Survivin-targeting cancer vaccine and uses thereof

Abstract

To provide nucleic acid molecules comprising one or more nucleic acid sequences that encode synthetic consensus Survivin antigens.SOLUTION: Vectors, compositions, and vaccines comprising one or more nucleic acid sequences that encode synthetic consensus Survivin antigens are disclosed. Methods of treating a subject with a Survivin-expressing tumor and methods of preventing a Survivin-expressing tumor are disclosed. Synthetic consensus Survivin antigens are disclosed.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 62 / 598,267, filed December 13, 2017, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on December 13, 2018, is named 104409_000448_sequence_listing.txt, and is 8,797 bytes in size.

[0003] The present invention relates to a survivin antigen and a nucleic acid molecule encoding the same. The present invention also relates to a vaccine comprising such a survivin antigen and / or nucleic acid molecule. The present invention further relates to a method of using the vaccine to induce an immune response and to prevent and / or treat subjects with cancer cells and / or tumors expressing survivin. [Background technology]

[0004] Cancer is one of the leading causes of death worldwide. In the United States, cancer is the second leading cause of death, accounting for nearly one in four deaths. Cancer develops from a single cell that transforms from a normal cell into a cancerous cell. Such transformation is often a multi-step process that progresses from a precancerous lesion to a malignant tumor. Multiple factors contribute to this progression, including aging, genetic contributions, and exposure to external agents such as physical carcinogens (e.g., ultraviolet and ionizing radiation), chemical carcinogens (e.g., asbestos, components of tobacco smoke), and biological carcinogens (e.g., certain viruses, bacteria, parasites).

[0005] Cancer prevention, diagnosis, and treatment can take many different forms. Prevention includes screening for predisposing factors (e.g., certain genetic variants), behavior modification (e.g., smoking, diet, and amount of physical activity), and vaccination against viruses (e.g., human papillomavirus, hepatitis B virus, etc.). Treatment can include chemotherapy, radiation therapy, and surgical removal of tumors or cancerous tissue. Despite the availability of numerous prevention and treatment methods, such methods often have limited success in effectively preventing and / or treating cancer.

[0006] Survivin, also known as baculovirus inhibitor of apoptosis repeat-containing protein 5 (BIRC5), is an apoptosis inhibitor that blocks caspase function, thereby preventing programmed cell death. In addition to its role in apoptosis, survivin clearly has an essential, evolutionarily conserved role in mitosis. (Li, F. et al. Control of apoptosis and mitotic spindle checkpoint by Survivin. Nature 396, 580-584, doi:10.1038 / 25141(1998)). Overexpression of survivin is associated with tumor cell proliferation, progression, angiogenesis, therapeutic resistance, and poor prognosis. In healthy cells and tissues, survivin expression is either absent or present at low levels. However, survivin is a member of the inhibitor of apoptosis protein (IAP) family, and IAP genes are highly expressed in a variety of cancer cells and primary tumor biopsies. Among the IAPs, survivin shows the most dramatic overexpression in tumors and fetal tissues. In multiple studies of ovarian cancer, the number of patient samples testing positive for survivin expression ranged from 74 to 92%.(Cohen, C., Lohmann, CM, Cotsonis, G., Lawson, D. & Santoianni, R. Survivin expression in ovarian carcinoma: correlation with apoptotic markers and prognosis. Modern pathology: an official journal of the United States and Canadian Academy of Pathology, Inc. 16,574-583, doi:10.1097 / 01.MP.0000073868.31297.B0(2003), Felisiak-Golabek,A.et al.Nuclear Survivin expression is a positive prognostic factor in taxane-platinum-treated ovarian cancer patients.Journal of ovarian research 4,20,doi:10.1186 / 1757-2215-4-20(2011)). Survivin's contribution to tumorigenesis, combined with its restricted pattern of expression and overexpression in various tumors, makes it an attractive target for cancer immunotherapy.

[0007] Survivin is the smallest member of the IAP family. It is a 16.3 kD protein consisting of 142 amino acids and is characterized by the presence of a single BIR repeat. Also, its protein structure lacks a RING finger domain at the carboxyl terminus. (Chen, X., Duan, N., Zhang, C. & Zhang, W. Survivin and Tumorigenesis: Molecular Mechanisms and Therapeutic Strategies. Journal of Cancer 7, 314-323, doi:10.7150 / jca.13332 (2016)). Several survivin isoforms have been identified, with survivin isoform 1 being the major transcript. Survivin is expressed during fetal development but not in fully differentiated tissues. However, it is highly expressed in many cancer cells. Thus, survivin is a potential target antigen for the treatment of cancer. Summary of the Invention [Problem to be solved by the invention]

[0008] Vaccines for the treatment and prevention of cancer, especially epithelial ovarian cancer (EOC), are of interest.However, existing vaccines that target tumor cell antigens are limited by poor antigen expression in vivo.Therefore, there is still a need in the art for safe and effective vaccines and their use methods for preventing and / or treating cancer and reducing the mortality of subjects suffering from cancer. [Means for solving the problem]

[0009] (a) a nucleic acid sequence encoding amino acids 19 to 159 of SEQ ID NO: 2; (b) a nucleic acid sequence encoding amino acids 19 to 210 of SEQ ID NO: 4; (c) a nucleic acid sequence encoding amino acids 19 to 232 of SEQ ID NO: 8; (d) a nucleic acid sequence encoding a fragment comprising at least 90% of the full length of amino acids 19 to 159 of SEQ ID NO: 2; (e) a nucleic acid sequence encoding a fragment comprising at least 90% of the full length of amino acids 19 to 210 of SEQ ID NO: 4; (f) a nucleic acid sequence encoding a fragment comprising at least 90% of the full length of amino acids 19 to 232 of SEQ ID NO: 8; (g) a nucleic acid sequence encoding a protein that is at least 95% identical to amino acids 19 to 159 of SEQ ID NO: 2; (h) a nucleic acid sequence encoding a protein that is at least 95% identical to amino acids 19 to 210 of SEQ ID NO: 4. (i) a nucleic acid sequence encoding a protein that is at least 95% identical to amino acids 19 to 232 of SEQ ID NO:8; (j) a nucleic acid sequence encoding a fragment comprising at least 90% of the full length of a protein that is at least 95% identical to amino acids 19 to 159 of SEQ ID NO:2; (k) a nucleic acid sequence encoding a fragment comprising at least 90% of the full length of a protein that is at least 95% identical to amino acids 19 to 210 of SEQ ID NO:4; and (l) a nucleic acid sequence encoding a fragment comprising at least 90% of the full length of a protein that is at least 95% identical to amino acids 19 to 232 of SEQ ID NO:8.

[0010] The nucleic acid molecule comprises one or more nucleic acid sequences selected from the group consisting of: (a) nucleotides 55-423 of SEQ ID NO:1; (b) nucleotides 55-636 of SEQ ID NO:3; (c) a fragment comprising at least 90% of the entire length of nucleotides 55-423 of SEQ ID NO:1; (d) a fragment comprising at least 90% of the entire length of nucleotides 55-636 of SEQ ID NO:3; (e) a fragment that is at least 95% identical to nucleotides 55-423 of SEQ ID NO:1; (f) a fragment that is at least 95% identical to nucleotides 55-636 of SEQ ID NO:3; (g) a fragment that comprises at least 90% of a nucleic acid sequence that is at least 95% identical to nucleotides 55-423 of SEQ ID NO:1; and (h) a fragment that comprises at least 90% of a nucleic acid sequence that is at least 95% identical to nucleotides 55-636 of SEQ ID NO:3.

[0011] The nucleic acid molecule comprises one or more nucleic acid sequences selected from the group consisting of: (a) a nucleic acid sequence encoding SEQ ID NO:2; (b) a nucleic acid sequence encoding SEQ ID NO:4; (c) a nucleic acid sequence encoding SEQ ID NO:8; (d) a nucleic acid sequence encoding a fragment comprising at least 90% of the full length of SEQ ID NO:2; (e) a nucleic acid sequence encoding a fragment comprising at least 90% of the full length of SEQ ID NO:4; (f) a nucleic acid sequence encoding a fragment comprising at least 90% of the full length of SEQ ID NO:8; (g) a nucleic acid sequence encoding a protein that is at least 95% identical to SEQ ID NO:2; (h) a nucleic acid sequence encoding a protein that is at least 95% identical to SEQ ID NO:4; (i) a nucleic acid sequence encoding a protein that is at least 95% identical to SEQ ID NO:8; (j) a nucleic acid sequence encoding a fragment comprising at least 90% of the full length of a protein that is at least 95% identical to SEQ ID NO:2; (k) a nucleic acid sequence encoding a fragment comprising at least 90% of the full length of a protein that is at least 95% identical to SEQ ID NO:4; and (l) a nucleic acid sequence encoding a fragment comprising at least 90% of the full length of a protein that is at least 95% identical to SEQ ID NO:8.

[0012] The nucleic acid molecule comprises one or more nucleic acid sequences selected from the group consisting of: (a) SEQ ID NO:1, (b) SEQ ID NO:3, (c) a fragment comprising at least 90% of the entire length of SEQ ID NO:1, (d) a fragment comprising at least 90% of the entire length of SEQ ID NO:3, (e) a fragment that is at least 95% identical to SEQ ID NO:1, (f) a fragment that is at least 95% identical to SEQ ID NO:3, (g) a fragment that comprises at least 90% of the entire length of a nucleic acid sequence that is at least 95% identical to SEQ ID NO:1, and (h) a fragment that comprises at least 90% of the entire length of a nucleic acid sequence that is at least 95% identical to SEQ ID NO:3.

[0013] The nucleic acid molecule comprises the nucleic acid sequence shown in SEQ ID NO:1.

[0014] The nucleic acid molecule comprises the nucleic acid sequence shown in SEQ ID NO:3.

[0015] The nucleic acid molecules described herein find use as pharmaceutical agents.

[0016] The nucleic acid molecules described herein find use as agents in the treatment of cancer.

[0017] The nucleic acid molecules described herein find use in the preparation of a medicament.

[0018] The nucleic acid molecules described herein are used in the preparation of a medicament for the treatment of cancer.

[0019] The vector comprises a nucleic acid molecule described herein.

[0020] The vector includes a plasmid or a viral vector.

[0021] The compositions comprise one or more of the nucleic acid molecules described herein.

[0022] The compositions described herein include a pharma- ceutically acceptable carrier.

[0023] The compositions described herein include one or more of the vectors described herein.

[0024] The protein is selected from the group consisting of (a) amino acids 19 to 159 of SEQ ID NO:2, (b) amino acids 19 to 210 of SEQ ID NO:4, (c) amino acids 19 to 232 of SEQ ID NO:8, (d) a fragment comprising at least 90% of the full length of amino acids 19 to 159 of SEQ ID NO:2, (e) a fragment comprising at least 90% of the full length of amino acids 19 to 210 of SEQ ID NO:4, (f) a fragment comprising at least 90% of the full length of amino acids 19 to 232 of SEQ ID NO:8, (g) an amino acid sequence that is at least 95% identical to amino acids 19 to 159 of SEQ ID NO:2, (h) an amino acid sequence that is at least 95% identical to amino acids 19 to 210 of SEQ ID NO:4, % identical to amino acids 19 to 232 of SEQ ID NO:8, (i) an amino acid sequence that is at least 95% identical to amino acids 19 to 232 of SEQ ID NO:8, (j) a fragment comprising at least 90% of the entire length of an amino acid sequence that is at least 95% identical to amino acids 19 to 159 of SEQ ID NO:2, (k) a fragment comprising at least 90% of the entire length of an amino acid sequence that is at least 95% identical to amino acids 19 to 210 of SEQ ID NO:4, and (l) a fragment comprising at least 90% of the entire length of an amino acid sequence that is at least 95% identical to amino acids 19 to 232 of SEQ ID NO:8.

