encoding the immunogenic fragments presented herein and immunogenic protein fragments with similar percentage identities as stated above to the FSHR proteins presented herein are also provided.

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

Some embodiments of the invention relate to fragments of SEQ ID NO: 61. The fragments may be at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% , at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the sequence, set forth in SEQ ID NO: 61. The fragments may be at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous to the fragments of SEQ ID NO: 61. Fragments may be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, according to 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: 61. In some In embodiments of the invention, the fragments contain sequences that encode 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 FSHR consensus protein is SEQ ID NO: 62. The amino acid sequence of the FSHR consensus protein associated with the IgE leader sequence is SEQ ID NO: 62. The amino acid sequence of the FSHR consensus protein associated with the IgE leader sequence may be connected to the HA mark.

Some embodiments of the invention relate to proteins that are homologous to SEQ ID NO: 62. Some embodiments of the invention relate to immunogenic proteins that have 95% homology to the consensus protein sequences presented in SEQ ID NO: 62. Some embodiments of the invention relate to immunogenic proteins that have 96% homology to the consensus protein sequences presented in SEQ ID NO: 62. Some embodiments of the invention relate to immunogenic proteins that have 97% homology to the consensus protein sequences presented in SEQ ID NO: 62. Some embodiments The inventions relate to immunogenic proteins that have 98% homology to the consensus protein sequences presented in SEQ ID NO: 62. Some embodiments of the invention relate to immunogenic proteins that have 99% homology to the consensus protein sequences presented in SEQ ID NO: 62 .

Some embodiments of the invention provide proteins that are identical to SEQ ID NO: 62. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 80% identical to the full-length consensus amino acid sequence presented in SEQ ID NO: 62. Some embodiments embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 85% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 62. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 90% identical to the full-length consensus amino acid sequence shown in SEQ ID NO: 62. Some variations

- 101 043982 embodiments of the invention relate to immunogenic proteins that have an amino acid sequence that is 91% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 62. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 92% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 62. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 93% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 62. Some embodiments Embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 94% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 62. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 95% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 62. Some embodiments of the invention relate to immunogenic proteins that have an amino acid sequence that is 96% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 62. Some embodiments of the invention relate to immunogenic proteins that have an amino acid sequence that is 97% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 62. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 98% identical to the full-length consensus amino acid sequence, set forth in SEQ ID NO: 62. Some embodiments of the invention provide immunogenic proteins that have an amino acid sequence that is 99% identical to the full-length consensus amino acid sequence set forth in SEQ ID NO: 62.

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 may be provided SEQ ID NO: 62. 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%, according 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 presented in SEQ ID NO: 62. 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 homologous to the immunogenic fragments of SEQ ID NO: 62. Such immunogenic fragments may contain at least 10%, at least 15%, at least 20%, at least 25%, at least at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70 %, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of a protein that is 95% or more homologous to the sequence set forth in SEQ ID NO: 62. Some embodiments of the invention provide immunogenic fragments that have 96% homology to the immunogenic fragments of the consensus protein sequences presented herein. Some embodiments of the invention provide immunogenic fragments that have 97% homology to immunogenic fragments of the consensus protein sequences presented herein. Some embodiments of the invention provide immunogenic fragments that have 98% homology to immunogenic fragments of the consensus protein sequences presented herein. Some embodiments of the invention provide immunogenic fragments that have 99% homology to immunogenic fragments of the consensus protein sequences presented herein. In some embodiments, the fragments comprise a leader sequence, such as, for example,

- 102 043982 nuclear immunoglobulin 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: 62. Such immunogenic fragments may contain at least 10%, at least 15%, at least 20%, at least 25%, at least 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: 62. In some embodiments The fragments of the invention 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 stated herein with respect to the attachment of a signal peptide or leader sequence to the N-terminus of a protein, the signal peptide/leader sequence replaces the N-terminal methionine of the protein, which is encoded by the start codon of the nucleic acid sequence, as opposed to protein coding sequences without signal peptides.

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

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

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

(18) gp100.

The immunogenic composition of the present invention may comprise cancer antigen glycoprotein 100 (gp100; also known as Trp2 and premelanosome protein (PMEL)), a fragment thereof, or a variant thereof. gp100 is encoded by the PMEL gene. It is a 70 kDa type 1 transmembrane glycoprotein consisting of 661 amino acids that plays a central role in melanosome biogenesis as it is involved in melanosome maturation from stage I to stage II. gp100 controls the formation of grooves in multivesicular bodies and is directly involved in the biogenesis of premelanosomes. gp100 is present in greater abundance in premelanosomes compared to mature melanosomes, but is overexpressed by proliferating neonatal melanocytes and during tumor growth. The gp100 protein contains multiple immunogenic epitopes that are recognized by cytotoxic T lymphocytes from the peripheral blood of melanoma patients and from tumor-infiltrating lymphocytes.

The gp100 antigen can induce an antigen-specific T cell response and/or high antibody titers, thereby eliciting or inducing an immune response that targets or responds to the cancer or tumor expressing the antigen. In some embodiments, the induced or evoked immune response may be a cellular, humoral, or both cellular and humoral immune response. 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

- 103 043982 or more immunosuppressive factors that promote the growth of a tumor or cancer expressing an antigen, for example, but not limited to, factors that suppress MHC presentation, factors that increase levels of antigen-specific regulatory T cells (Treg), PD-L1, FasL, cytokines such as IL-10 and TFG-β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by immunosuppressive cells, CTLA-4, PD-1, MDSCs, MCP-1 and immune checkpoint molecule, which are described in more detail below.

(19) 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, as described below.

The viral antigen can induce an antigen-specific T cell response and/or high titers of antibodies, thereby eliciting or inducing an immune response that targets or responds to the cancer or tumor expressing the antigen. In some embodiments, the induced or evoked immune response may be a cellular, humoral, or both cellular and humoral immune response. 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 evoked immune response may reduce or inhibit one or more immunosuppressive factors that promote the growth of a tumor or cancer expressing an antigen, such as, but not limited to, factors that suppress MHC presentation, factors that increase levels of antigen-specific regulatory T cells (Treg), PD-L1, FasL, cytokines such as IL-10 and TFG-β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by immunosuppressive cells, CTLA-4, PD-1 , MDSCs, MCP-1 and immune checkpoint molecule, which are described in more detail below.

Hepatitis B virus antigen.

The viral antigen may be a hepatitis B virus (HBV) antigen, 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 encodes one or more 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 to enhance expression. Genetic modifications, including codon optimization, RNA optimization, and addition of a high-performance immunoglobulin leader sequence to enhance the immunogenicity of the constructs, can be made to 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 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 genotype A HBV core consensus nucleic acid sequence construct, an IgE leader sequence fused to a genotype A HBV core protein consensus sequence, or a genotype A HBV core consensus protein sequence.

In other embodiments, the HBV antigen may be a genotype B HBV core consensus nucleic acid sequence construct, an IgE leader sequence fused to a genotype B HBV core protein consensus sequence, or a genotype B HBV core consensus protein sequence.

In other embodiments, the HBV antigen may be a genotype C HBV core consensus nucleic acid sequence construct, the leader sequence

- 104 043982 IgE activity linked to the HBV genotype C consensus core protein sequence, or the HBV genotype C consensus core protein sequence.

In some embodiments, the HBV antigen may be a genotype D HBV core consensus nucleic acid sequence construct, an IgE leader sequence fused to a genotype D HBV core protein consensus sequence, or a genotype D HBV core consensus protein sequence.

In other embodiments, the HBV antigen may be a genotype E HBV core consensus nucleic acid sequence construct, an IgE leader sequence fused to a genotype E HBV core protein consensus sequence, or a genotype E HBV core consensus protein sequence.

In some embodiments, the HBV antigen may be a genotype F HBV core consensus nucleic acid sequence construct, an IgE leader sequence fused to a genotype F HBV core protein consensus sequence, or a genotype F HBV core consensus protein sequence.

In other embodiments, the HBV antigen may be a genotype G HBV core consensus nucleic acid sequence construct, an IgE leader sequence fused to a genotype G HBV core protein consensus sequence, or a genotype G HBV core consensus protein sequence.

In some embodiments, the HBV antigen may be a genotype H HBV core consensus nucleic acid sequence construct, an IgE leader sequence fused to a genotype H HBV core protein consensus sequence, or a genotype H HBV core consensus protein sequence.

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

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

In other embodiments, the HBV antigen may be a genotype C HBV surface consensus nucleic acid sequence construct, an IgE leader sequence fused to a genotype C HBV surface protein consensus sequence, or a genotype C HBV consensus surface protein sequence.

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

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

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

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

In other embodiments, the HBV antigen may be a genotype H HBV surface consensus nucleic acid sequence construct, an IgE leader sequence fused to a genotype H HBV surface protein consensus sequence, or a genotype H HBV consensus surface protein sequence.

Hepatitis C virus antigen.

The viral antigen may be a hepatitis C virus (HCV) antigen, a fragment thereof, or a variant thereof. HCV antigen may be associated with or cause liver cancer. In some embodiments, the HCV antigen may be a heterologous nucleic acid molecule(s), such as 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

- 105 043982 cation to enhance expression. Genetic modifications, including codon optimization, RNA optimization, and addition of a high-performance immunoglobulin leader sequence to enhance the immunogenicity of the constructs, can be made to 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.

The 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, a variant thereof, or a combination thereof.

Human papillomavirus.

The viral antigen may be an HPV antigen, a fragment thereof, or a variant thereof. The HPV antigen can be 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 can be HPV types 6 and/or 11, which are the cause of genital warts and are known to cause head and neck cancer. The HPV antigen can be HPV types 16 and/or 18, which are the cause of cervical cancer. The HPV antigen can be HPV types 6, 11 and/or 16, which are the cause of RRP and anal cancer. HPV cancer antibodies may 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 Patent Application U.S. No. 61/777198, filed March 12, 2013, each of which is incorporated by reference in its entirety.