[0025] The protein comprises an amino acid sequence selected from the group consisting of: (a) SEQ ID NO:2, (b) SEQ ID NO:4, (c) SEQ ID NO:8, (d) a fragment comprising at least 90% of the entire length of SEQ ID NO:2, (e) a fragment comprising at least 90% of the entire length of SEQ ID NO:4, (f) a fragment comprising at least 90% of the entire length of SEQ ID NO:8, (g) an amino acid sequence that is at least 95% identical to SEQ ID NO:2, (h) an amino acid sequence that is at least 95% identical to SEQ ID NO:4, (i) an amino acid sequence that is at least 95% identical to SEQ ID NO:8, (j) a fragment comprising at least 90% of the entire length of an amino sequence that is at least 95% identical to SEQ ID NO:2, (k) a fragment comprising at least 90% of the entire length of an amino sequence that is at least 95% identical to SEQ ID NO:4, and (l) a fragment comprising at least 90% of the entire length of an amino sequence that is at least 95% identical to SEQ ID NO:8.

[0026] The protein comprises the amino acid sequence shown in SEQ ID NO:2.

[0027] The protein comprises the amino acid sequence shown in SEQ ID NO:4.

[0028] The protein comprises the amino acid sequence shown in SEQ ID NO:8.

[0029] The vaccine comprises the nucleic acid molecules described herein.

[0030] The vaccine comprises the vector described herein.

[0031] The vaccines described herein further comprise a pharma- ceutically acceptable excipient.

[0032] The vaccines described herein further comprise an adjuvant.

[0033] In the vaccines described herein, the adjuvant is IL-12, IL-15, IL-28, or RANTES.

[0034] A method of treating a subject having cancerous cells that express survivin comprises administering a therapeutically effective amount of a vaccine described herein.

[0035] In the methods described herein, the administering comprises an electroporation step.

[0036] The methods described herein are performed at one or more sites in a subject.

[0037] A method of vaccinating a subject against cancerous cells expressing survivin comprises administering an effective amount of a vaccine described herein to induce a humoral or cellular immune response. [Brief description of the drawings]

[0038] The summary and the following detailed description will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, there are shown in the drawings exemplary embodiments of the invention; however, the invention is not limited to the specific methods, compositions, and devices disclosed. Drawings: [Figure 1] 1 shows a schematic representation of the synthetic consensus survivin antigen isoform 1. Asterisks indicate mutations essential for abolishing anti-apoptotic activity. [Diagram 2] Shown are the overall structures of synthetic consensus survivin antigen isoform 1 monomer A (left) and monomer B (right). Changes relative to native survivin are indicated by spheres. [Diagram 3] A schematic representation of the synthetic consensus survivin antigen isoform 1T3 is shown. The asterisk indicates the mutations essential for abolishing anti-apoptotic activity. The dark grey region on the right (3') represents the truncated survivin isoform 3 (T3) region. [Figure 4] 1 shows the structure of pGX1428, which contains the synthetic consensus survivin antigen isoform 1. [Diagram 5] 1 shows the structure of pGX1429, which contains the synthetic consensus survivin antigen isoform 1T3. [Figure 6] Immunoblots show the expression of synthetic consensus survivin antigen 1 and synthetic consensus survivin antigen 1T3 proteins in human rhabdomyosarcoma (RD) cells transfected with pGX1428 and pGX1429, respectively. Protein bands of the expected molecular weight were detected in pGX1428 (17.5 kD) and pGX1429 (25.3 kD). β-actin was used as a loading control. [Figure 7A-C]Immunogenicity of synthetic consensus survivin antigen 1 and synthetic consensus survivin antigen 1T3. Female CB6F1 were immunized three times at 3-week intervals with the indicated doses of survivin 1 (pGX1428) (Figures 7A-7D), survivin 1T3 pGX1429 (E-H) (n=8 / group), or pGX0001 (empty vector) (n=4). Figure 7A: Survivin 1-specific IFNγ responses by ELISpot in the indicated doses of pGX1428. Figure 7B: Survivin 1-specific CD4+ T cell responses. Figure 7C. Survivin 1-specific CD8+ T cell responses. [Fig. 7D-F] Figure 7D: Survivin 1T3-specific IFNγ responses by ELISpot in the indicated doses of pGX1429. Figure 7E: Survivin 1T3-specific CD4+ T cell responses. Figure 7F: Survivin 1T3-specific CD8+ T cell responses. [Figure 7G] FIG. 7G: Cytokine profile of survivin-1 specific CD4+ and CD8+ T cell responses. [Figure 7H] FIG. 7H: Cytokine profile of survivin1T3-specific CD4+ and CD8+ T cell responses. [Figure 8] Flow cytometry gating strategy is shown. [Figure 9] The relative frequencies of CD4+ and CD8+ T cells are shown. The cellular immune responses induced by pGX1428 and pGX1429 were predominantly in the CD4+ T cell compartment compared to the CD8+ T cell compartment. [Figure 10A] Cytolytic potential of survivin-specific T cells. Cytolytic potential of antigen-specific T cells induced by survivin-1 and survivin-1T3. FIG. 10A: Frequency of survivin-1 specific CD4+CD107a+ T cells. [Figure 10B] FIG. 10B: Frequency of survivin1T3-specific CD4+CD107a+ T cells. [Figure 10C] FIG. 10C: Cytokine profile of CD4+CD107a+ T cells. [Figure 10D] FIG. 10D: Frequency of survivin 1-specific CD8+CD107a+ T cells. [Figure 10E] FIG. 10E: Frequency of survivin1T3-specific CD8+CD107a+ T cells. [Figure 10F] FIG. 10F: Cytokine profile of CD4+CD107a+ T cells. [Figure 11A] Epitope mapping of the survivin IFN-γ response. Breadth of IFNγ responses induced by survivin 1 and survivin 1T3. Figure 11A: Survivin 1 peptide pool following treatment with 50 μg of pGX1428, pGX1429, or pGX0001. [Figure 11B] Figure 11B: Survivin T3 peptide pool after treatment with 50 μg of pGX1429 or pGX0001. Position of matrix pool indicated by dashed box and sequence comparison with pGX1428 and pGX1429. [Figure 11C] Figure 11C: Sequence comparison of pGX1428 and pGX1429. Amino acids in red text indicate epitopes identified by matrix mapping. Underlined text represents sequence portions added to the synthetic consensus survivin antigen: the N-terminus indicates the IgELS and RGRKRRS furin cleavage sites. [Figure 11D] Figure 11D: Design of the survivin 1 and survivin T3 matrices. Peptides that elicited responses are indicated by dashed boxes. [Figure 12] 1 shows survivin-specific IFNγ SFU / 106 PBMCs from individual non-human primates immunized with embodiments of the disclosure, including synthetic consensus survivin 1T3. [Figure 13] 1 shows survivin-specific IFNγ SFU / 106 PBMCs from individual non-human primates immunized using an embodiment of the disclosure comprising synthetic consensus survivin 1T3 with low dose IL-12 (0.04 mg). [Figure 14] 1 shows survivin-specific IFNγ SFU / 106 PBMCs from individual non-human primates immunized using an embodiment of the disclosure including synthetic consensus survivin 1T3 with high dose IL-12 (0.20 mg). [Figure 15A] The results of individual animals from the NHP study are shown, further illustrating a comparison of immunization with the synthetic consensus survivin 1 construct and the synthetic consensus survivin 1T3 construct. FIG. 15A shows the results for Group 1 animals. [Figure 15B] FIG. 15B shows the results for Group 2 animals. [Figure 15C] FIG. 15C shows the results for Group 3 animals. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] The present invention relates to a vaccine comprising a synthetic consensus survivin antigen. Survivin is expressed in many tumors. Thus, the vaccine provides treatment for cancer or cancer-based tumors that express survivin.

[0040] The synthetic consensus survivin antigen may be a consensus survivin antigen derived from the sequence of survivin from different species or different isoforms within a species, and thus the synthetic consensus survivin antigen is non-natural. The consensus survivin antigen may be further modified by introducing one or more mutations into the consensus sequence to generate a synthetic consensus sequence. The mutations may block or modify a specific functional domain of the natural survivin sequence, thereby destroying or enhancing the structure or function of the functional domain. In some embodiments, additional sequences are added to the synthetic consensus survivin antigen sequence to introduce new structures or functions. For example, the synthetic consensus survivin antigen sequence may have a furin cleavage site. The synthetic consensus survivin antigen sequence may include additional localization signals to enhance the extracellular transport of the final protein product. The additional localization signals may be IgELS or other cellular transport sequences.

[0041] The synthetic consensus survivin antigen can induce antigen-specific T cell and / or high titer antibody responses, thereby inducing or eliciting an immune response that is direct or reactive against the cancer or tumor expressing the antigen. In some embodiments, the induced or elicited immune response can be a cellular, humoral, or both a cellular and humoral immune response. In some embodiments, the induced or elicited cellular immune response can include induction or secretion of interferon-gamma (IFN-γ) and / or tumor necrosis factor alpha (TNF-α) and / or interleukin 2 (IL-2). In other embodiments, the induced or elicited immune response can reduce or inhibit one or more immunosuppressive factors that promote the growth of an antigen-expressing tumor or cancer, including, but not limited to, factors that downregulate MHC presentation, antigen-specific regulatory T cells (Tregs), cytokines such as PD-L1, FasL, IL-10 and TFG-β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by immunosuppressive cells, CTLA-4, PD-1, MDSC, MCP-1, and factors that upregulate immune checkpoint molecules.

[0042] The vaccines of the present invention can provide any combination of specific cancer antigens for the specific prevention or treatment of cancer in a subject in need of treatment.

[0043] One way to design the nucleic acid of a recombinant cancer antigen and its encoded amino acid sequence is by introducing mutations that change specific amino acids in the overall amino acid sequence of a native cancer antigen. Although the introduction of mutations does not modify the cancer antigen much and cannot be universally applied across mammalian subjects, preferably human or canine subjects, the resulting amino acid sequence changes enough to break tolerance or be considered a foreign antigen to generate an immune response. Another way can be to create a consensus recombinant cancer antigen that has at least 85% to up to 99% amino acid sequence identity, preferably at least 90% to up to 98% sequence identity, more preferably at least 93% to up to 98% sequence identity, or even more preferably at least 95% to up to 98% sequence identity, compared to its corresponding native cancer antigen. In some cases, the recombinant cancer antigen has 95%, 96%, 97%, 98%, or 99% amino acid sequence identity compared to its corresponding native cancer antigen. A native cancer antigen is an antigen that is normally associated with a particular cancer or cancer tumor. Depending on the cancer antigen, the consensus sequence of the cancer antigen can span across mammalian species, or within a subtype of a species, or across viral strains, or serotypes. Some cancer antigens do not differ significantly from the wild-type amino acid sequence of the cancer antigen. Some cancer antigens have divergent nucleic acid / amino acid sequences across species, making it impossible to generate a consensus sequence. In these cases, recombinant cancer antigens are generated to break tolerance and generate an immune response with at least 85% to up to 99% amino acid sequence identity, preferably at least 90% to up to 98% sequence identity, more preferably at least 93% to up to 98% sequence identity, or even more preferably at least 95% to up to 98% sequence identity, compared to the corresponding native cancer antigen. In some cases, the recombinant cancer antigen has 95%, 96%, 97%, 98%, or 99% amino acid sequence identity compared to the corresponding native cancer antigen. The aforementioned approaches can be combined to make the final recombinant cancer antigen have the percentage similarity to the native cancer antigen amino acid sequence discussed above.

[0044] The vaccines can be further combined with antibodies against checkpoint inhibitors such as PD-1 and PDL-1 to enhance the stimulation of both cellular and humoral immune responses. Using anti-PD-1 or anti-PDL-1 antibodies prevents PD-1 or PDL-1 from suppressing T cell and / or B cell responses. Overall, designing cancer antigens to be recognized by the immune system helps to overcome other forms of immune suppression by tumor cells, and these vaccines can be used in combination with suppressive or inhibitory therapies (such as anti-PD-1 and anti-PDL-1 antibody therapies) to further enhance T cell and / or B cell responses.

[0045] The vaccine can increase tumor free survival by 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, and 45%. The vaccine can reduce tumor mass by 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, and 60% after immunization. The vaccine can prevent and block the increase in monocyte chemoattractant protein 1 (MCP-1), a cytokine secreted by myeloid-derived suppressor cells. The vaccine can enhance 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%.

[0046] The vaccine can enhance the cellular immune response in a vaccinated subject by about 50-fold to about 6000-fold, about 50-fold to about 5500-fold, about 50-fold to about 5000-fold, about 50-fold to about 4500-fold, about 100-fold to about 6000-fold, about 150-fold to about 6000-fold, about 200-fold to about 6000-fold, about 250-fold to about 6000-fold, or about 300-fold to about 6000-fold, compared to the cellular immune response in a subject not administered the vaccine. In some embodiments, the vaccine enhances the cellular immune response in a vaccinated subject by about 50 fold, 100 fold, 150 fold, 200 fold, 250 fold, 300 fold, 350 fold, 400 fold, 450 fold, 500 fold, 550 fold, 600 fold, 650 fold, 700 fold, 750 fold, 800 fold, 850 fold, 900 fold, 950 fold, 1000 fold, 1100 fold, 1200 fold, 1300 fold, 1400 fold, 1500 fold, 1600 fold, 1700 fold, 1800 fold, 1900 fold, 2000 fold, 2100 fold, 220 fold, or more than about 50 fold, 100 fold, 150 fold, 200 fold, 300 fold, 400 fold, 500 fold, 600 fold, 700 fold, 800 fold, 850 fold, 900 fold, 950 fold, 1000 fold, 1100 fold, 1200 fold, 1300 fold, 1400 fold, 1500 fold, 1600 fold, 1700 fold, 1800 fold, 1900 fold, 2000 fold, 2100 fold, 220 fold, or more than about 50 fold, 100 fold, 1500 fold, 2500 fold, 3000 fold, 3500 fold, 4000 fold, The amplification may be 0x, 2300x, 2400x, 2500x, 2600x, 2700x, 2800x, 2900x, 3000x, 3100x, 3200x, 3300x, 3400x, 3500x, 3600x, 3700x, 3800x, 3900x, 4000x, 4100x, 4200x, 4300x, 4400x, 4500x, 4600x, 4700x, 4800x, 4900x, 5000x, 5100x, 5200x, 5300x, 5400x, 5500x, 5600x, 5700x, 5800x, 5900x, or 6000x.

[0047] The vaccine can enhance interferon gamma (IFN-γ) levels in a vaccinated subject by about 50-fold to about 6000-fold, about 50-fold to about 5500-fold, about 50-fold to about 5000-fold, about 50-fold to about 4500-fold, about 100-fold to about 6000-fold, about 150-fold to about 6000-fold, about 200-fold to about 6000-fold, about 250-fold to about 6000-fold, or about 300-fold to about 6000-fold, compared to IFN-γ levels in a non-vaccinated subject. In some embodiments, the vaccine increases IFN-γ levels in a vaccinated subject by about 50 fold, 100 fold, 150 fold, 200 fold, 250 fold, 300 fold, 350 fold, 400 fold, 450 fold, 500 fold, 550 fold, 600 fold, 650 fold, 700 fold, 750 fold, 800 fold, 850 fold, 900 fold, 950 fold, 1000 fold, 1100 fold, 1200 fold, 1300 fold, 1400 fold, 1500 fold, 1600 fold, 1700 fold, 1800 fold, 1900 fold, 2000 fold, 2100 fold, 2200 fold, 2300 fold, 2400 fold, 2500 fold, 2600 fold, 2700 fold, 2800 fold, 2900 fold, 3000 fold, 3100 fold, 3200 fold, 3300 fold, 3400 fold, 3500 fold, 3600 fold, 3700 fold, 3800 fold, 3900 fold, 4000 fold, 4100 fold, 4200 fold, 4300 fold, 4400 fold, 4500 fold, 4600 fold, 4700 fold, 4800 fold, 4900 fold, 5000 fold, 5100 fold, 5200 fold, 5300 fold, 5400 In one embodiment, the compound can be augmented by 00x, 2300x, 2400x, 2500x, 2600x, 2700x, 2800x, 2900x, 3000x, 3100x, 3200x, 3300x, 3400x, 3500x, 3600x, 3700x, 3800x, 3900x, 4000x, 4100x, 4200x, 4300x, 4400x, 4500x, 4600x, 4700x, 4800x, 4900x, 5000x, 5100x, 5200x, 5300x, 5400x, 5500x, 5600x, 5700x, 5800x, 5900x, or 6000x.

[0048] As described in more detail below, the vaccine may further comprise one or more inhibitors of one or more immune checkpoint molecules (i.e., immune checkpoint inhibitors). Immune checkpoint molecules are described in more detail below. An immune checkpoint inhibitor is any nucleic acid or protein that prevents the suppression of any component of 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 immune cell proliferation and / or differentiation. As described in more detail below, the vaccine can be further combined with antibodies against checkpoint inhibitors, such as PD-1 and PDL-1, to enhance the stimulation of both cellular and humoral immune responses. Using anti-PD-1 or anti-PDL-1 antibodies prevents PD-1 or PDL-1 from suppressing T cell and / or B cell responses.

[0049] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. In case of conflict, the present document including definitions shall prevail. Preferred methods and materials are described below, however, methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are merely illustrative and are not intended to be limiting. The terms used herein are for the purpose of describing specific embodiments only and are not intended to be limiting.

[0050] As used herein, the terms "comprise(s)", "include(s)", "having", "has", "can", "contain(s)", and variations thereof are intended to be open-ended transitional phrases, terms, or words that do not exclude the possibility of additional acts or structures. The singular forms "a", "and", and "the" include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments that "comprise", "consist of", and "consist essentially of" the embodiments or elements presented herein, whether or not expressly stated.

[0051] For the recitation of numerical ranges herein, each intervening value is expressly contemplated with the same degree of precision as the minimum and maximum values ​​of the recited range. For example, in the range of 6 to 9, the values ​​7 and 8 are contemplated in addition to 6 and 9, and in the range of 6.0 to 7.0, the values ​​6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are expressly contemplated.

[0052] As used herein, "adjuvant" means any molecule added to the vaccines described herein to enhance the immunogenicity of an antigen.

[0053] As used herein, "antibody" refers to an antibody of the class IgG, IgM, IgA, IgD, or IgE, or a fragment or derivative thereof, including Fab, F(ab') 2 , Fd, and single chain antibodies, diabodies, bispecific antibodies, bifunctional antibodies, and derivatives thereof. The antibody may be an antibody isolated from a mammalian serum sample, a polyclonal antibody, an affinity purified antibody, or any mixture thereof, which exhibits sufficient binding specificity for the desired epitope or sequence derived therefrom.

[0054] An "antigen" is (a) amino acids 19 to 159 of SEQ ID NO:2, (b) amino acids 19 to 210 of SEQ ID NO:4, (c) amino acids 19 to 232 of SEQ ID NO:8, (d) a fragment comprising at least 90% of amino acids 19 to 159 of SEQ ID NO:2, (e) a fragment comprising at least 90% of amino acids 19 to 210 of SEQ ID NO:4, (f) a fragment comprising at least 90% of amino acids 19 to 232 of SEQ ID NO:8, (g) a protein that is at least 95% identical to amino acids 19 to 159 of SEQ ID NO:2, or (h) a protein that is at least 95% identical to amino acids 19 to 210 of SEQ ID NO:4. (i) a protein that is at least 95% identical to amino acids 19 to 232 of SEQ ID NO:8, (j) a fragment that comprises at least 90% of a protein that is at least 95% identical to amino acids 19 to 159 of SEQ ID NO:2, (k) a fragment that comprises at least 90% of a protein that is at least 95% identical to amino acids 19 to 210 of SEQ ID NO:4, and (l) a fragment that comprises at least 90% of a protein that is at least 95% identical to amino acids 19 to 232 of SEQ ID NO:8. "Antigen" also refers to a protein having a synthetic consensus survivin antigen amino acid sequence, including: (a) SEQ ID NO:2, (b) SEQ ID NO:4, (c) SEQ ID NO:8, (d) a fragment comprising at least 90% of the full length of SEQ ID NO:2, (e) a fragment comprising at least 90% of the full length of SEQ ID NO:4, (f) a fragment comprising at least 90% of the full length of SEQ ID NO:8, (g) a protein that is at least 95% identical to SEQ ID NO:2, (h) a protein that is at least 95% identical to SEQ ID NO:4, (i) a protein that is at least 95% identical to SEQ ID NO:8, (j) a fragment comprising at least 90% of the full length of a protein that is at least 95% identical to SEQ ID NO:2, (k) a fragment comprising at least 90% of the full length of a protein that is at least 95% identical to SEQ ID NO:4, and (l) a fragment comprising at least 90% of the full length of a protein that is at least 95% identical to SEQ ID NO:8. Antigens may optionally include signal peptides, such as those from other proteins.

[0055] As used herein, "coding sequence" or "encoding nucleic acid" refers to a nucleic acid (RNA or DNA molecule) that comprises a nucleotide sequence that codes for a protein. The coding sequence may further comprise initiation and termination signals operably linked to control elements, including a promoter and polyadenylation signal, capable of directing expression in the cells of a subject or mammal to which the nucleic acid is administered.

[0056] "Complement" or "complementary" as used herein with respect to nucleic acids can mean Watson-Crick (e.g., AT / U and CG) or Hoogsteen base pairing between nucleotides or nucleotide analogs of a nucleic acid molecule.

[0057] As used herein, "consensus" or "consensus sequence" refers to a polypeptide sequence based on an analysis of an alignment of multiple sequences for the same gene from different organisms or different isoforms within an organism. Nucleic acid sequences encoding consensus polypeptide sequences can be prepared.

[0058] "Constant current" as used herein refers to the current received or experienced by a tissue or cells defining the tissue over the duration of an electrical pulse delivered to the same tissue. The electrical pulse is delivered from an electroporation device as described herein. The electroporation device provided herein preferably has a feedback element with instantaneous feedback, so that this current remains at a constant amperage in the tissue over the life of the electrical pulse. The feedback element measures the resistance of the tissue (or cells) over the duration of the pulse and allows the electroporation device to change its electrical energy output (e.g., increase the voltage) so that the current in the same tissue remains constant over the entire electrical pulse (on the order of microseconds) and between pulses. In some embodiments, the feedback element includes a controller.