HPV antigens may be the HPV E6 or E7 domains of 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 visus antigen may be an antigen selected from the group consisting of CMV, HSV1, HSV2, VZV, CeHV1, EBV, roseolovirus, Kaposi's sarcoma-associated herpesvirus, and MuHV.

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).

Nucleic acid sequences include coding sequences 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. Immunogenic compositions may contain one or more nucleic acid sequences that encode one or more consensus versions of immunogenic proteins selected from this group of sequences designed to optimize stability and expression in humans.

Nucleic acid sequence encoding the HCMV-gB consensus protein (SEQ ID NO: 25), Nucleic acid sequence encoding the HCMV-gM consensus protein (SEQ ID NO: 27), Nucleic acid sequence encoding the HCMV-gN consensus protein (SEQ ID NO : 29) nucleic acid sequence encoding the HCMV-gH consensus protein (SEQ ID NO: 31), nucleic acid sequence encoding the HCMVgL consensus protein (SEQ ID NO: 33), nucleic acid sequence encoding the HCMV-gO consensus protein (SEQ ID NO: 35), nucleic acid sequence encoding the HCMV-UL128 consensus protein (SEQ ID NO: 37), nucleic acid sequence encoding the HCMV-UL130 consensus protein (SEQ ID NO: 39), nucleic acid sequence encoding the HCMV- consensus protein UL-131A (SEQ ID NO: 41), a nucleic acid sequence encoding the HCMV-UL-83 consensus protein (pp65) (SEQ ID NO: 43). The nucleic acid sequence may further have a sequence encoding an IgE leader sequence linked to the 5' end.

In view of the evolutionary divergence from clinical isolates and the extensive genetic differences between common circulating human strains, consensus amino acid sequences were obtained for each of the immunogenic proteins. Consensus amino acid sequences for gB, gB, gH, gL, gE, gI, gK, gC, gD, UL128, UL130, UL-131A, and UL-83 (pp65) were based on sequences of human clinical isolates. Due to great evolution

- 106 043982 onic divergence of the gN protein consensus sequence was generated from only one (gN-4c) of the seven serotypes, which represents the most common serotype (gN-4). Similarly, for gO, consensus amino acid sequences were generated from one (gO-5) of the eight serotypes due to specific serotypes being reported to be associated 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 connected to the N-terminus. As described herein, when reference is made to a signal peptide linked to the N-terminus of a consensus sequence, it is intended that embodiments of the invention are specifically included in which the N-terminal Xa residue of the consensus sequences is replaced by a signal peptide. Thus, as used herein, Xaa refers to any amino acid or lack of amino acid. Proteins that contain the consensus sequence presented herein at SEQ ID NO: 26, 28, 30, 32, 34, 36, 38, 40, 42, 44 may contain those sequences that do not contain Xaa at the N-terminus.

Amino acid sequences were obtained, which in each case contained 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 through proteolytic cleavage sites. The furin proteolytic cleavage site is an example of a proteolytic cleavage site that can join herpes virus antigens in a fusion protein expressed by the construct. The herpes family viral cancer antigens may further be any antigen disclosed in US Patent Application No. 13/982,457, the contents of which are incorporated by reference in their entirety.

4. An immunogenic composition in combination with an immune checkpoint inhibitor.

The immunogenic composition 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 inhibition of any component in the immune system, such as MHC class 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.

Such an inhibitor may be a nucleic acid sequence, an amino acid sequence, a small molecule, or a combination thereof. The nucleic acid sequence may be DNA, RNA, cDNA, a variant thereof, a fragment thereof, or a combination thereof. The nucleic acid sequence may also contain additional sequences that encode linker or tag sequences that are linked to the immune checkpoint inhibitor by a peptide bond. The small molecule may have a low molecular weight, such as less than 800 Daltons, and may be an organic or inorganic compound that may serve as an enzyme substrate, a ligand (or analog thereof), a bound 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.

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

Immune checkpoint molecule.

The immune checkpoint molecule may be a nucleic acid sequence, an amino acid sequence, a small molecule, or a combination thereof. The nucleic acid sequence may be DNA, RNA, cDNA, a variant thereof, a fragment thereof, or a combination thereof.

The nucleic acid sequence may also contain additional sequences that encode linker or tag sequences that are linked to the immune checkpoint inhibitor by a peptide bond. The small molecule may have a low molecular weight, such as less than 800 Daltons, and may be an organic or inorganic compound that may serve as an enzyme substrate, a ligand (or analog thereof), a bound 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. The immune checkpoint molecule may be an inhibitor of at least one immune checkpoint protein. Immune checkpoint proteins include, but are not limited to

- 107 043982 are PD-1, PD-L1, CTLA4, TIM3 and LAG3.

Antibody against 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 anti-immune checkpoint molecule antibody. The antibody may be encoded by a nucleic acid 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), and 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. Starting from 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 region hypervariable regions. Starting from 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.

An antibody may comprise a set of complementarity determining regions (CDRs) of a light chain and a heavy chain, and are accordingly located between a framework (FR) set of a heavy chain and a light chain, which provides support for the CDRs and defines the spatial relationship of the CDRs relative to each other. A set of CDRs may contain three hypervariable regions of the heavy or light chain V region. Starting from the N-terminus of the heavy or light chain, these regions are designated CDR1, CDR2 and CDR3, respectively. Therefore, the antigen binding site may contain six CDRs containing a set of CDRs from each heavy and light chain V region.

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 to form multiple fragments, two of which (F(ab) fragments) each contain a covalent heterodimer that contains an intact antigen-binding site. The enzyme pepsin is capable of cleaving IgG molecules to form several fragments, including the F(ab') ₂ fragment, which contain 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. The Fab heavy chain polypeptide may contain a VH region and a CH1 region. The Fab light chain may comprise 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 of a non-human species that binds to a desired antigen, having one or more complementarity determining regions (CDRs) from a non-human species and framework regions of a human immunoglobulin molecule.

(1) Anti-PD-1 antibody.

The anti-immune checkpoint molecule antibody may be an anti-PD-1 antibody (also referred to herein as an anti-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 can inhibit the activity of PD-1, thereby eliciting, inducing or enhancing an immune response against a tumor or cancer and reducing tumor growth.

(2) Anti-PD-L1 antibody.

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

- 108 043982 (3) Antibody against CTLA4.

The anti-immune checkpoint molecule antibody may be an anti-CTLA4 antibody (also referred to herein as an anti-CTLA4 antibody), a variant thereof, a fragment thereof, or a combination thereof. An anti-CTLA4 antibody can inhibit the activity of CTLA4, thereby eliciting, inducing or enhancing an immune response against a tumor or cancer and reducing tumor growth.

(4) Antibody against TIM3.

The anti-immune checkpoint molecule antibody may be an anti-TIM3 antibody (also referred to herein as an anti-TIM3 antibody), a variant thereof, a fragment thereof, or a combination thereof. An anti-TIM3 antibody can inhibit the activity of TIM3, thereby eliciting, inducing or enhancing an immune response against a tumor or cancer and reducing tumor growth.

(5) Anti-LAG3 antibody.

The anti-immune checkpoint molecule antibody may be an anti-LAG3 antibody (also referred to herein as an anti-LAG3 antibody), a variant thereof, a fragment thereof, or a combination thereof. An anti-LAG3 antibody can inhibit the activity of LAG3, thereby eliciting, inducing or enhancing an immune response against a tumor or cancer and reducing tumor growth.

5. Constructs and plasmids.

The immunogenic composition may contain nucleic acid constructs or plasmids that encode the above-described antigens and/or antibodies. Nucleic acid constructs or plasmids may have or contain one or more heterologous nucleic acid sequences. Provided herein are genetic constructs that may contain a nucleic acid sequence that encodes the above-described antigens and/or antibodies. The genetic construct may be present in the cell as a functioning extrachromosomal molecule. The genetic construct may be a linear minichromosome containing centromeres, telomeres, or plasmids or cosmids. Genetic constructs may have or contain one or more heterologous nucleic acid sequences.

The genetic construct may be useful for transfecting cells with a nucleic acid encoding the above-described antigens and/or antibodies, which the transformed host cell is cultured and maintained under conditions in 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 resulting, for example, from intramolecular binding.

The genetic constructs may be in the form of plasmids expressing the antigens and/or antibodies described above in any order.

Expression vectors.

The vector may be a circular plasmid or a linear nucleic acid. The circular plasmid and linear nucleic acid are capable of directing the expression of a specific nucleotide sequence in a corresponding cell of a subject. The vector may have a promoter operably linked to an antigen-encoding nucleotide sequence, which may be operably linked to termination signals. The vector may also contain sequences necessary for proper translation of the nucleotide sequence. The vector containing the nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous to at least one of its other components. Expression of a nucleotide sequence in an expression cassette can be controlled by a constitutive promoter or an inducible promoter, which initiates transcription only when the host cell is exposed to a particular external stimulus. In the case of a multicellular organism, the promoter may also be specific to a particular tissue or organ or developmental stage.

RNA Vectors.

In one embodiment of the invention, the nucleic acid is an RNA molecule. Accordingly, in one embodiment, the invention provides an RNA molecule encoding one or more MAYV antigens. The RNA may be a plus strand. Accordingly, in some embodiments, the RNA molecule can be translated by cells without the need for any intermediate replication steps, such as reverse transcription. The RNA molecule used in the invention may have a 5' cap (eg, 7-methylguanosine). This cap can enhance RNA translation in vivo. The 5' nucleotide of the RNA molecule used in the invention may have a 5'-triphosphate group. In capped RNA it can be linked to 7-methylguanosine through a 5'-5' bridge. An RNA molecule may have a 3' polyA tail. It may also contain a polyA polymerase recognition sequence (eg, AAUAAA) near its 3' end. The RNA molecule used in the invention may be single-stranded. In some embodiments, the RNA molecule is a naked RNA molecule. In one embodiment of the invention, the RNA molecule is contained in a vector.

- 109 043982

In one embodiment of the invention, the RNA has 5' and 3' UTRs. In one embodiment, the 5'-UTR is between zero and 3000 nucleotides in length. The length of the 5' and 3' UTR sequences to be added to the coding region can be varied in various ways, including, but not limited to, selecting PCR primers that anneal to different regions of the UTR. Using this approach, one skilled in the art can modify the lengths of the 5' and 3' UTRs as needed to achieve optimal translation efficiency after transfection of the transcribed RNA.

The 5' and 3' UTRs may be naturally occurring endogenous 5' and 3' UTRs for the gene of interest. Alternatively, UTR sequences that are not endogenous to the gene of interest can be added by including UTR sequences in the forward and reverse primers or by any other modification of the template. The use of UTR sequences that are not endogenous to the gene of interest may be useful to alter the stability and/or translational efficiency of RNA. For example, it is known that AU-rich elements in 3' UTR sequences can reduce RNA stability. Therefore, 3'-UTRs can be selected or designed to increase the stability of the transcribed RNA based on the properties of the UTRs, which are well known in the art.

In one embodiment, the 5'-UTR may comprise a Kozak sequence of an endogenous gene.

Alternatively, when a 5' UTR that is not endogenous to the gene of interest is introduced by PCR as described above, the Kozak consensus sequence can be modified by adding a 5' UTR sequence. Kozak sequences can improve the translation efficiency of some RNA transcripts, but do not appear to require all RNAs to ensure efficient translation. The requirement for Kozak sequences for many RNAs is known in the art. In other embodiments, the 5'-UTR may be derived from an RNA virus whose RNA genome is stable in cells. In other embodiments, various nucleotide analogs may be used in the 3' or 5' UTR to interfere with exonuclease degradation of the RNA.

In one embodiment, the RNA has both a cap at the 5' end and a 3' poly(A) tail, which determine ribosome binding, translation initiation, and stability of the RNA in the cell.

In one embodiment of the invention, the RNA is nucleoside-modified RNA. Nucleoside-modified RNA has particular advantages over unmodified RNA, including, for example, increased stability, low or no inherent immunogenicity, and enhanced translation.

Ring and linear vectors.

The vector may be a circular plasmid that can transform a target cell by integration into the cellular genome or exist extrachromosomal (eg, an autonomously replicating plasmid with an origin of replication).

The vector may be pVAX, pcDNA3.0 or provax, or any other expression vector capable of expressing DNA encoding an antigen and allowing the cell to translate the sequence into an antigen that is recognized by the immune system.

Also provided herein is a linear nucleic acid immunogenic composition or linear expression cassette (LEC) that is capable of being efficiently delivered to a subject by electroporation and expressing one or more desired antigens. LEC can be any linear DNA lacking any phosphate backbone. DNA may encode one or more antigens. The LEC may contain a promoter, intron, stop codon and/or polyadenylation signal. Antigen expression can be controlled by a promoter. LEC may not contain antibiotic resistance and/or phosphate backbone genes. The LEC may not contain other nucleotide sequences not associated with the expression of the desired antigen gene.

LEC can be obtained from any plasmid capable of linearization. The plasmid may be capable of expressing an antigen. The plasmid may be pNP (Puerto Rico/34) or pM2 (New Caledonia/99). The plasmid may be WLV009, pVAX, pcDNA3.0 or provax or any other expression vector capable of expressing DNA encoding the antigen and allowing the cell to translate the sequence into an antigen that is recognized by the immune system.

The LEC may be pcrM2. The LEC may be pcrNP. pcrNP and pcrMR can be obtained from pNP (Puerto Rico/34) and pM2 (New Caledonia/99), respectively.

Promoter, intron, stop codon and polyadenylation signal.

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 before one or more antigen coding sequences, and a stop codon, which may be located after the coding sequence(s) described above. antigens and/or antibodies.

The vector may have a promoter. The promoter can be any promoter that

- 110 043982 is capable of controlling gene expression and regulating the expression of isolated nucleic acid. Such a promoter is a cis-acting sequence element required for transcription by a DNA-dependent RNA polymerase that transcribes the antigen sequence described herein. The choice of promoter used for direct expression of a heterologous nucleic acid depends on the specific application. The promoter may be located at approximately the same distance from the transcription start site in the vector as from the transcription start site in its natural position. However, changing this distance can be accomplished without loss of promoter function.

The initiation and termination codon may be in frame with the coding sequence(s) of the above-described antigens and/or antibodies. The vector may also contain a promoter that is operably linked to the coding sequence(s) of the above-described antigens and/or antibodies. 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 a bovine immunodeficiency virus (BIV) long terminal repeat (LTR), Moloney virus promoter, avian leukemia virus (ALV) promoter, cytomegalovirus (CMV) promoter such as the CMV immediate early promoter, Epstein-Barr virus (EBV) promoter, or virus promoter Rous sarcoma (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. US 20040175727, the contents of which are incorporated herein in their entirety.

The vector may also contain a polyadenylation signal, which may be located after 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 the polyadenylation signal of the pCEP4 vector (Invitrogen, San Diego, Calif.).

The vector may also contain an enhancer in front of the above-described antigens and/or antibodies. The enhancer may be required for expression. The enhancer may be a human actin enhancer, human myosin enhancer, human hemoglobin enhancer, human muscle creatine enhancer, or a viral enhancer such as a CMV, HA, RSV, or EBV enhancer. Polynucleotide enhancers of function are described in US Patent Nos. 5,593,972, 5,962,428 and WO 94/016737, the contents of each of which are incorporated by reference in their entirety.

The vector may contain an enhancer and an intron with functional splice donor and acceptor sites. The vector may contain a transcription termination region after the structural gene to ensure efficient termination. The termination region may be derived from the same gene as the promoter sequence or may be derived from other genes.

Multiple vectors.

The immunogenic composition may contain multiple copies of a single nucleic acid molecule, such as a single plasmid, or multiple copies of two or more different nucleic acid molecules, such as two or more different plasmids. For example, an immunogenic composition may contain a plurality of two, three, four, five, six, seven, eight, nine or ten or more different nucleic acid molecules. Such compositions may contain a plurality of two, three, four, five, six or more different plasmids.

Immunogenic compositions may contain nucleic acid molecules, such as plasmids, which collectively contain the coding sequence for a single antigen. In one embodiment of the invention, the antigen is FAP. Immunogenic compositions may contain nucleic acid molecules, such as plasmids, which collectively contain the coding sequence for multiple antigens. As an example, in one embodiment, the antigens are multiple antigens selected from TERT and an additional cancer antigen. In one exemplary embodiment of the invention, the antigens are WT-1 and TERT. In one exemplary embodiment of the invention, the antigens are PSMA and TERT. In another exemplary embodiment, the antigens are TERT, WT-1 and PSMA. Immunogenic compositions may contain nucleic acid molecules, such as plasmids, which collectively contain the coding sequence of one or more antigens and one or more cancer antigens.

Replication start point.

The vector may also contain a mammalian origin of replication to maintain the extrachromosomal vector and create multiple copies of the vector in the cell. The vector can be

- 111 043982 pVAXl, pCEP4 or pREP4 from Invitrogen (San Diego, CA), which may contain the Epstein-Barr virus origin of replication and the coding region of the EBNA-1 nuclear antigen, which can allow high copy number episomal replication without integration. The vector may be pVAX1 or a variant of pVax1 with modifications, 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 CA). The CMV promoter is located at base positions 137-724.

The T7 promoter/priming site is located at base positions 664-683. Multiple insertion sites are located at base positions 696-811. The bovine polyadenylation signal is located at base positions 829-1053. The kanamycin resistance gene is located at base positions 1226-2020. The pUC transcription start point is located at base positions 2320-2993.

Based on the pVAX1 sequence available from Invitrogen, the following mutations were discovered in the pVAX1 sequence, which was used as the basis for plasmids 1-6 presented here:

C>G241 in the CMV promoter.

C>T 1942 scaffold after bovine growth hormone polyadenylation signal (bGHpolyA).

A> - 2876 frame after the kanamycin resistance gene.

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

G>C 3753 at the very end of pUC Ori preceding the RNASeH 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 the mammalian or human cell into which the vector is introduced. One or more cancer antigen sequences disclosed herein may contain a codon that may allow 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 strains of the yeast Saccharomyces cerevisiae. The vector may also be a complete baculovirus MAHBAS™ expression system (Invitrogen, San Diego, Calif.), which can be used to produce protein in insect cells. The vector may also be pcDNA I or pcDNA3 (Invitrogen, San Diego, Calif.), which can be used to produce the protein in mammalian cells, such as Chinese hamster ovary (CHO) cells. The vector may be expression vectors or protein production systems using conventional methods and readily available starting materials, including Sambrook et al., Molecular Cloning and Laboratory Manual, Second Ed., Cold Spring Harbor (1989), which is incorporated by reference in its entirety.