[0059] As used herein, "current feedback" or "feedback" may be used interchangeably and may refer to the active response of the provided electroporation device, which includes measuring the current in the tissue between the electrodes and modifying the energy output provided by the EP device accordingly to maintain the current at a constant level. This constant level is preset by the user before the start of the pulse sequence or electrical therapy. The feedback may be achieved by the electroporation components of the electroporation device, e.g., a controller, since the electrical circuitry continuously monitors the current in the tissue between the electrodes, compares the monitored current (or the current in the tissue) to a preset current, and continuously makes energy output adjustments to maintain the monitored current at the preset level. The feedback loop may be instantaneous, since it is an analog closed-loop feedback.

[0060] As used herein, "distributed current" may refer to a pattern of current delivered from the various needle electrode arrays of the electroporation devices described herein, where the pattern minimizes, or preferably eliminates, the occurrence of electroporation-associated thermal stress in any region of the tissue being electroporated.

[0061] "Electroporation," "electropermeabilization," or "electrokinetic enhancement" ("EP"), as used interchangeably herein, refers to the use of transmembrane electric field pulses to induce microscopic pathways (pores) in biological membranes, the presence of which allows the passage of biological molecules, such as plasmids and vectors, oligonucleotides, siRNA, drugs, ions, water, and the like, from one side of the cell membrane to the other.

[0062] A "fragment" as used herein with respect to a nucleic acid sequence means a nucleic acid sequence or a portion thereof that encodes a polypeptide capable of eliciting an immune response in a mammal that cross-reacts with an antigen disclosed herein. The fragment may be a DNA fragment selected from at least one of the various nucleotide sequences encoding protein fragments shown below. The fragment 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 one or more of the nucleic acid sequences shown below, excluding an added heterologous signal peptide. The fragment may comprise at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of one or more of the nucleic acid sequences shown below, and optionally further comprises a sequence encoding a heterologous signal peptide, which need not be included when calculating percent identity. The fragment may further comprise a coding sequence for a signal peptide, such as an immunoglobulin signal peptide, e.g., an IgE or IgG signal peptide. A coding sequence encoding an N-terminal methionine and / or a signal peptide may be linked to the fragment of the coding sequence.

[0063] In some embodiments, a fragment may comprise at least 20 or more nucleotides, at least 30 or more nucleotides, at least 40 or more nucleotides, at least 50 or more nucleotides, at least 60 or more nucleotides, at least 70 or more nucleotides, at least 80 or more nucleotides, at least 90 or more nucleotides, at least 100 or more nucleotides, at least 150 or more nucleotides, at least 200 or more nucleotides, at least 250 or more nucleotides, at least 300 or more nucleotides, at least 350 or more nucleotides, at least 400 or more nucleotides, at least 450 or more nucleotides, at least 500 or more nucleotides, at least 550 or more nucleotides, at least 600 or more nucleotides, or at least 650 or more nucleotides of at least one of the nucleic acid sequences set forth below.

[0064] "Fragment" or "immunogenic fragment" with respect to a polypeptide sequence means a polypeptide capable of eliciting an immune response in a mammal that cross-reacts with an antigen disclosed herein. The fragment may be a polypeptide fragment selected from at least one of the following various amino acid sequences. A fragment of a consensus protein 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, excluding any added heterologous signal peptides. A fragment may comprise at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of one or more of the amino acid sequences shown below, and optionally further comprises a heterologous signal peptide, which need not be included when calculating percent identity. A fragment may further comprise a signal peptide, such as an immunoglobulin signal peptide, e.g., an IgE or IgG signal peptide.

[0065] In some embodiments, a fragment of a consensus protein can comprise at least 20 amino acids or more, at least 30 amino acids or more, at least 40 amino acids or more, at least 50 amino acids or more, at least 60 amino acids or more, at least 70 amino acids or more, at least 80 amino acids or more, at least 90 amino acids or more, at least 100 amino acids or more, at least 110 amino acids or more, at least 120 amino acids or more, at least 130 amino acids or more, at least 140 amino acids or more, at least 150 amino acids or more, at least 160 amino acids or more, at least 170 amino acids or more, at least 180 amino acids or more, at least 200 amino acids or more, or at least 220 amino acids or more of a protein sequence disclosed herein.

[0066] As used herein, the term "genetic construct" refers to a DNA or RNA molecule that comprises a nucleotide sequence that codes for a protein. The coding sequence comprises an initiation and termination signal that is operably linked to regulatory elements, including a promoter and a polyadenylation signal that can direct expression in the cells of a subject to which the nucleic acid molecule is administered. As used herein, the term "expressible form" refers to a genetic construct that comprises the necessary regulatory elements that are operably linked to the coding sequence that codes for a protein, so that the coding sequence is expressed when present in the cells of a subject.

[0067] The term "homology" as used herein refers to a degree of complementarity. There can be partial or complete homology (i.e., identity). Partially complementary sequences that at least partially inhibit a completely complementary sequence from hybridizing to a target nucleic acid are referred to using the functional term "substantially homologous". When used in reference to a double-stranded nucleic acid sequence, such as a cDNA or genomic clone, the term "substantially homologous" 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 to (i.e., is the complement of) a single-stranded nucleic acid template sequence under low stringency conditions.

[0068] "Identical" or "identity" as used herein in the context of two or more nucleic acid or polypeptide sequences means that the sequences have a certain percentage of residues that are the same over a specified region. The percentage can be calculated by optimally aligning the two sequences, comparing the two sequences over a specified region, determining the number of positions where identical residues occur in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying the result by 100 to obtain the percentage of sequence identity. If the two sequences are of different lengths or the alignment produces one or more staggered ends and only a single sequence is included in the specified comparison region, the residues of the single sequence are included in the denominator but not in the numerator of the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. Identity can be performed manually or using a computer sequence algorithm such as BLAST or BLAST 2.0.

[0069] As used herein, "impedance" may be used when discussing feedback mechanisms and can be converted to a current value according to Ohm's law, thus allowing comparison to a preset current.

[0070] As used herein, "immune response" refers to the activation of a host's immune system, e.g., a mammalian immune system, in response to the introduction of an antigen. The immune response can be in the form of a cellular or humoral response, or both.

[0071] As used herein, "nucleic acid" or "oligonucleotide" or "polynucleotide" refers to at least two nucleotides covalently linked to each other. The depiction of a single strand also defines the sequence of the complementary strand. Thus, the nucleic acid also encompasses the complementary strand of the depicted single strand. Many variants of nucleic acids can be used for the same purpose as a given nucleic acid. Thus, the nucleic acid also encompasses substantially identical nucleic acids and their complements. The single strand provides a probe that can hybridize to a target sequence under stringent hybridization conditions. Thus, the nucleic acid also encompasses a probe that hybridizes under stringent hybridization conditions.

[0072] Nucleic acids can be single-stranded or double-stranded, or can contain portions of both double-stranded and single-stranded sequences. Nucleic acids can be DNA, RNA, or hybrids, both genomic and cDNA, and can contain combinations of deoxyribonucleotides and ribonucleotides, with base combinations including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine. Nucleic acids can be obtained by chemical synthesis or recombinant methods.

[0073] As used herein, "operably linked" means that the expression of a gene is under the control of the promoter to which it is spatially connected. The promoter can be located 5' (upstream) or 3' downstream) of the gene under its control. The distance between the promoter and the gene can be approximately the same as the distance between the promoter and the gene it controls in the gene from which it is derived. As is known in the art, variations in this distance can be accommodated without loss of promoter function.

[0074] As used herein, "peptide," "protein," or "polypeptide" may mean a linked sequence of amino acids, which may be natural, synthetic, or a modified or combination of natural and synthetic.

[0075] As used herein, "promoter" refers to a synthetic or naturally derived molecule that can confer, activate, or enhance the expression of a nucleic acid in a cell. A promoter can include one or more specific transcription control sequences to further enhance the expression of a nucleic acid in a cell and / or to modify the spatial and / or temporal expression. A promoter can also include distal enhancer or repressor elements that can be located as far away as several thousand base pairs from the start site of transcription. Promoters can be derived from sources including viruses, bacteria, fungi, plants, insects, and animals. A promoter can constitutively or differentially control the expression of gene components with respect to the cell, tissue, or organ in which expression occurs, or with respect to the developmental stage in which expression occurs, or in response to external stimuli such as physiological stress, pathogens, metal ions, or inducers. Representative examples of promoters include a bacteriophage T7 promoter, a bacteriophage T3 promoter, an SP6 promoter, a lac operator-promoter, a tac promoter, an SV40 late promoter, an SV40 early promoter, an RSV-LTR promoter, a CMV IE promoter, an SV40 early promoter, or an SV40 late promoter, and a CMV IE promoter.

[0076] "Signal peptide" and "leader sequence" are used interchangeably herein and refer to an amino acid sequence that may be linked at the amino terminus of a protein described herein. A signal peptide / leader sequence typically directs the localization of a protein. A signal peptide / leader sequence as used herein preferably facilitates the secretion of a protein from the cell in which it is produced. Upon secretion from the cell, the signal peptide / leader sequence is often cleaved from the remainder of the protein, often referred to as the mature protein. The signal peptide / leader sequence is linked at the amino terminus (i.e., N-terminus) of a protein.

[0077] As used herein, "stringent hybridization conditions" refers to conditions under which a first nucleic acid sequence (e.g., a probe) hybridizes to a second nucleic acid sequence (e.g., a target), such as in a complex mixture of nucleic acids. Stringent conditions are sequence-dependent and will be different in different circumstances. Stringent conditions may be selected to be about 5-10°C lower than the thermal melting point (Tm) of the specific sequence at a defined ionic strength pH. Tm may be the temperature (under defined ionic strength, pH, and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (the target sequence is present in excess, so that at Tm, 50% of the probes are occupied at equilibrium). Stringent conditions can be those in which the salt concentration is less than about 1.0 M sodium ion, e.g., about 0.01 to 1.0 M sodium ion concentration (or other salt) at pH 7.0 to 8.3, and the temperature is at least about 30° C. for short probes (e.g., about 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g., more than about 50 nucleotides). Stringent conditions can also be achieved by the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal can be at least 2 to 10 times background hybridization. Exemplary stringent hybridization conditions include: 50% formamide, 5xSSC, and 1% SDS, incubation at 42° C., or 5xSSC, 1% SDS, incubation at 65° C., washing with 0.2xSSC, and 0.1% SDS at 65° C.

[0078] As used herein, "subject" may refer to a mammal that desires or needs to be immunized with a vaccine described herein. The mammal may be a human, chimpanzee, dog, cat, horse, cow, mouse, or rat.

[0079] As used herein, "substantially complementary" refers to a sequence that is substantially complementary to a first sequence over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 180, 270, 360, 450, 540 or more nucleotides or amino acids. It means at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the complement of a second sequence, or that the two sequences hybridize under stringent hybridization conditions.

[0080] As used herein, "substantially identical" refers to a sequence in which the first and second sequences are at least 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 apart. "complementary" means at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical over the acid region, or with respect to nucleic acids, that a first sequence is substantially complementary to the complement of a second sequence.

[0081] As used herein, "treat", "treatment" or "treating" may mean protecting an animal from a disease by preventing, suppressing, arresting, or completely eliminating the disease. Prevention of the disease involves administering a vaccine of the invention to an animal before the onset of the disease. Suppression of the disease involves administering a vaccine of the invention to an animal after induction of the disease but before its clinical appearance. Prevention of the disease involves administering a vaccine of the invention to an animal after the clinical appearance of the disease.

[0082] "Variant," as used herein with respect to a nucleic acid, means (i) a portion or fragment of a reference nucleotide sequence; (ii) the complement of a reference nucleotide sequence or a portion thereof; (iii) a nucleic acid that is substantially identical to a reference nucleic acid or its complement; or (iv) a nucleic acid that hybridizes under stringent conditions to a reference nucleic acid, its complement, or a sequence substantially identical thereto.

[0083] "Variant" as used herein with respect to a peptide or polypeptide refers to a peptide or polypeptide that differs in amino acid sequence by insertion, deletion, or conservative substitution of amino acids, but retains at least one biological activity. Variant can also refer to a protein with an amino acid sequence that is substantially identical to a reference protein with an amino acid sequence that retains at least one biological activity. Conservative substitution of amino acids, i.e., replacing an amino acid with an amino acid with a different amino acid of similar properties (e.g., hydrophilicity, degree, and distribution of charged regions), is recognized in the art as typically involving minor changes. These minor changes can be identified, in part, by considering the hydropathic index of an amino acid, as understood in the art. Kyte et al., J. Mol. Biol. 157:105-132 (1982). The hydropathic index of an amino acid is based on considering its hydrophobicity and charge. It is known in the art that amino acids with similar hydropathic indexes can be substituted and still retain protein function. In one embodiment, amino acids with hydropathic indexes of ±2 are substituted. The hydrophilicity of amino acids can also be used to identify substitutions that will result in proteins that retain biological function. In the context of a peptide, consideration of the hydrophilicity of amino acids allows calculation of the maximum local average hydrophilicity of the peptide, a useful tool that has been reported to correlate well with antigenicity and immunogenicity. U.S. Patent No. 4,554,101, fully incorporated herein by reference. Substitution of amino acids with similar hydrophilicity values ​​can result in peptides that retain biological activity, such as immunogenicity, as understood in the art. Substitutions can be made with amino acids that have hydrophilicity values ​​within ±2 of each other. Both the hydropathic index and hydrophilicity value of an amino acid are influenced by the particular side chain of that amino acid. Consistent with that observation, it is understood that amino acid substitutions that are compatible with biological function depend on the relative similarity of amino acids and particularly their side chains, as revealed by hydrophobicity, hydrophilicity, charge, size, and other properties.

[0084] A variant may be a nucleic acid sequence that is substantially identical over the full length of the complete gene sequence or a fragment thereof. The nucleic acid sequence may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the full length of the gene sequence or a fragment thereof. A variant may be an amino acid sequence that is substantially identical over the full length of the amino acid sequence or a fragment thereof. The amino acid sequence may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the full length of the amino acid sequence or a fragment thereof.

[0085] "Vector" as used herein means a nucleic acid sequence that contains a replication origin. A vector can be a virus vector, a bacteriophage, a bacterial artificial chromosome, or a yeast artificial chromosome. A vector can be a DNA or RNA vector. A vector can be a self-replicating extrachromosomal vector, preferably a DNA plasmid. A vector can contain or include one or more heterologous nucleic acid sequences.

[0086] vaccine Provided herein is a vaccine comprising the synthetic consensus survivin antigen disclosed herein, the nucleic acid molecule encoding the antigen, the nucleic acid molecule encoding a fragment of the antigen, the nucleic acid molecule encoding a variant of the antigen, or a combination thereof.The vaccine can be capable of generating an immune response against the antigen in a subject.The immune response can be a therapeutic or preventive immune response.The vaccine can comprise one vector or multiple vectors, as described in more detail below.

[0087] In some embodiments, the vaccine comprises a nucleic acid molecule. In some embodiments, the nucleic acid molecule encodes a synthetic consensus survivin antigen. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding SEQ ID NO:3, a nucleic acid sequence encoding a fragment comprising at least 90% of the length of SEQ ID NO:3, a nucleic acid sequence encoding a protein that is at least 95% identical to SEQ ID NO:3, or a nucleic acid sequence encoding a fragment comprising at least 90% of the full length of a protein that is at least 95% identical to SEQ ID NO:3. In some embodiments, the nucleic acid molecule comprises SEQ ID NO:1, a fragment comprising at least 90% of the full length of SEQ ID NO:1, a fragment that is at least 95% identical to SEQ ID NO:1, or a fragment that comprises at least 90% of the full length of a nucleic acid sequence that is at least 95% identical to SEQ ID NO:1.

[0088] In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding SEQ ID NO:4, a nucleic acid sequence encoding a fragment comprising at least 90% of the length of SEQ ID NO:4, a nucleic acid sequence encoding a protein that is at least 95% identical to SEQ ID NO:4, or a nucleic acid sequence encoding a fragment comprising at least 90% of the full length of a protein that is at least 95% identical to SEQ ID NO:4. In some embodiments, the nucleic acid molecule comprises SEQ ID NO:2, a fragment comprising at least 90% of the full length of SEQ ID NO:2, a fragment that is at least 95% identical to SEQ ID NO:2, or a fragment that comprises at least 90% of the full length of a nucleic acid sequence that is at least 95% identical to SEQ ID NO:2.

[0089] In some embodiments, the vaccine comprises a synthetic consensus survivin antigen, wherein the antigen comprises SEQ ID NO:3, a fragment comprising at least 90% of the length of SEQ ID NO:3, an amino acid sequence that is at least 95% identical to SEQ ID NO:3, or a fragment that comprises at least 90% of the full length of a protein that is at least 95% identical to SEQ ID NO:3.

[0090] In some embodiments, the vaccine comprises a synthetic consensus survivin antigen, wherein the antigen comprises SEQ ID NO:4, a fragment comprising at least 90% of the length of SEQ ID NO:4, an amino acid sequence that is at least 95% identical to SEQ ID NO:4, or a fragment that comprises at least 90% of the full length of a protein that is at least 95% identical to SEQ ID NO:4.

[0091] The vaccine can be used to protect against cancer, for example, cancer or tumor that expresses survivin. The vaccine can be used in subjects in need thereof to prevent and / or treat tumor that expresses survivin. The vaccine can induce cellular and / or antibody responses against survivin and tumor that expresses survivin.

[0092] In one embodiment, the vaccine can be used to protect, prevent and / or treat, or induce a cellular and / or antibody response against ovarian cancer cells that express survivin, particularly epithelial ovarian cancer cells that express survivin, particularly serous ovarian cancer cells that express survivin.

[0093] The development of the cancer vaccine described herein involves identifying a cancer antigen, e.g., survivin, that is not recognized by the immune system and is a tumor-associated ("cancer / testis", "C / T") antigen. The identified cancer antigen is modified from a self antigen to a foreign antigen in order to be recognized by the immune system. Redesigning the nucleic acid and amino acid sequence of the recombinant cancer antigen from a self to a foreign antigen breaks the tolerance of the antigen by the immune system. To break the tolerance, several redesign measures can be applied to the cancer antigen, as described below.

[0094] The recombinant cancer antigens of the vaccine are not recognized as self, thus breaking tolerance. Breaking tolerance induces antigen-specific T cell and / or high titer antibody responses, thereby inducing or eliciting an immune response that is direct or reactive against the cancer or tumor expressing the antigen. In some embodiments, the induced or elicited immune response can be cellular, humoral, or both cellular and humoral immune responses. In some embodiments, the induced or elicited cellular immune response can include induction or secretion of interferon-gamma (IFN-γ) and / or tumor necrosis factor alpha (TNF-α) and / or interleukin 2 (IL-2). In other embodiments, the induced or elicited immune response can reduce or inhibit one or more immunosuppressive factors that promote the growth of an antigen-expressing tumor or cancer, including, but not limited to, factors that downregulate MHC presentation, antigen-specific regulatory T cells (Tregs), cytokines such as PD-L1, FasL, IL-10 and TFG-β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by immunosuppressive cells, CTLA-4, PD-1, MDSC, MCP-1, and factors that upregulate immune checkpoint molecules.

[0095] In a particular embodiment, the vaccine comprises: (1) CD8 + and / or CD107a + The clearance or proliferation of tumor cells can be mediated by increasing cytotoxic T lymphocytes such as (CTL) to attack and kill tumor cells, (2) increasing T helper cell responses, and / or (3) increasing the inflammatory response via IFN-γ, IL-2, and TNF-α, or preferably all of the foregoing.

[0096] Vaccine can be DNA vaccine.DNA vaccine is disclosed in U.S. Patent No. 5,593,972, U.S. Patent No. 5,739,118, U.S. Patent No. 5,817,637, U.S. Patent No. 5,830,876, U.S. Patent No. 5,962,428, U.S. Patent No. 5,981,505, U.S. Patent No. 5,580,859, U.S. Patent No. 5,703,055 and U.S. Patent No. 5,676,594, which are fully incorporated herein by reference.DNA vaccine can further comprise an element or reagent that inhibits it from being integrated into chromosome.

[0097] The vaccine may comprise RNA encoding a cancer antigen. The RNA vaccine may be introduced into a cell.

[0098] The vaccines can be live attenuated vaccines, vaccines using recombinant vectors to deliver antigens, subunit vaccines, and glycoprotein vaccines, for example, but not limited to, vaccines described in U.S. Pat. Nos. 4,510,245; 4,797,368; 4,722,848; 4,790,987; 4,920,209; 5,017,487; 5,077,044; 5,110,587; 5,112,749; 5,174,993; 5,223,424; 5,225,336; 5,240,703; 5,242,829; 5,294,441; No. 5,294,548, No. 5,310,668, No. 5,387,744, No. 5,389,368, No. 5,424,065, No. 5,451,4 No. 99, No. 5,453,364, No. 5,462,734, No. 5,470,734, No. 5,474,935, No. 5,482,713, No. 5, Nos. 5,955,088, 6,034,298, 6,042,836, 6,156,319, and 6,589,529, each of which is incorporated herein by reference.

[0099] In some embodiments, the nucleic acid vaccine may further comprise a coding sequence for a molecular adjuvant, in some cases the molecular adjuvant may be IL-12, IL-15, IL-28, IL-31, IL-33, and / or RANTES, in some cases the molecular adjuvant is a checkpoint inhibitor, including anti-cytotoxic T-lymphocyte antigen 4 (CTLA-4), anti-programmed death receptor 1 (PD-1), and anti-lymphocyte activation gene (LAG-3). The coding sequence for IL-12, IL-15, IL-28, IL-31, IL-33, and / or RANTES may be included in one or more nucleic acid molecules that include a coding sequence for one or more antigens. The coding sequence for IL-12, IL-15, IL-28, IL-31, IL-33, and / or RANTES may be included in one or more separate nucleic acid molecules, such as one or more separate plasmids or vectors, and administered in combination with the nucleic acid vaccine.

[0100] The vaccines of the present invention can have the characteristics required for an effective vaccine, such as being safe so that the vaccine itself does not cause disease or death, protecting against disease, inducing neutralizing antibodies, inducing protective T cell responses, providing ease of administration, few side effects, biological stability, low cost per dose, etc. The vaccine can achieve some or all of these characteristics by including a nucleic acid molecule(s) encoding a cancer antigen, as discussed below.