6. Pharmaceutical compositions.

The immunogenic composition may be in the form of a pharmaceutical composition. The pharmaceutical composition may contain an immunogenic composition. Pharmaceutical compositions may contain from about 5 ng to about 10 mg of a nucleic acid molecule encoding an antigen of the invention. In some embodiments, the pharmaceutical compositions of this invention contain from about 25 ng to about 5 mg of nucleic acid. In some embodiments, the pharmaceutical compositions contain from about 50 ng to about 1 mg of nucleic acid. In some embodiments, the pharmaceutical compositions contain from about 0.1 to about 500 μg of nucleic acid. In some embodiments, the pharmaceutical compositions contain from about 1 to about 350 μg of nucleic acid. In some embodiments, the pharmaceutical compositions contain from about 5 to about 250 μg of nucleic acid. In some embodiments, the pharmaceutical compositions contain from about 10 to about 200 μg of nucleic acid. In some embodiments, the pharmaceutical compositions contain from about 15 to about 150 μg of nucleic acid. In some embodiments, the pharmaceutical compositions contain from about 20 to about 100 μg of nucleic acid. In some embodiments, the pharmaceutical compositions contain from about 25 to about 75 μg of nucleic acid. In some embodiments, the pharmaceutical compositions contain from about 30 to about 50 μg of nucleic acid. In some embodiments, the pharmaceutical compositions contain from about 35 to about 40 μg of nucleic acid. In some embodiments, the pharmaceutical compositions contain from about 100 to about 200 μg of nucleic acid. In some

- 112 043982 embodiments of the invention, the pharmaceutical compositions contain from about 10 μg to about 100 μg of nucleic acid. In some embodiments, the pharmaceutical compositions contain from about 20 μg to about 80 μg of nucleic acid. In some embodiments, the pharmaceutical compositions contain from about 25 μg to about 60 μg of nucleic acid. In some embodiments, the pharmaceutical compositions contain from about 30 ng to about 50 μg of nucleic acid. In some embodiments, the pharmaceutical compositions contain from about 35 ng to about 45 μg of nucleic acid. In some embodiments, the pharmaceutical compositions contain from about 0.1 to about 500 μg of nucleic acid. In some embodiments, the pharmaceutical compositions contain from about 1 to about 350 μg of nucleic acid. In some embodiments, the pharmaceutical compositions contain from about 25 to about 250 μg of nucleic acid. In some embodiments, the pharmaceutical compositions contain from about 100 to about 200 μg of nucleic acid.

In some embodiments, the pharmaceutical compositions of this invention contain at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 ng of nucleic acid. 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, 21 5, 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 μg of nucleic acid. 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 nucleic acid.

In other embodiments, the pharmaceutical composition may contain up to and including 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 ng of nucleic acid. In some embodiments, the pharmaceutical composition may contain up to and including 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,100 , 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225 ,

230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 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 μg of nucleic acid. In some embodiments, the pharmaceutical composition may contain up to and including 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5 , 8, 8.5, 9, 9.5 or 10 mg of nucleic acid.

The pharmaceutical composition may further contain other agents for formulation purposes in accordance with the route of administration used. Where the pharmaceutical compositions are injectable pharmaceutical compositions, they are sterile, pyrogen-free and particulate-free. In one embodiment of the invention, an isotonic composition 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 used. Stabilizers include gelatin and albumin. In some embodiments, a vasoconstrictor is added to the preparation.

The immunogenic composition may further comprise a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient may be functional molecules 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 squalene and squalene , hyaluronic acid, lipids, liposomes, calcium ions, viral proteins, polyanions, polycations or nanoparticles or other known agents that facilitate transfection.

The transfection facilitating agent is a polyanion, polycation including poly-Lglutamate (LGS) or lipid.

The transfection facilitating agent is poly-L-glutamate. In one embodiment, poly-L-glutamate is present in the immunogenic composition at a concentration of less than 6 mg/ml. An agent that facilitates

- 113 043982 transfection, which may also include surfactants such as immunostimulating complexes (ISCOMS), Freund's incomplete adjuvant, LPS analog including monophosphoryl lipid A, muramyl peptides, quinone analogs and vesicles such as squalene and squalene, and hyaluronic acid. In some embodiments, the immunogenic composition may also contain a transfection facilitating agent such as lipids, liposomes, including lecithin liposomes or other liposomes known in the art, in the form of a DNA-liposome mixture (see, for example, WO 9324640), calcium ions, viral proteins, polyanions, polycations or nanoparticles or other known agents that facilitate transfection. In one embodiment of the invention, the transfection facilitating agent is a polyanion, polycation, including poly-L-glutamate (LGS), or lipid. The concentration of the transfection agent in the immunogenic composition 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 0.050 mg/ml or less than 0.010 mg/ml.

The pharmaceutically acceptable excipient may be an adjuvant. The adjuvant may be other genes that are expressed on an alternative plasmid or delivered as proteins in combination with the above plasmid in an immunogenic composition. 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), thymus-expressed epithelial chemokine (TECK), mucosal-associated epithelial chemokine (MEC), IL-12, IL-15, MHC, CD80, CD86, including IL- 15 with the signal sequence removed and, optionally, the IgE signal peptide.

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, IL10, IL-12, IL-18 or a combination thereof. In an exemplary embodiment of the invention, the adjuvant is IL-12.

Other genes that may be useful adjuvants include those encoding: MCP1, MIP-1a, MIP-1e, 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, ICE caspase, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, inactive NIK, SAP K, SAP-1, JNK, interferon-sensitive 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.

7. Combination immunogenic compositions for the treatment of certain types of cancer.

The immunogenic composition may be in the form of various combinations of a TERT antigen with one or more cancer antigens, as described above, for the treatment of a particular cancer disease or tumors. Depending on the combination of one or more cancer antigens, the immunogenic composition may target different cancers or other tumor types. These cancers may include melanoma, blood cancer (eg, leukemia, lymphoma, myeloma), lung cancer, squamous cell cancer of the esophagus, bladder cancer, colorectal cancer, esophageal cancer, stomach cancer, hepatocarcinoma, head and neck cancer, brain cancer, anal cancer, non-small cell lung cancer, pancreatic cancer, synovial cancer, prostate cancer, testicular cancer, liver cancer, cervical cancer, recurrent respiratory papillomatosis, skin cancer and stomach cancer.

Melanoma.

The immunogenic composition may combine a TERT antigen and one or more cancer antigens, such as tyrosinase, PRAME, or GP100-Trp2, to treat or prevent melanoma. The immunogenic composition may further combine one or more tyrosinase, PRAME, or GP100-Trp2 cancer antigens with any one or more NY-ESO-1, MAGE-A1, or WTI cancer antigens for the treatment or prevention of melanoma. Other combinations of cancer antigens may also be used to treat or prevent melanoma.

Head and neck cancer.

The immunogenic composition may comprise a combination of a TERT antigen and one or more HPV 16 E6/E7 cancer antigens for the treatment or prevention of head and neck cancer. The immunogenic composition may further combine the HPV 16 E6/E7 cancer antigen with any one or more of the NY-ESO-1, MAGE-A1, or WTI 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 immunogenic composition may combine a TERT antigen and one or more cancer antigens

- 114 043982 new with one or more cancer antigens, such as HPV 6, HPV 11 or HPV 16, for the treatment or prevention of recurrent respiratory papillomatosis or anal cancer. The immunogenic composition may further combine one or more cancer antigens HPV 6, HPV11 or HPV16 with one or more cancer antigens NY-ESO-1, MAGE-A1 or WTI for the treatment or prevention of 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 immunogenic composition may combine a TERT antigen and one or more cancer antigens with one or more cancer antigens, such as HPV 16 E6/E7 or HPV 18 E6/E7, for the treatment or prevention of cervical cancer. The immunogenic composition may further combine one or more cancer antigens, such as HPV 16 E6/E7 or HPV 18 E6/E7, with one or more cancer antigens NY-ESO-1, MAGE-A1 or WTI for the treatment or prevention of cervical cancer. Other combinations of cancer antigens may also be used to treat or prevent cervical cancer.

Liver cancer.

The immunogenic composition may combine a TERT antigen and 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. The immunogenic composition 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 cancer antigens NY-ESO-1, MAGE-A1 or WTI for treatment or prevention liver cancer. Other combinations of cancer antigens may also be used to treat or prevent liver cancer.

Glioblastoma.

The immunogenic composition may contain a TERT antigen and one or more CMV cancer antigens for the treatment or prevention of glioblastoma. The immunogenic composition may further combine CMV with one or more cancer antigens NY-ESO-1, MAGE-A1 or WTI for the treatment or prevention of glioblastoma. Other combinations of cancer antigens may also be used to treat or prevent glioblastoma.

Prostate cancer.

The immunogenic composition may combine a TERT antigen and one or more cancer antigens, such as PSA, PSMA or STEAP, for the treatment or prevention of prostate cancer. The immunogenic composition may further combine one or more PSA, PSMA or STEAP cancer antigens with one or more NY-ESO-1, MAGE-A1 or WTI cancer antigens for the treatment or prevention of prostate cancer. Other combinations of cancer antigens may also be used to treat or prevent prostate cancer.

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

The immunogenic composition may combine a TERT antigen and one or more cancer antigens, such as PRAME and WT-1, for the treatment or prevention of blood cancers such as leukemia, lymphoma and myeloma. The immunogenic composition may further combine one or more cancer antigens PRAME and WT-1 with one or more cancer antigens NY-ESO-1 or MAGE-A1 for the treatment or prevention of 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 cancer.

Ovarian cancer.

The immunogenic composition may combine a TERT cancer antigen and one or more cancer antigens, such as a follicle-stimulating hormone receptor (FSHR) antigen, for the treatment or prevention of ovarian cancer. The immunogenic composition may further combine FSHR with one or more cancer antigens NY-ESO-1 or MAGE-A1 for the treatment or prevention of ovarian cancer. Other combinations of cancer antigens may also be used to treat or prevent ovarian cancer.

Other oncological diseases.

The immunogenic composition may combine a TERT antigen and one or more cancer antigens, such as FAP, for the treatment or prevention of cancer. The immunogenic composition may further combine FAP with one or more cancer antigens NY-ESO-1 or MAGE-A1 for the treatment or prevention of cancer. Other combinations of cancer antigens may also be used to treat or prevent cancer.

8. Method of vaccination.

Provided herein is a method of treating or preventing cancer using pharmaceutical preparations to provide genetic constructs and proteins of one or more cancer antigens, as described above, that contain epitopes that make them particularly effective immunogens against which an immune response can be induced to one or more more cancerous

- 115 043982 new antigens. A method of administering an immunogenic composition 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 immunogenic composition can be administered to an individual to modulate the activity of the mammal's immune system and enhance the immune response. Administration of the immunogenic composition may be the transfection of one or more cancer antigens disclosed herein into a nucleic acid molecule that is expressed in the cell and thereby delivered to the surface of the cell, at which the immune system recognizes and induces cellular, humoral or cellular and humoral response. Administration of an immunogenic composition can be used to induce or elicit an immune response in a mammal to one or more cancer antigens described herein by administering the immunogenic composition described herein to the mammal.

Upon administration of the immunogenic composition to a mammal and, subsequently, the vector into cells of the mammal, the transfected cells will express and secrete one or more cancer antigens disclosed herein. These secreted proteins or synthetic antigens will be recognized by the immune system as foreign, resulting in an immune response that may include: antibodies produced against one or more cancer antigens, and a T cell response specifically against one or more cancer antigens. In some examples, a mammal to which an immunogenic composition discussed herein is administered will have a primed immune system, and when exposed to one or more of the cancer antigens disclosed herein, the primed immune system will enable rapid clearance of additional cancer antigens disclosed herein. either through humoral, cellular, or both cellular and humoral immune responses. The immunogenic composition can be administered to an individual to modulate the activity of the individual's immune system, thereby enhancing the immune response.

Methods for administering an immunogenic composition 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 to induce 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.

The dose of the immunogenic composition may be from 1 μg to 10 mg of active component/kg body weight/time and may be from 20 μg to 10 mg of component/kg body weight/time. The immunogenic composition 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 doses of the immunogenic composition for effective treatment may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more doses.

A method for generating an immune response using an immunogenic composition.

The immunogenic composition 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 target one or more of the cancer antigens disclosed herein. Such antibodies and T cells can be isolated.

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

Method for treating cancer using an immunogenic composition.

The immunogenic composition can be used to generate or induce an immune response in a mammal that responds to or targets a cancer or tumor (e.g., melanoma, head and neck cancer, cervical, liver, prostate, blood cancers, esophageal squamous cell cancer, gastric cancer) in a mammal or subject in need of it. The immune response generated can prevent cancer or tumor growth.

The elicited immune response can prevent and/or reduce metastasis of cancer or tumor cells. Accordingly, the immunogenic composition can be used in a method that treats and/or prevents cancer or tumors in a mammal or subject to which the immunogenic composition is administered. Depending on the antigen used in the immunogenic composition, the cancer or tumor growth to be treated may be any

- 116 043982 fight type of cancer such as, but not limited to, melanoma, blood cancer (eg leukemia, lymphoma, myeloma), lung cancer, squamous cell cancer of the esophagus, bladder cancer, colorectal cancer, esophageal cancer, stomach cancer, hepatocarcinoma , head and neck cancer, brain cancer, anal cancer, non-small cell lung cancer, pancreatic cancer, synovial cancer, prostate cancer, testicular cancer, liver cancer, cervical cancer, recurrent respiratory papillomatosis, skin cancer and stomach cancer.

In some embodiments, the administered immunogenic composition may mediate the clearance or prevent the growth of tumor cells by inducing (1) humoral immunity through B cell responses to generate antibodies that block the production of monocyte chemoattractant protein-1 (MCP-1), thereby slowing the development myeloid-derived suppressor cells (MDSC) and suppressing tumor growth; (2) increasing levels of cytotoxic T lymphocytes such as CD8 ⁺ (CTL) to attack and kill tumor cells; (3) enhancing T-helper responses; (4) and enhancement of inflammatory responses by IFN-γ and TFN-α or a combination 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 neurological system, etc.) in the subject , who was administered an immunogenic composition.

In some embodiments, the administered immunogenic composition may increase tumor survival, reduce tumor burden, increase tumor survival, or a combination thereof in a subject. The administered immunogenic composition 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 subject. The administered immunogenic composition can reduce tumor weight 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 immunogenic composition can prevent and block the increase in monocyte chemoattractant protein 1 (MCP-1), a cytokine secreted by myeloid-derived suppressor cells in a subject. In some embodiments, the administered immunogenic composition can prevent and block the increase of MCP1 in cancer or tumor tissue in a subject, thereby reducing the vascularization of the cancer or tumor tissue in the subject.

The administered immunogenic composition 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, 64, 65 , 66, 67, 68, 69 and 70% in the subject after immunization. In some embodiments, the immunogenic composition can be administered peripherally (as described in more detail below) to generate an antigen-specific immune response targeting cancer or tumor cells or tissue to eliminate or eliminate a cancer or tumor expressing one or more cancer antigens, without damaging or causing illness or death in a subject to whom the immunogenic composition is administered.

The administered immunogenic composition may enhance the cellular immune response in a subject by about 50-6000-fold, about 50-5500-fold, about 50-fold to 5000-fold, about 50-fold to 4500-fold, about 100-fold to 6000-fold, about 150 to 6000 times, about 200 to 6000 times, about 250 to 6000 times, or about 300 to 6000 times. In some embodiments, the administered immunogenic composition may enhance the cellular immune response in a subject by about 50-fold, 100-fold, 150-fold, 200-fold, 250-fold, 300-fold, 350-fold, 400-fold, 450-fold times, 500 times, 550 times, 600 times, 650 times, 700 times, 750 times, 800 times, 850 times, 900 times, 950 times, 1000 times, 1100 times, 1200 times, 1300 times, 1400 times, 1500 times, 1600 times, 1700 times, 1800 times, 1900 times, 2000 times, 2100 times, 2200 times, 2300 times, 2400 times times, 2500 times, 2600 times, 2700 times, 2800 times, 2900 times, 3000 times, 3100 times, 3200 times, 3300 times, 3400 times, 3500 times, 3600 times, 3700 times, 3800 times, 3900 times, 4000 times, 4100 times, 4200 times, 4300 times, 4400 times, 4500 times, 4600 times, 4700 times, 4800 times, 4900 times times, 5000 times, 5100 times, 5200 times, 5300 times, 5400 times, 5500 times, 5600 times, 5700 times, 5800 times, 5900 times or 6000 times.

The administered immunogenic composition can increase interferon gamma (IFN-γ) levels in a subject by about 50-6000-fold, by about 50-5500-fold, by about 50-5000-fold, by about 50-fold to 4500-fold, by about 100 to 6000 times, about 150 to 6000 times, about 200 to 6000 times, about 250 to 6000 times, or about 300 to 6000 times. In some embodiments, the administered immunogenic composition may increase interferon gamma (IFN-γ) levels in a subject by about 50-fold, 100-fold, 150-fold, 200-fold, 250-fold, 300-fold, 350-fold, 400 times, 450 times, 500 times, 550 times, 600 times, 650 times, 700 times, 750 times, 800 times, 850 times, 900 times, 950 times, 1000 times , 1100 times, 1200 times, 1300 times, 1400 times, 1500 times, 1600 times, 1700 times, 1800 times, 1900 times, 2000 times, 2100 times, 2200 times, 2300 times, 2400 times, 2500 times, 2600 times, 2700 times, 2800 times, 2900 times, 3000 times,

- 117 043982

3100 times, 3200 times, 3300 times, 3400 times, 3500 times, 3600 times, 3700 times, 3800 times, 3900 times, 4000 times, 4100 times, 4200 times, 4300 times , 4400 times, 4500 times, 4600 times, 4700 times, 4800 times, 4900 times, 5000 times, 5100 times, 5200 times, 5300 times, 5400 times, 5500 times, 5600 times, 5700 times, 5800 times, 5900 times or 6000 times.

The dose of the immunogenic composition may be from 1 μg to 10 mg of active component/kg body weight/time and may be from 20 μg to 10 mg of component/kg body weight/time. The immunogenic composition 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 doses of the immunogenic composition for effective treatment may be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

(1) Combination therapy with immune checkpoint antibodies.

This invention also relates to a method of enhancing the immune response in a mammal using the immunogenic composition described above. The immunogenic composition described above may contain a cancer antigen and an anti-PD1 antibody and/or an anti-PDL1 antibody described above. The combination may be in one composition or may be separate and administered sequentially (either the cancer antigen first and then the immune checkpoint antibody or the immune checkpoint antibody 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 min, 45 min, 50 min, 55 min, 60 min, 0.25 h, 0.5 h, 0.75 h, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours , 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, 96 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks before administering the immune checkpoint antibody to the subject. In other embodiments, the immune checkpoint 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 min, 45 min, 50 min, 55 min, 60 min, 0.25 h, 0.5 h, 0.75 h, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, 21 h, 22 h, 23 h, 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 antibody to the subject.

The combination of a cancer antigen and an immune checkpoint antibody induces the immune system more effectively than an immunogenic composition containing only the cancer antigen. This more effective immune response provides increased effectiveness in the treatment and/or prevention of a particular cancer. Depending on the antigen used in the immunogenic composition combined with the immune checkpoint antibody, the cancer or tumor growth being treated may be any type of cancer, such as, but not limited to, melanoma, blood cancer (e.g., leukemia, lymphoma, myeloma), lung cancer, squamous cell cancer of the esophagus, bladder cancer, colorectal cancer, esophageal cancer, stomach cancer, hepatocarcinoma, head and neck cancer, brain cancer, anal cancer, non-small cell lung cancer, pancreatic cancer, synovial cancer, cancer prostate, testicular cancer, liver cancer, cervical cancer, recurrent respiratory papillomatosis, skin cancer and stomach cancer.

In some embodiments, the immune response may be enhanced by about 0.5 to 15 times, by about 0.5 to about 10 times, or by about 0.5 to about 8 times. Alternatively, the immune response in a subject administered the immunogenic composition 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 administered the immunogenic composition may be enhanced by about 50% to about 1500%, from about 50% to about 1000%, or from about 50% to about 800%. Alternatively, the immune response in a subject to whom the immunogenic composition is administered may be enhanced by at least about 50%, at least about 100%, at least about 150%, at least about 200%, at least by about 250%, by at least by about 300%, by at least by about 350%, by at least

- 118 043982 about 400%, at least about 450%, at least about 500%, at least about 550%, at least about 600%, at least about 650%, at least by about 700%, by at least about 750%, by at least about 800%, by at least about 850%, by at least about 900%, by at least about 950%, by 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% , by at least about 1350%, by at least about 1450%, or by at least about 1500%.