[0101] Vaccines combined with immune checkpoint inhibitors The vaccine may further comprise one or more inhibitors of one or more immune checkpoint molecules (i.e., immune checkpoint inhibitors). Immune checkpoint molecules are described in more detail below. An immune checkpoint inhibitor is any nucleic acid or protein that prevents the suppression of any component of 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 immune cell proliferation and / or differentiation.

[0102] Such inhibitors can be nucleic acid sequences, amino acid sequences, small molecules, or combinations thereof. The nucleic acid sequences can be DNA, RNA, cDNA, variants thereof, fragments thereof, or combinations thereof. The nucleic acid can also include additional sequences that code for linker or tag sequences that are linked to the immune checkpoint inhibitor by peptide bonds. The small molecule can be an organic or inorganic compound with low molecular weight, e.g., less than 800 daltons, that functions as an enzyme substrate, a ligand (or analog thereof) bound by a protein or nucleic acid, or a regulator of a biological process. The amino acid sequence can be a protein, a peptide, variants thereof, fragments thereof, or combinations thereof.

[0103] In some embodiments, the 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.

[0104] Immune Checkpoint Molecules The immune checkpoint molecule can be a nucleic acid sequence, an amino acid sequence, a small molecule, or a combination thereof. The nucleic acid sequence can be DNA, RNA, cDNA, a variant thereof, a fragment thereof, or a combination thereof. The nucleic acid can also include an additional sequence that codes for a linker or tag sequence that is linked to the immune checkpoint inhibitor by a peptide bond. The small molecule can be an organic or inorganic compound with a low molecular weight, for example, less than 800 daltons, an enzyme substrate, a ligand (or its analogue) bound by a protein or nucleic acid, or a regulator of a biological process. The amino acid sequence can be a protein, a peptide, a variant thereof, a fragment thereof, or a combination thereof.

[0105] PD-1 and PD-L1 The immune checkpoint molecule can be programmed cell death protein 1 (PD-1), programmed cell death ligand 1 (PD-L1), a fragment thereof, a variant thereof, or a combination thereof. PD-1 is a cell surface protein encoded by the PDCD1 gene. PD-1 is a member of the immunoglobulin superfamily and is expressed on T cells and pro-B cells, thus contributing to the fate and / or differentiation of these cells. In particular, PD-1 is a type 1 membrane protein of the CD28 / CTLA-4 family of T cell regulators, which negatively regulates T cell receptor (TCR) signals, thereby negatively regulating immune responses. PD-1 can negatively regulate CD8+ T cell responses, thus inhibiting CD8-mediated cytotoxicity and promoting tumor growth.

[0106] PD-1 has two ligands, PD-L1 and PD-L2, which are members of the B7 family. PD-L1 is upregulated on macrophages and dendritic cells (DCs) in response to LPS and GM-CSF treatment, and on T cells and B cells upon TCR and B cell receptor signaling. PD-L1 is expressed on many tumor cell lines, including myeloma, mast cell tumor, and melanoma.

[0107] Anti-immune checkpoint molecule antibodies As described above, the immune checkpoint inhibitor can be an antibody. The antibody can bind to or react with an antigen (i.e., the immune checkpoint molecule described above). Thus, the antibody can be considered as an anti-immune checkpoint molecule antibody or an immune checkpoint molecule antibody. The antibody can be encoded by a nucleic acid sequence that contains it.

[0108] The antibody may comprise a heavy chain polypeptide and a light chain polypeptide. The heavy chain polypeptide may comprise a variable heavy chain (VH) region and / or at least one constant heavy chain (CH) region. The at least one constant heavy chain region may comprise constant heavy chain region 1 (CH1), constant heavy chain region 2 (CH2), and constant heavy chain region 3 (CH3), and / or a hinge region.

[0109] In some embodiments, the heavy chain polypeptide can comprise a VH region and a CH1 region. In other embodiments, the heavy chain polypeptide can comprise a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region.

[0110] A heavy chain polypeptide can include a set of complementarity determining regions ("CDRs"). A CDR set can include three hypervariable regions of the VH region. From the N-terminus of the heavy chain polypeptide, these CDRs are designated as "CDR1", "CDR2" and "CDR3", respectively. CDR1, CDR2 and CDR3 of the heavy chain polypeptide can contribute to antigen binding or recognition.

[0111] A light chain polypeptide can include a variable light (VL) region and / or a constant light (CL) region. A light chain polypeptide can include a set of complementarity determining regions ("CDRs"). A set of CDRs can include three hypervariable regions of the VL region. From the N-terminus of the light chain polypeptide, these CDRs are designated as "CDR1", "CDR2" and "CDR3", respectively. CDR1, CDR2 and CDR3 of a light chain polypeptide can contribute to antigen binding or recognition.

[0112] An antibody may comprise a set of heavy and light chain complementarity determining regions ("CDRs"), each interposed between a set of heavy and light chain frameworks ("FRs") that provide support for the CDRs and define the spatial relationship of the CDRs to each other. A set of CDRs may comprise three hypervariable regions of a heavy or light chain V region. From the N-terminus of the heavy or light chain, these regions are designated as "CDR1", "CDR2", and "CDR3", respectively. Thus, an antigen-binding site may comprise six CDRs, including a set of CDRs from each of the heavy and light chain V regions.

[0113] The antibody can be an immunoglobulin (Ig). The Ig can be, for example, IgA, IgM, IgD, IgE, and IgG. The immunoglobulin can include a heavy chain polypeptide and a light chain polypeptide. The heavy chain polypeptide of the immunoglobulin can include a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region. The light chain polypeptide of the immunoglobulin can include a VL region and a CL region.

[0114] Additionally, the proteolytic enzyme papain preferentially cleaves IgG molecules to generate several fragments, two of which (F(ab) fragments) each contain a covalent heterodimer that contains an intact antigen-binding site. The enzyme pepsin cleaves IgG molecules to generate F(ab') fragments. 2 Some fragments can be provided, including fragments that contain both antigen-binding sites. Thus, an antibody can be a Fab or F(ab') fragment. 2 The Fab can comprise a heavy chain polypeptide and a light chain polypeptide. The heavy chain polypeptide of the Fab can comprise a VH region and a CH1 region. The light chain of the Fab can comprise a VL region and a CL region.

[0115] The antibody may be a polyclonal or monoclonal antibody. The antibody may be a chimeric antibody, a single chain antibody, an affinity matured antibody, a human antibody, a humanized antibody, or a fully human antibody. A humanized antibody may be an antibody from a non-human species that binds to a desired antigen having one or more complementarity determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule.

[0116] PD-1 antibody The anti-immune checkpoint molecule antibody can be an anti-PD-1 antibody (also referred to herein as "PD-1 antibody"), a variant thereof, a fragment thereof, or a combination thereof. The PD-1 antibody can be nivolumab. The anti-PD-1 antibody can inhibit PD-1 activity, thereby inducing, eliciting, or increasing an immune response against tumors or cancer, and reducing tumor growth.

[0117] PD-L1 antibody The anti-immune checkpoint molecule antibody can be an anti-PD-L1 antibody (also referred to herein as a "PD-L1 antibody"), a variant thereof, a fragment thereof, or a combination thereof. The anti-PD-L1 antibody can inhibit PD-L1 activity, thereby inducing, eliciting, or increasing an immune response against tumors or cancer, and reducing tumor growth.

[0118] antigen As described above, the vaccine may comprise an antigen or a nucleic acid encoding an antigen. The antigen may be survivin, a fragment thereof, a variant thereof, or a combination of fragments and variants thereof.

[0119] Thus, the vaccine can be used to treat subjects suffering from cancer or tumors that express survivin. In some embodiments, the cancer is ovarian cancer. In some embodiments, the ovarian cancer is epithelial ovarian cancer. The ovarian cancer can be serous epithelial ovarian cancer. The vaccine can also be used to treat subjects with cancer or tumors that express survivin to prevent the development of such tumors in the subject. The synthetic consensus survivin antigen can be different from the natural survivin gene, thus providing therapy or prevention against tumors that express the synthetic consensus survivin antigen. Thus, provided herein are synthetic consensus survivin antigen sequences (i.e., mutated or synthetic survivin genes or sequences) that differ from the natural survivin gene.

[0120] The transcription product of natural survivin gene is processed into various mRNAs.Specific survivin mRNA isoforms can be selected based on their expression in cancer cells, for example.In certain embodiments, survivin isoforms are selected based on their expression in ovarian cancer cells.The synthetic consensus survivin antigen sequence described herein avoids selective processing and produces one full-length transcription product, resulting in stronger induction of effector T and B cell responses.

[0121] An isolated nucleic acid molecule is provided that comprises the heterologous sequence described above. An isolated nucleic acid molecule is provided that consists of the heterologous sequence described above. The isolated nucleic acid molecule that comprises the heterologous sequence described above can be incorporated into a vector, such as a plasmid, a viral vector, and other forms of nucleic acid molecules as described below. A nucleic acid sequence encoding a synthetic consensus survivin antigen is provided herein. The coding sequence encoding the synthetic consensus survivin antigen has a sequence as described above.

[0122] Protein molecules are provided that comprise the heterologous amino acid sequences described above. Protein molecules are provided that consist of the heterologous amino acid sequences described above. Proteins and polypeptides having the sequences described above are provided herein. The proteins and polypeptides may be referred to as synthetic consensus survivin antigens and survivin immunogens. The synthetic consensus survivin antigen is capable of eliciting an immune response against tumors expressing survivin.

[0123] In one aspect, it is desirable for a synthetic consensus survivin antigen to provide improved transcription and translation, including one or more of a low GC-content leader sequence to increase transcription, mRNA stability and codon optimization, and, whenever possible, elimination of cis-acting sequence motifs (i.e., internal TATA boxes).

[0124] The synthetic consensus survivin antigen can be a consensus antigen (or immunogen) sequence derived from two or more species, isoforms, or variants. In one embodiment, the consensus sequence is generated from survivin isoforms of different species. The consensus sequence is derived from survivin sequences collected from GenBank or other similar DNA or protein sequence databases. In some embodiments, the consensus antigen can include a portion of a first isoform combined with a portion of a second isoform, for example, the portion of the second isoform is non-homologous to any portion of the first isoform. The synthetic consensus survivin antigen can include consensus sequences and / or modifications to improve expression. Modifications include codon optimization, RNA optimization, adding a Kozak sequence (e.g., GCC ACC) to enhance translation initiation, and / or adding an immunoglobulin leader sequence to enhance the immunogenicity of the synthetic consensus survivin antigen. The synthetic consensus survivin antigen can include a signal peptide, such as, but not limited to, an immunoglobulin signal peptide, for example, an immunoglobulin E (IgE) or immunoglobulin G (IgG) signal peptide. In some embodiments, the synthetic consensus survivin antigen can include a localization signal sequence, for example, a mutation or deletion to a nuclear localization signal to disrupt nuclear localization upon translation. In some embodiments, the survivin consensus antigen can include a hemagglutinin (HA) tag. The survivin consensus antigen can be designed to elicit a stronger and broader cellular and / or humoral immune response than the corresponding non-codon-optimized survivin antigen.