The dose of the immunogenic composition may be from 1 μg to 10 mg of active component/kg body weight/time and may be from 20 μg to 10 mg of component/kg body weight/time. The immunogenic composition 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 doses of the immunogenic composition for effective treatment may be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

(2) Oncological disease.

The immunogenic composition can be used to generate or induce an immune response in a mammal that is reactive to or targets a tumor in the mammal or subject in need thereof. The resulting immune response can prevent tumor growth. The resulting immune response can reduce tumor growth. The elicited immune response can prevent and/or reduce metastasis of cancer or tumor cells. Accordingly, the immunogenic composition can be used in a method that treats and/or prevents cancer in a mammal or subject to which the immunogenic composition is administered.

In some embodiments, the administered immunogenic composition may mediate the clearance or prevent the growth of tumor cells by inducing (1) humoral immunity through B cell responses to generate antibodies that block the production of monocyte chemoattractant protein-1 (MCP-1), thereby slowing the development myeloid-derived suppressor cells (MDSC) and suppressing the growth of melanoma; (2) increasing levels of cytotoxic T lymphocytes such as CD8 ⁺ (CTL) to attack and kill melanoma cells; (3) enhancing T-helper responses; (4) and enhancement of inflammatory responses by IFN-γ and TFN-α or a combination of the above.

In some embodiments, the administered immunogenic composition may increase tumor-free survival, reduce tumor burden, increase tumor-bearing survival, or a combination thereof in a subject. The administered immunogenic composition can increase tumor-free survival by 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43%, 44% and 45% in a subject. The administered immunogenic composition can reduce tumor weight 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% in the subject after immunization. The administered immunogenic composition may reduce the vascularization of tumor tissue in a subject. The administered immunogenic composition 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% in the subject.

9. Routes of administration.

The immunogenic composition or pharmaceutical composition can be administered by various routes, including oral, parenteral, sublingual, transdermal, rectal, transmucosal, topical, inhalation, buccal, intrapleural, intravenous, intraarterial, intraperitoneal, subcutaneous, intramuscular, intranasal, intrathecal and intraarticular, or their combinations. For veterinary use, the composition can be administered in a suitable acceptable preparation 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 immunogenic composition can be administered using traditional syringes, needleless injection devices, biobalistically using microparticles or other physical methods such as electroporation (EP), hydrodynamic method or ultrasound.

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

Electroporation.

The immunogenic composition or pharmaceutical composition can be administered by electroporation. Administration of an immunogenic composition by electroporation can be accomplished using electroporation devices that can be configured to deliver to the desired mammalian tissue a pulse of energy effective to form reversible pores in cell membranes. In one embodiment of the invention, the energy pulse is represented by

- 119 043982 is a constant current similar to the user-specified current. The electroporation device may comprise an electroporation component and an electrode assembly or a handle assembly. An electroporation component may comprise and comprise 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 message element, a communication port, a memory component, a power supply, and 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 electroporation methods that may facilitate administration of the immunogenic compositions of this invention include those described in U.S. Patent No. 7,245,963 to Draghia-Akli, et al., U.S. Patent Publication 2005/0052630 filed by Smith et al., the contents of which are incorporated herein in their entirety by reference. Other electroporation devices and electroporation methods that may be used to facilitate administration include those disclosed in co-pending U.S. Patent Application No. 11/874,072, filed Oct. 17, 2007, which claims priority under 35 USC § 119(e) based on US Provisional Applications No. 60/852149, filed October 17, 2006, and US No. 60/978982, filed October 10, 2007, all of which are incorporated herein in their entirety.

In US Patent No. 7245963, Draghia-Akli, et al. modular electrode systems and their use to facilitate the introduction of a biomolecule into the cells of a selected tissue of an organism or plant are described. Modular electrode systems can contain multiple needle electrodes; hypodermic needle; an electrical connector that provides conductive communication from the programmable DC pulse controller to the plurality of needle electrodes; and power supply. The operator can take a variety 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 DC pulse controller is activated and a DC electrical pulse is applied to a plurality of needle electrodes. The applied DC electrical pulse facilitates the introduction of the biomolecule into the cell between multiple electrodes. The entire contents of US Pat. No. 7,245,963 are incorporated herein in their entirety by reference.

US Patent Publication 2005/0052630 filed 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 body or plant tissue. The electroporation device contains an electrokinetic device (EKD device), the operation of which is determined by software or firmware. The EKD device generates a series of programmable DC pulse patterns between electrodes in an array based on user control and input of pulse parameters and allows current waveform data to be stored and retrieved. The electroporation device also includes a replaceable electrode disk having a row of needle electrodes, a central channel for an injection needle, and a removable guide disk. The entire contents of US Patent Publication 2005/0052630 are incorporated herein in their entirety by reference.

The electrode arrays and methods described in US Patent No. 7245963 and US Patent Publication 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 selected 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. In one embodiment, the electrodes are 20 mm long and 21 gauge, as described in US Patent No. 7,245,963 and US Patent Publication 2005/005263.

Additionally, certain embodiments that include electroporation devices and their use include electroporation devices that are described in the following patents: US Patent 5,273,525 issued December 28, 1993, US Patents 6,110,161 issued August 29, 2000, 6,261,281 issued July 17, 2001, and 6,958,060 issued October 25, 2005, and US Patent 6,939,862 issued September 6, 2005. Also discussed herein are patents covering the subject matter of US Patent No. 6,697,669, issued February 24, 2004, which relates to the introduction of DNA using any of a variety of devices, and US Patent No. 7,328,064, issued February 5, 2008. , which refers to the method of injecting DNA. The above patents are incorporated by reference in their entirety.

10. Method for preparing an immunogenic composition.

Presented herein are methods for preparing nucleic acid molecules that contain the immunogenic compositions discussed herein. Nucleic acid molecules can be used to inoculate cell culture in large-scale fermentation

- 120 043982 nom tank using methods known in the art.

Nucleic acid molecules for use with the EP devices of this invention can be prepared or manufactured using a combination of known devices and technologies. In one embodiment, they are manufactured using optimized plasmid manufacturing technology, which is described in US Patent Application Published No. 20090004716, filed May 23, 2007. In some examples, the nucleic acid molecules used in these studies can be prepared in concentrations greater than or equal to 10 mg/ml. The manufacturing techniques also include or cover various devices and protocols that are generally known to those skilled in the art, in addition to those described in US patent application 60/939,792, and including those described in the patent subject to the license, the patent US No. 7238522, issued July 3, 2007. The above application and patent, US Patent Application No. 60/939792 and US Patent No. 7238522, respectively, are incorporated herein in their entirety.

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

11. Examples.

Example 1.

Construction of pTyr.

As shown 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 shown 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 nucleic acid sequence encoding the consensus Tyr was adapted to contain an IgE leader sequence. Specifically, the IgE leader sequence was fused in frame to the Tyr consensus nucleic acid sequence (Fig. 1B). The resulting sequence was then inserted into the pVax1 expression vector to create a Tyrosinase or plasmid (pTyr) construct such that the Kozak sequence preceded the nucleotide sequence encoding the IgE leader sequence and the consensus Tyr.

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

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

Example 2.

Vaccination with pTyr induced a cellular immune response.

The pTyr described above was used to vaccinate mice to evaluate whether a cellular immune response was induced by pTyr. C57/B6 mice were immunized using the immunization strategy shown in 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) 5 μg pTyr dosage; (2) dosage 20 μg pTyr; (3) dosage 30 μg pTyr; and (4) dosage of 60 μg pTyr.

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

The cellular immune response to pTyr was further 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 vaccination with pTyr (data not shown).

Example 3.

Vaccination with pTyr increased the levels of the cytokines IFN-γ and TNF-α.

Cytokine production was studied in mice immunized with pTyr and pVax1. Mice were immunized using the strategy shown in FIG. 2A. On the 35th day of the stratum

- 121 043982 immunization cells isolated from immunized mice were stimulated overnight with Tyr peptides. After stimulation, analysis of multifunctional responses was measured using FACS. Specifically, 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 the 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 vaccination with pTyr.

The humoral immune response was studied in mice vaccinated with pTyr. Specifically, C57BI/6 mice (n=4) were immunized three times at 2-week intervals with either pTyr or pVax1. Each immunization was 20 μg/IM followed by electroporation with MID-EP. After the third immunization (i.e., day 35), serum was collected from mice and antibody titers were measured by ELISA using all HRP-labeled IgG-specific secondary antibodies. Sera were diluted as indicated in Fig. 4A. As shown in FIG. 4A, specific antibodies against Tyr were produced in 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. An increase in peak Tyr-specific titer compared to preimmune serum was found for all immunized groups of mice at days 90 and 120 of retesting. Representative results from three independent experiments at each serial dilution point are depicted in FIG. 4B. These data also indicate that immunization with pTyr induces the production of specific antibodies against Tyr in mice immunized with pTyr.

Example 5.

Mice Vaccinated with pTyr Have Increased Survival Following Tumor Challenge pTyr was further analyzed to determine whether vaccination with pTyr could provide protection against tumors. Specifically, C57BI/6 mice (10 per group) were immunized at 2-week intervals with either pTyr or pVax1. Each immunization was 20 μg/IM followed by electroporation with MID-EP. One week after the third immunization (ie, on day 35), immunized mice were injected 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 shown in FIG. 5A (Kaplan-Meier survival curve), Mice immunized with pTyr had increased tumor-free survival (i.e., 40% at day 40 and post-tumor challenge) compared to mice immunized with pVax1 (p=0 ,05), who were all dead 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). For both figs. 5A and 5B, mice immunized with pVax1 are represented by filled squares and mice immunized with pTyr are represented by filled circles. Accordingly, these data demonstrated that vaccination with pTyr provided protection against melanoma, namely increased tumor-free survival and decreased tumor volume.

Example 6.

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

MDSC populations were examined in mice immunized with pTyr and in nonimmunized mice to examine whether vaccination with pTyr altered MDSC levels in tumors from the respective groups of mice. Specifically, the percentage of Gr-1+ and CD11b+ cells was examined in immunized and nonimmunized mice.

As shown in FIG. 6 and 7, MDSC levels were significantly reduced in tumors from mice immunized with pTyr compared with nonimmunized 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 demonstrated that immunization with pTyr reduced the MDSC population in tumors of pTyr-vaccinated mice.

Example 7.

MCP-1 levels are reduced by pTyr vaccination.

MDSCs can secrete the cytokine MCP-1, which promotes angiogenesis or vascularization through endothelial cell migration. Considering the effect of vaccination described above with

- 122 043982 pTyr on MDSC levels in tumors, MCP-1 levels were examined after vaccination with pTyr.

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

As shown 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. However, MCP-1 levels in mice immunized with pTyr were about 3-fold lower than in mice immunized with pVax1. Accordingly, these data demonstrated that vaccination with pTyr reduced the level of MCP-1 secretion by MDSCs in tumors of mice immunized with pTyr.

Example 8.

Construction of pPRAME.

A consensus sequence for PRAME was generated, and the nucleotide sequence encoding the 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 referred to 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 antigen and the PRAME consensus antigen staining, is depicted in FIG. 10V. This staining 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 the transfected cells was used to confirm the expression of the PRAME consensus antigen in the 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 an application control. In summary, staining of transfected cells and Western blotting of lysates from transfected cells demonstrated that the pPRAME vector mediates 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 evaluate whether a cellular immune response was induced by pPRAME. C57BL/6 mice were divided into groups. The first group was native 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 using the IFN-γ ELISpot assay. As shown in FIG. 11A and 11B, each dose of pPRAME induced IFN-γ production or secretion in contrast to naïve negative control mice. Specifically, IFN-γ levels were increased by about 3000-fold to about 4500-fold in vaccinated mice compared to unvaccinated mice. Accordingly, these data demonstrated 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 generate pGX1409 ( also referred to herein as pNY-ESO-1) (see FIG. 12A).

To confirm that pNY-ESO-1 resulted in expression of the NY-ESO1 consensus antigen, pVAX and pNY-ESO-1 were transfected into cells. DAPI was used to stain nuclei, and the NY-ESO-1 consensus antigen was also fluorescently stained. This staining, along with a fusion of the DAPI antigen and the NY-ESO-1 consensus antigen staining, is depicted in FIG. 12V. This staining 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

- 123 043982 (i.e. negative control).

In addition, Western blot analysis of lysates from transfected 293T and RD cells was used to confirm expression of the NY-ESO-1 consensus antigen in 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 an application control. In summary, staining of transfected cells and Western blotting of lysates from transfected cells demonstrated that the pNY-ESO-1 vector mediates expression of the NY-ESO-1 consensus antigen in cells.

Example 11.

Interferon gamma response to vaccination with pNY-ESO-1.

The pNY-ESO-1 described above was used to vaccinate mice to evaluate whether a cellular immune response was induced by pNY-ESO-1. C57BL/6 mice were divided into groups. The first group was native 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 using the IFN-γ ELISpot assay. As shown in FIG. 13, each dose of pPRAME induced IFN-γ production or secretion in contrast to naïve negative control mice. Specifically, IFN-γ levels were increased by about 700-fold to about 1100-fold in vaccinated mice compared to unvaccinated mice. Accordingly, these data demonstrated 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 vaccination with pNY-ESO-2.

The NY-ESO-2 consensus sequence was obtained, 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 referred to herein as pVAX1) to generate pNY-ESO-2.

This pNY-ESO-2 was used to vaccinate mice to evaluate whether a cellular immune response was induced by pNY-ESO-2. C57BL/6 mice were divided into groups. The first group was native 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 interferon gamma (IFN-γ) induction using the IFN-γ ELISpot assay. As shown in FIG. 14, each dose of pNY-ESO-2 induced IFN-γ production or secretion in contrast to naïve negative control mice. Specifically, IFN-γ levels were increased by about 400-fold to about 500-fold in vaccinated mice compared to unvaccinated mice. Accordingly, these data demonstrated 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 above detailed description and the accompanying examples are illustrative only and should not be construed as limiting the scope of the invention, which is defined solely by the 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 but not limited to those relating to the chemical structures, substituents, derivatives, intermediates, syntheses, compositions, formulations or methods of use of the invention, may be made without departing from the spirit and scope of the invention.

Example 13.

Synthetic consensus hTERT.

The experiments presented herein were designed to evaluate the ability of synthetic consensus hTERT, designed and manufactured using the methods described herein, to break tolerance in non-human primates. A synthetic consensus hTERT (pGX1434) was developed that is approximately 96% identical to rhesus monkey TERT. Rhesus monkeys were either immunized with a plasmid encoding the synthetic consensus hTERT or with a plasmid encoding native RhTERT in the presence of IL12 adjuvant. The nucleotide sequence that encodes the synthetic consensus hTERT (pGX1434) is shown in SEQ ID NO: 45. The amino acid sequence of the synthetic consensus hTERT (pGX1434) is shown in SEQ ID NO: 46. Monkeys were immunized four times, and cellular immune responses were assessed using ELISpot assay . It was noted that compared to native RhTERT

- 124 043982 immunization with synthetic consensus hTERT induced stronger immune responses (1200 SFU/10 ⁶ vs. 400 SFU/10 ⁶ ).

A synthetic hTERT consensus was developed for human TERT with 5 mutations to eliminate its function. Native RhTERT encodes rhesus TERT with 2 mutations. Both designs have been optimized. A schematic representation of TERT designs is shown in FIG. 16 (top). In fig. 16 also depicts the protein homology between optimized TERT and native TERT identified in human, rhesus and mouse (FIG. 16 (bottom)).

Experiments were performed to characterize TERT-specific IFN-γ responses in mice against native rhesus peptides. Mice (n=8 per group) were immunized at 0, 2 and 4 weeks with native RhTERT and synthetic consensus hTERT via IM/electroporation. The immunization scheme with the DNA vaccine is shown in FIG. 17A. The frequency of TERT-specific IFN-γ spot-forming units (SFU) per million splenocytes isolated from vaccinated mice was determined by assay.

ELISpot IFN-γ using Rh peptides. It was observed that immunization with RhTERT and synthetic consensus hTERT induced a strong cellular immune response (Fig. 17B).

Experiments were performed to characterize TERT-specific IFN-γ responses in rhesus. Rhesus monkeys (n=5 per group) were immunized at 0, 4, 8, and 12 weeks with native RhTERT or synthetic consensus hTERT, and with rhIL-12 via IM/electroporation. The immunization scheme with the DNA vaccine is shown in FIG. 18A. The frequency of TERT-specific IFN-γ spot-forming units (SFU) per million splenocytes isolated from vaccinated mice was determined by ELISpot IFN-γ assay using native Rh peptides and SynCon hTERT peptide. It was noted that the synthetic consensus hTERT exhibited better ability to break tolerance in non-human primates compared to RhTERT (Fig. 18B and 18C).

Further experiments were performed to evaluate the effects of synthetic consensus hTERT (pGX1434) on T cell responses and tumor growth. Mice received 3 immunizations 2 weeks apart with 0, 20, 40, or 60 μg of pGX1434. T cell responses in mice were assessed after 5 weeks, demonstrating that pGX1434 induced a robust T cell response (Fig. 19 (left). To assess tumor growth, mice were implanted with 25 x 10 ⁴ TC-1 cells. The mice then received 4 immunizations at intervals of 1 week with 25 μg pGX1434 Tumor volume was measured over 32 days and it was determined that mice treated with pGX1434 had reduced tumor volume compared to untreated mice (Figure 19 (right), Figures 20 and 21) .

The experiments presented here demonstrate that synthetic consensus hTERT is able to break tolerance, induce a strong cellular immune response, and reduce tumor growth. Synthetic consensus hTERT is superior to native autoantigen in its ability to induce a neo-antigen-like immune response to break tolerance.

Example 14.

SynCon hTERT breaks tolerance in NHP.

Experiments were performed to evaluate the immune responses induced by pGX1434 in NHP +/- IL-12 (FIG. 22B, groups 1 and 2) and to evaluate the immune responses induced by pGX1434 in the Wave 1 antigen combination (FIG. 22B, groups 1 and 2). group 3). Experiments were further designed to determine whether the SynCon construct allowed tolerance breaking (Figure 22B, group 4) and to evaluate the ability of SynCon hTERT to break tolerance in NHP (Figure 22B, groups 1-4).

The experimental design is shown in Fig. 22A. The preparation used for these experiments was as follows: 3.0 mg/construct in 1.0 ml 20X SSC with an injection volume of 1 ml.

The experimental results are presented in Fig. 23-26. Responses to native TERT were detected by PD3 and PD4 in the pGX1434+pGX6006 group (Fig. 23). pGX1406 induces more robust TERT responses compared to pGX1434 when combined in a Wave 1 multivalent formulation with 0.20 mg pGX6006 (Figure 25). The specific responses of SynCon PSMA and SynCon WT-1 PD4 are similar for groups containing pGX1434 and pGX1406.

Multivalent formulation of WT-1, TERT and PSMA induced robust immune responses in mice when assayed by IFNy ELISpot and in the CD8+ T cell compartment (Figure 27), and mice immunized with a combination of WT1, PSMA and consensus mTERT showed reduced tumor burden and increased survival (Fig. 28).