[0125] The consensus survivin sequence can be mutated to disrupt and / or enhance certain structures and / or functions of natural survivin to generate a synthetic consensus survivin antigen sequence. In one embodiment, mutations are introduced to eliminate the anti-apoptotic activity of survivin. In a specific embodiment, T34A, T48A, and C84A mutations are introduced into the consensus survivin isoform 1 sequence to eliminate anti-apoptotic function. (Muchmore,SWet al.Crystal structure and mutagenic analysis of the inhibitor-of-apoptosis protein Survivin.Molecular cell 6,173-182(2000);O'Connor,DSet al.Regulation of apoptosis at cell division by p34cdc2 phosphorylation of Survivin.Proceedings of the National Academy of Sciences of the United States of America 97,13103-13107,doi:10.1073 / pnas.240390697 (2000);Barrett,RM,Colnaghi,R.&Wheatley,SPThreonine 48 in the BIR domain of Survivin is critical to its mitotic and anti-apoptotic activities and can be phosphorylated by CK2 in vitro.Cell cycle (Georgetown, Tex.) 10, 538-548 (2011).

[0126] In some embodiments, the synthetic consensus survivin antigen sequence can be generated from one isoform, for example, the predominant survivin isoform, or the consensus sequence can include a combination of a portion of a first isoform and a portion of a second isoform, or a truncated portion of a second isoform. In one embodiment, the synthetic consensus sequence is derived from survivin isoform 1 (survivin 1). In another embodiment, the synthetic consensus sequence is derived from survivin isoform 3 (survivin 3). In one embodiment, the synthetic consensus survivin antigen 3 sequence is a truncated portion of survivin 3 (survivin 3T). In one embodiment, the synthetic consensus sequence is a combination of survivin 1 and survivin T3, or survivin 1T3.

[0127] In a preferred embodiment, the synthetic consensus survivin antigen sequence shares 95.0% or greater identity with SEQ ID NO:1 or SEQ ID NO:3. In this embodiment, the nucleic acid sequence of SEQ ID NO:1 or SEQ ID NO:3 encodes the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:8, respectively. In other embodiments, the synthetic consensus survivin antigen sequence shares 95.0% or greater identity, 95.2% or greater identity, 95.4% or greater identity, 95.6% or greater identity, 95.8% or greater identity, 96.0% or greater identity, 96.2% or greater identity, 96.4% or greater identity, 96.6% or greater identity, 96.8% or greater identity, 97.0% or greater identity, 97.2% or greater identity, or a combination thereof with SEQ ID NO:1 or SEQ ID NO:3. or 97.4% or more identity, 97.6% or more identity, 97.8% or more identity, 98.0% or more identity, 98.2% or more identity, 98.4% or more identity, or 98.6% or more identity, 98.8% or more identity, 99.0% or more identity, 99.2% or more identity, 99.4% or more identity, 99.6% or more identity, 99.8% or more identity, or 100% identity.

[0128] vector The vaccine may include one or more vectors that include a heterologous nucleic acid that encodes a synthetic consensus survivin antigen. The one or more vectors may be capable of expressing the antigen in an amount effective to induce an immune response in a mammal. The vector may include a heterologous nucleic acid that encodes the antigen. The vector may have a nucleic acid sequence that includes an origin of replication. The vector may be a plasmid, a bacteriophage, a bacterial artificial chromosome, or a yeast artificial chromosome. The vector may be either a self-replicating extrachromosomal vector or a vector that integrates into a host genome.

[0129] One or more vectors may be expression constructs, which are generally plasmids used to introduce specific genes into target cells. Once the expression vector enters the cell, the protein encoded by the gene is produced by the cellular transcription and translation machinery ribosome complex. Plasmids are often designed to contain regulatory sequences that function as enhancer and promoter regions, leading to efficient transcription of the gene carried on the expression vector. The vectors of the present invention express large amounts of stable messenger RNA and thus proteins.

[0130] The vector may have expression signals such as a strong promoter, a strong stop codon, adjustment of the distance between the promoter and the cloned gene, and insertion of a transcription termination sequence and a PTIS (portable translation initiation sequence).

[0131] The vector can be a circular plasmid or a linear nucleic acid. Circular plasmids and linear nucleic acids are capable of directing the expression of a particular nucleotide sequence in appropriate target cells. The vector can have a promoter operably linked to the nucleotide sequence encoding the antigen and can be operably linked to a termination signal. The vector can also include sequences necessary for proper translation of the nucleotide sequence, as well as sequences for cloning and subcloning the vector and its fragments. The vector containing the nucleotide sequence of interest can be chimeric, meaning that at least one of its components is heterologous with respect to at least one of the other components. The expression of the nucleotide sequence in the expression cassette can be under the control of a constitutive promoter or an inducible promoter, which only initiates transcription when the host cell is exposed to some particular external stimulus. In the case of a multicellular organism, the promoter can also be specific to a particular tissue or organ or developmental stage. In a preferred embodiment, the plasmid vector is pGX1428 as described herein, further comprising the nucleic acid sequence of SEQ ID NO: 1, or pGX1429 as described herein, further comprising the nucleic acid sequence of SEQ ID NO: 3.

[0132] The vector may be a plasmid. The plasmid may be useful for transfecting cells with a nucleic acid encoding the antigen. The transformed host cells may be cultured and maintained under conditions in which expression of the antigen occurs.

[0133] The plasmid may contain a nucleic acid sequence encoding one or more of the various antigens disclosed above, including coding sequences encoding consensus antigens, fragments of such proteins, variants of such proteins, variants or fragments of fusion proteins capable of eliciting an immune response against synthetic antigens, which are composed of combinations of consensus proteins and / or fragments of consensus proteins and / or variants of consensus proteins and / or fragments of variants of consensus proteins.

[0134] A single plasmid may contain the coding sequence for a single antigen, the coding sequence for two antigens, the coding sequence for three antigens, or the coding sequence for four antigens.

[0135] In some embodiments, the plasmids may further comprise coding sequences encoding CCR20, either alone or as part of these plasmids. Similarly, the plasmids may further comprise coding sequences for IL-12, IL-15, and / or IL-28.

[0136] The plasmid may further comprise a start codon, which may be upstream of the coding sequence, and a stop codon, which may be downstream of the coding sequence. The start and stop codons may be in frame with the coding sequence.

[0137] The plasmid may also include a promoter operably linked to the coding sequence. The promoter operably linked to the coding sequence may be a promoter from a human immunodeficiency virus (HIV) promoter, such as a simian virus 40 (SV40), mouse mammary tumor virus (MMTV) promoter, a bovine immunodeficiency virus (BIV) long terminal repeat (LTR) promoter, a Moloney virus promoter, an avian leukosis virus (ALV) promoter, a cytomegalovirus (CMV) promoter, such as a CMV immediate early promoter, an Epstein-Barr virus (EBV) promoter, or a Rous sarcoma virus (RSV) promoter. The promoter 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 or skin specific promoter, natural or synthetic. Examples of such promoters are described in U.S. Patent Application Publication No. US2004 / 0175727, the contents of which are incorporated herein in their entirety.

[0138] The plasmid may also include a polyadenylation signal, which may be downstream of the coding sequence. The polyadenylation signal may be an SV40 polyadenylation signal, an LTR polyadenylation signal, a bovine growth hormone (bGH) polyadenylation signal, a human growth hormone (hGH) polyadenylation signal, or a human β-globin polyadenylation signal. The SV40 polyadenylation signal may be the polyadenylation signal from pCEP4 plasmid (Invitrogen, San Diego, CA).

[0139] The plasmid may also contain an enhancer upstream of the coding sequence.The enhancer may be human actin, human myosin, human hemoglobin, human muscle creatine, or a viral enhancer such as that from CMV, FMDV, RSV, or EBV.Polynucleotide function enhancers are described in U.S. Patent Nos. 5,593,972, 5,962,428, and WO94 / 016737, the contents of each of which are fully incorporated by reference.

[0140] The plasmid may also contain a mammalian origin of replication to maintain the plasmid extrachromosomally and produce multiple copies of the plasmid in cells. The plasmid may be pVAXI, pCEP4 or pREP4 from Invitrogen (San Diego, CA), which contains the Epstein-Barr virus origin of replication and the nuclear antigen EBNA-1 coding region, and may generate high-copy episomal replication without integration. The backbone of the plasmid may be pA V0242. The plasmid may be a replication-deficient adenovirus type 5 (Ad5) plasmid.

[0141] The plasmid may also contain regulatory sequences that may be suitable for gene expression in a cell into which the plasmid is administered. The coding sequence may contain codons that may allow for more efficient transcription of the coding sequence in the host cell.

[0142] The coding sequence may also include an Ig leader sequence. The leader sequence may be 5' of the coding sequence. The consensus antigen encoded by this sequence may include an N-terminal Ig leader followed by the consensus antigen protein. The N-terminal Ig leader may be IgE or IgG.

[0143] The plasmid may be pSE420 (Invitrogen, San Diego, Calif.), which may be used for protein production in Escherichia coli (E. coli). The plasmid may also be YES2 (Invitrogen, San Diego, Calif.), which may be used for protein production in Saccharomyces cerevisiae strains of yeast. The plasmid may also be of the MAXBAC™ Complete Baculovirus Expression System (Invitrogen, San Diego, Calif.), which may be used for protein production in insect cells. The plasmid may also be pcDNA I or pcDNA3 (Invitrogen, San Diego, Calif.), which may be used for protein production in mammalian cells, such as Chinese Hamster Ovary (CHO) cells.

[0144] Vectors can be circular plasmids (eg, autonomously replicating plasmids having an origin of replication) which transform target cells by integrating into the cellular genome, or which can exist extrachromosomally.

[0145] The vector can 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 recognized by the immune system.

[0146] Also provided herein is a linear nucleic acid vaccine or linear expression cassette ("LEC") that can be efficiently delivered to a subject by electroporation and express one or more desired antigens. The LEC can be any linear DNA that lacks any phosphate backbone. The DNA can encode one or more antigens. The LEC can contain a promoter, an intron, a stop codon, and / or a polyadenylation signal. The expression of the antigen can be controlled by a promoter. The LEC may not contain any antibiotic resistance gene and / or a phosphate backbone. The LEC may not contain other nucleic acid sequences unrelated to the desired antigen gene expression.

[0147] The LEC can be derived from any plasmid that can be localized. The plasmid can be capable of expressing the antigen. The plasmid can be pNP (Puerto Rico / 34) or pM2 (New Caledonia / 99). The plasmid can be WLV009, pVAX, pcDNA3.0, or provax, or any other expression vector that can express DNA encoding an antigen and allow the cell to translate the sequence into an antigen that is recognized by the immune system.

[0148] The LEC can be pcrM2. The LEC can be pcrNP. The pcrNP and pcrMR can be derived from pNP (Puerto Rico / 34) and pM2 (New Caledonia / 99), respectively.

[0149] The vector may have a promoter. The promoter may be any promoter capable of driving gene expression and controlling the expression of isolated nucleic acid. Such promoter is a cis-acting sequence element required for transcription via DNA-dependent RNA polymerase that transcribes the antigen sequence described herein. The choice of promoter used to direct the expression of heterologous nucleic acid depends on the specific application. The promoter may be located at approximately the same distance from the transcription start in the vector as it is from the transcription start site in its natural setting. However, this difference in distance may be adjusted without losing the function of the promoter.

[0150] The promoter can be operably linked to nucleic acid sequences encoding antigens and signals required for efficient polyadenylation of the transcript, a ribosome binding site, and translation termination.

[0151] The promoter can be the CMV promoter, the SV40 early promoter, the SV40 late promoter, the metallothionein promoter, the mouse mammary tumor virus promoter, the Rous sarcoma virus promoter, the polyhedrin promoter, or another promoter shown to be effective for expression in eukaryotic cells.

[0152] The vector may contain an enhancer and an intron with functional splice donor and acceptor sites. The vector may contain a transcription termination region downstream of the structural gene to provide efficient termination. The termination region may be obtained from the same gene as the promoter sequence or may be obtained from a different gene.