The experiments presented here demonstrate that synthetic consensus hTERT is capable of breaking tolerance and inducing a strong cellular immune response in non-human primates.

- 125 043982

Example 15.

Sequences.

SEQ ID NO: Antigen Type Plasmid a 1 Consensus tyrosinase Nucleic acid 2 Consensus tyrosinase Amino acid h Consensus tyrosinase- Nucleic dependent protein 1 acid 4 Consensus tyrosinase- dependent protein 1 Amino acid 5 Consensus tyrosinase- Nucleic dependent protein 2 acid 6 Consensus tyrosinase- dependent protein 2 Amino acid 7 Consensus melanoma- Nucleic associated antigen 4 acid 8 Consensus melanoma- associated antigen 4 Amino acid 9 Consensus somatotropin- Nucleic releasing hormone acid 10 Consensus somatotropin- releasing hormone Amino acid eleven Consensus Melan-A Nucleic acid 12 Consensus Melan-A Amino acid 13 Consensus NY-ESO-1 Nucleic acid 14 Consensus NY-ESO-1 Amino acid 15 Consensus NY-ESO-2 Nucleic acid 16 Consensus NY-ESO-2 Amino acid 17 Consensus FRAME Nucleic acid 18 Consensus FRAME Amino acid 19 Con WT1-L with modified zinc pins Nucleic acid pGX1404 20 Con WT1-L with modified zinc pins Amino acid pGX1404 21 Con WT1-S without zinc fingers Nucleic

- 126 043982

acid 22 Con WT1-S without zinc fingers Amino acid 23 hTERT is functionally related to IgE Nucleic acid pGX1406 24 hTERT is functionally related to IgE Amino acid pGX1406 25 Consensus gB Nucleic acid 26 Consensus dV Amino acid 27 Consensus DM Nucleic acid 28 Consensus DM Amino acid 29 Consensus gN Nucleic acid thirty Consensus gN Amino acid 31 Consensus dN Nucleic acid 32 Consensus dN Amino acid 33 Consensus gL Nucleic acid 34 Consensus gL Amino acid 35 Consensus before Nucleic acid 36 Consensus before Amino acid 37 Consensus UL128 Nucleic acid 38 Consensus UL128 Amino acid 39 Consensus UL130 Nucleic acid 40 Consensus UL130 Amino acid 41 Consensus UL131a Nucleic acid 42 Consensus UL131a Amino acid 43 Consensus UL83 Nucleic acid

- 127 043982

44 Consensus UL83 Amino acid 45 Synthetic Consensus hTERT functionally linked to IgE Nucleic acid pGX1434 46 Synthetic Consensus hTERT functionally linked to IgE Amino acid pGX1434 47 Synthetic Consensus hTERT Nucleic acid PGX1434 48 Synthetic Consensus hTERT Amino acid PGX1434 49 Synthetic Consensus mTERT functionally linked to IgE Nucleic acid PGX1418 50 Synthetic Consensus mTERT functionally linked to IgE Amino acid PGX1418 51 Synthetic Consensus mTERT Nucleic acid PGX1418 52 Synthetic Consensus mTERT Amino acid PGX1418 53 Synthetic Consensus rhTERT functionally linked to IgE Nucleic acid PGX1473 54 Synthetic Consensus rhTERT functionally linked to IgE Amino acid PGX1473 55 Synthetic Consensus rhTERT Nucleic acid PGX1473 56 Synthetic Consensus rhTERT Amino acid PGX1473 57 mut rhTERT Nucleic acid PGX1447 58 mut rhTERT Amino acid PGX1447 59 Consensus FAP Nucleic

- 128 -

Claims (25)

60 61 Consensus Consensus KAR FSHR acid Amino acid Nucleic acid 62 Consensus FSHR Amino acid 63 Consensus P.S.A. Nucleic acid 64 Consensus P.S.A. Amino acid 65 Consensus PSMA Nucleic acid pGX1108 66 Consensus PSMA Amino acid pGX1108 67 Consensus STEAP Nucleic acid 68 Consensus STEAP Amino acid 69 Consensus PSCA Nucleic acid 70 Consensus PSCA Amino acid CLAIM 1. An immunogenic composition comprising an effective amount of a nucleic acid molecule encoding a TERT consensus antigen, wherein the TERT consensus antigen contains an amino acid sequence selected from the group consisting of: a) an amino acid sequence selected from the group consisting of SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54 and SEQ ID NO: 56, b) an amino acid sequence that is 98% or more identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54 and SEQ ID NO: 56, and c) a fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54 and SEQ ID NO: 56, wherein the fragment contains at least 98% of the full-length amino acid sequence. 2. The immunogenic composition according to claim 1, wherein the immunogenic composition further comprises one or more nucleotide sequences encoding one or more amino acid sequences selected from the group consisting of a) an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62 , SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70; b) an amino acid sequence that is 98% or more identical to an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, and c) a fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, wherein the fragment contains at least 98% of the full-length amino acid sequence. 3. The immunogenic composition according to claim 1, further containing a nucleic acid encoding one or more antigens selected from the group consisting of: MAGE A1, gp100, viral antigen and combinations thereof. 4. The immunogenic composition according to claim 3, wherein the viral antigen is an antigen of hepatitis B virus (HBV), hepatitis C virus (HCV) or human papillomavirus (HPV). 5. An immunogenic composition according to claim 4, wherein the HBV antigen is an HBV core antigen or an HBV surface antigen, or a combination thereof. 6. The immunogenic composition according to claim 5, wherein the HCV antigen is HCV NS34A antigen, HCV NS5A antigen, HCV NS5B antigen, HCV NS4B antigen, or a combination thereof. 7. An immunogenic composition according to claim 4, wherein the HPV antigen is an HPV type E6 antigen - 129 043982 6, HPV type 6 E7 antigen, HPV type 11 E6 antigen, HPV type 11 E7 antigen, HPV type 16 E6 antigen, HPV type 16 E7 antigen, HPV type 18 E6 antigen, HPV type 18 E7 antigen, or a combination thereof. 8. The immunogenic composition of claim 1, further comprising an immune checkpoint protein inhibitor. 9. An immunogenic composition according to claim 1, wherein the nucleic acid molecule contains a nucleotide sequence selected from the group consisting of a) a nucleotide sequence selected from the group consisting of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53 and SEQ ID NO: 55, b) a nucleotide sequence that is 98% or more identical to a nucleotide sequence selected from the group consisting of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53 and SEQ ID NO: 55, and c) a fragment of a nucleotide sequence selected from the group consisting of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53 and SEQ ID NO: 55, wherein the fragment contains at least 98% of the full-length nucleotide sequence. 10. The immunogenic composition according to claim 2, wherein the immunogenic composition contains one or more nucleotide sequences selected from the group consisting of a) a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO : 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61 , SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67 and SEQ ID NO: 69; b) a nucleotide sequence that is 98% or more identical to a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 , SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67 and SEQ ID NO: 69, and c) a fragment of a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67 and SEQ ID NO: 69, wherein the fragment contains at least 98% of the full-length nucleotide sequence. 11. An immunogenic composition according to claim 1, in which the nucleic acid is a plasmid. 12. An immunogenic composition according to claim 1, wherein the composition contains one or more plasmids. 13. Immunogenic composition according to claim 1, additionally containing an adjuvant. 14. The immunogenic composition according to claim 13, wherein the adjuvant is IL-12, IL-15, IL-28 or RANTES. 15. A method of treating or preventing cancer in a subject in need thereof, which includes administering to the subject an immunogenic composition according to claim 1. 16. The method according to claim 15, wherein the administration includes the step of electroporation. 17. The method of claim 15, further comprising administering to the subject an immune checkpoint protein inhibitor. 18. The method of claim 17, wherein the immunogenic composition and the immune checkpoint protein inhibitor are administered to the subject as a single formulation. 19. The method of claim 17, wherein the immunogenic composition and the immune checkpoint protein inhibitor are separately administered to the subject. 20. The method according to claim 15, in which the oncological disease is selected from the group consisting of melanoma, head and neck cancer, prostate cancer, liver cancer, cervical cancer, recurrent respiratory papillomatosis (RRP), anal cancer, blood cancer, ovarian cancer and their combinations. 21. A nucleic acid molecule containing one nucleotide sequence selected from the group consisting of: a) a nucleotide sequence selected from the group consisting of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53 and SEQ ID NO: 55, b) a nucleotide sequence that is 98% or more identical to a nucleotide sequence selected from the group consisting of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53 and SEQ ID NO: 55, and c) a fragment of a nucleotide sequence selected from the group consisting of SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53 and SEQ ID NO: 55, wherein the fragment contains at least 98% of the full-length nucleotide sequence. 22. The nucleic acid molecule according to claim 21, further comprising one or more nucleotide sequences selected from the group consisting of a) a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID - 130 043982 NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67 and SEQ ID NO: 69; b) a nucleotide sequence that is 98% or more identical to a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 , SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67 and SEQ ID NO: 69, and c) a fragment of a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67 and SEQ ID NO: 69, wherein the fragment contains at least 98% of the full-length nucleotide sequence. 23. The nucleic acid molecule according to claim 21, wherein the nucleic acid molecule is a plasmid. 24. A polypeptide containing one amino acid sequence selected from the group consisting of: a) an amino acid sequence selected from the group consisting of SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54 and SEQ ID NO: 56, b) an amino acid sequence that is 98% or more identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54 and SEQ ID NO: 56, and c) a fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54 and SEQ ID NO: 56, wherein the fragment contains at least 98% of the full-length amino acid sequence. 25. The polypeptide of claim 24, further comprising one or more amino acid sequences selected from the group consisting of: a) an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO : 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62 , SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70; b) an amino acid sequence that is 98% or more identical to an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, and c) a fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, wherein the fragment contains at least 98% of the full-length amino acid sequence. - 131 -