[0153] Methods for preparing vectors Provided herein are methods for preparing vectors containing nucleic acid molecules encoding the synthetic consensus survivin antigens discussed herein. The vectors, after a final subcloning step, can be used to inoculate cell cultures in large-scale fermentation tanks using methods known in the art.

[0154] The vectors used with the EP device can be formulated or manufactured using a combination of known devices and techniques, as described in more detail below, but are preferably manufactured using the optimized plasmid manufacturing techniques described in U.S. Provisional Application US 60 / 939,792, filed May 23, 2007 (see U.S. Patent Publication No. 2009 / 0004716). In some examples, the DNA vectors used in these studies can be formulated at concentrations of 10 mg / mL or higher. The manufacturing techniques also include or incorporate a variety of devices and protocols commonly known to those skilled in the art, in addition to those described in U.S. Patent 60 / 939,792, including those described in U.S. Patent 7,238,522, issued July 3, 2007. The above-referenced applications and patents, U.S. Patent 60 / 939,792 and U.S. Patent 7,238,522, are each incorporated herein in their entirety.

[0155] Vaccine excipients and other components The vaccine may further comprise a pharma- ceutically acceptable excipient. The pharma-ceutically acceptable excipient may be a functional molecule such as a vehicle, a carrier, or a diluent. The pharma-ceutically acceptable excipient may be a transfection-facilitating agent, and may comprise surfactants such as immune stimulating complexes (ISCOMS), Freund's incomplete adjuvant, LPS analogs including monophosphoryl lipid A, muramyl peptides, quinone analogs, vesicles such as squalene and squalene, hyaluronic acid, lipids, liposomes, calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other known transfection-facilitating agents.

[0156] The transfection facilitating agent is a polyanion, a polycation (including poly-L-glutamate (LGS)), or a lipid. The transfection facilitating agent is poly-L-glutamate, and poly-L-glutamate may be present in the vaccine at a concentration of less than 6 mg / ml. The transfection facilitating agent may also include surfactants such as immune stimulating complexes (ISCOMS), Freund's incomplete adjuvant, LPS analogs containing monophosphoryl lipid A, muramyl peptides, quinone analogs, and vesicles such as squalene and squalene, and hyaluronic acid administered with the gene construct may also be used. The DNA vector vaccine may also include transfection facilitating agents such as lipids, liposomes, including lecithin liposomes or other liposomes known in the art (see, for example, WO9324640), calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other known transfection facilitating agents, as DNA-liposome mixtures. The transfection facilitating agent is a polyanion, polycation (including poly-L-glutamate (LGS)), or lipid. The concentration of the transfection agent in the vaccine is less than 4 mg / ml, less than 2 mg / ml, less than 1 mg / ml, less than 0.750 mg / ml, less than 0.500 mg / ml, less than 0.250 mg / ml, less than 0.100 mg / ml, less than 0.050 mg / ml, or less than 0.010 mg / ml.

[0157] The pharma- ceutically acceptable excipient may be one or more adjuvants. The adjuvants may be other genes expressed in alternative vectors or delivered as proteins in combination with the vectors described above in the vaccine.The one or more adjuvants may be selected from the group consisting of CCL20, alpha-interferon (IFN-α), beta-interferon (IFN-β), gamma-interferon, platelet-derived growth factor (PDGF), tumor necrosis factor-like peptide (TNFα), tumor necrosis factor-beta (TNFβ), GM-CSF, epidermal growth factor (EGF), cutaneous T cell-attracting chemokine (CTACK), epithelial thymus-expressed chemokine (TECK), mucosa-associated epithelial chemokine (MEC), IL-12, IL-15, IL-28, MHC, CD80, CD86, IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-18, IL-33, MCP-1, MIP-1a, MIP-1, IL-8, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCA M-1, LFA-1, VLA-1, Mac-1, pl50.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, variant of IL-18, CD40, CD40L, vascular growth factor, fibroblast growth factor, IL-7, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Flt, Ap o-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DRS, KILLER, TRAIL-R2, TRICK2, DR6, caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, inactive NIK, SAP The signal sequence or coding sequence encoding a deleted signal sequence may be selected from the group consisting of K, SAP-I, JNK, interferon response genes, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, Ox40, Ox40 LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAPI, TAP2, IL-15 with a signal sequence or coding sequence encoding a deleted signal sequence and optionally including a different signal peptide such as from IgE, or a coding sequence encoding a different signal peptide such as from IgE, and functional fragments thereof, or combinations thereof.The adjuvant can be IL-12, IL-15, IL-28, CTACK, TECK, platelet-derived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermal growth factor (EGF), IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-18, or a combination thereof.

[0158] In some embodiments, the adjuvant may be one or more nucleic acid molecules encoding a protein selected from the group consisting of CCL-20, IL-12, IL-15, IL-28, CTACK, TECK, MEC, or RANTES. Examples of IL-12 constructs and sequences are disclosed in PCT Application No. PCT / US1997 / 019502 and corresponding U.S. Application No. 08 / 956,865, and U.S. Provisional Application No. 61 / 569600, filed December 12, 2011, each of which is incorporated herein by reference. Examples of IL-15 constructs and sequences are disclosed in PCT Application No. PCT / US04 / 18962 and corresponding US Application No. 10 / 560,650, PCT Application No. PCT / US07 / 00886 and corresponding US Application No. 12 / 160,766, and PCT Application No. PCT / USI0 / 048827, each of which is incorporated herein by reference. Examples of iL-28 constructs and sequences are disclosed in PCT Application No. PCT / US09 / 039648 and corresponding US Application No. 12 / 936,192, each of which is incorporated herein by reference. Examples of RANTES and other constructs and sequences are disclosed in PCT Application No. PCT / US1999 / 004332 and corresponding US Application No. 09 / 622452, each of which is incorporated herein by reference. Other examples of RANTES constructs and sequences are disclosed in PCT Application No. PCT / US11 / 024098, which is incorporated herein by reference. Examples of RANTES and other constructs and sequences are disclosed in PCT Application No. PCT / US1999 / 004332 and corresponding US Application No. 09 / 622452, each of which is incorporated herein by reference. Other examples of RANTES constructs and sequences are disclosed in PCT Application No. PCT / US11 / 024098, which is incorporated herein by reference. Examples of chemokine CTACK, TECK, and MEC constructs and sequences are disclosed in PCT Application No. PCT / US2005 / 042231 and corresponding US Application No. 11 / 719,646, each of which is incorporated herein by reference.Examples of OX40 and immunomodulators are disclosed in U.S. Application No. 10 / 560,653, which is incorporated herein by reference. Examples of DR5 and other immunomodulators are disclosed in U.S. Application No. 09 / 622452, which is incorporated herein by reference.

[0159] Other genes that may be useful as adjuvants include MCP-1, MIP-la, MIP-1p, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, pl50.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, mutated forms of IL-18, CD40, CD40L, vascular growth factor. , fibroblast growth factor, IL-7, IL-22, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Flt, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR 5, KILLER, TRAIL-R2, TRICK2, DR6, caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, inactive NIK, SAP These include those encoding K, SAP-1, JNK, interferon response genes, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, Ox40, Ox40 LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1, TAP2, and functional fragments thereof.

[0160] The vaccine may further comprise a genetic vaccine facilitator as described in US Ser. No. 021,579, filed Apr. 1, 1994, which is incorporated by reference in its entirety.

[0161] The vaccine may contain the plasmid in an amount of about 1 nanogram to about 100 milligrams, about 1 microgram to about 10 milligrams, or preferably about 0.1 microgram to about 10 milligrams, or more preferably about 1 milligram to about 2 milligrams. In some preferred embodiments, the vaccine according to the present invention contains about 5 nanograms to about 1000 macrograms of DNA. In some preferred embodiments, the vaccine may contain about 10 nanograms to about 800 macrograms of DNA. In some preferred embodiments, the vaccine may contain about 0.1 to about 500 macrograms of DNA. In some preferred embodiments, the vaccine may contain about 1 to about 350 macrograms of DNA. In some preferred embodiments, the vaccine may contain about 25 to about 250 micrograms, about 100 to about 200 micrograms, about 1 nanogram to 100 milligrams, about 1 microgram to about 10 milligrams, about 0.1 macrograms to about 10 milligrams, about 1 milligram to about 2 milligrams, about 5 nanograms to about 1000 micrograms, about 10 nanograms to about 800 micrograms, about 0.1 to about 500 micrograms, about 1 to about 350 micrograms, about 25 to about 250 micrograms, or about 100 to about 200 micrograms of the antigen or a plasmid encoding it.

[0162] The vaccine may be formulated according to the mode of administration used. The injectable vaccine pharmaceutical composition may be sterile, pyrogen-free, and particulate-free. An isotonic formulation or solution may be used. Additives for isotonicity may include sodium chloride, dextrose, mannitol, sorbitol, and lactose. The vaccine may include a vasoconstrictor. The isotonic solution may include phosphate buffered saline. The vaccine may further include a stabilizer including gelatin and albumin. The stabilizer including LGS or a polycation or polyanion may make the formulation stable at room or ambient temperature for an extended period of time.

[0163] Pharmaceutical Compositions of Vaccines The vaccine may be in the form of a pharmaceutical composition. The pharmaceutical composition may comprise the vaccine. The pharmaceutical composition may comprise about 5 nanograms (ng) to about 10 milligrams (mg) of the vaccine nucleic acid molecule(s). In some embodiments, the pharmaceutical composition according to the invention comprises about 25 ng to about 5 mg of the vaccine nucleic acid molecule(s). In some embodiments, the pharmaceutical composition comprises about 50 ng to about 1 mg of the vaccine nucleic acid molecule(s). In some embodiments, the pharmaceutical composition comprises about 0.1 to about 500 micrograms of the vaccine nucleic acid molecule(s). In some embodiments, the pharmaceutical composition comprises about 1 to about 350 micrograms of the vaccine nucleic acid molecule(s). In some embodiments, the pharmaceutical composition comprises about 5 to about 250 micrograms of the vaccine nucleic acid molecule(s). In some embodiments, the pharmaceutical composition comprises about 10 to about 200 micrograms of the vaccine nucleic acid molecule(s). In some embodiments, the pharmaceutical composition comprises about 15 to about 150 micrograms of the vaccine nucleic acid molecule(s). In some embodiments, the pharmaceutical composition comprises about 20 to about 100 micrograms of the vaccine nucleic acid molecule(s). In some embodiments, the pharmaceutical composition comprises about 25 to about 75 micrograms of the vaccine nucleic acid molecule(s). In some embodiments, the pharmaceutical composition comprises about 30 to about 50 micrograms of the vaccine nucleic acid molecule(s). In some embodiments, the pharmaceutical composition comprises about 35 to about 40 micrograms of the vaccine nucleic acid molecule(s). In some embodiments, the pharmaceutical composition comprises about 100 to about 200 micrograms of the vaccine nucleic acid molecule(s). In some embodiments, the pharmaceutical composition comprises about 10 micrograms to about 100 micrograms of the vaccine nucleic acid molecule(s). In some embodiments, the pharmaceutical composition comprises about 20 micrograms to about 80 micrograms of the vaccine nucleic acid molecule(s). In some embodiments, the pharmaceutical composition comprises about 25 micrograms to about 60 micrograms of the vaccine nucleic acid molecule(s). In some embodiments, the pharmaceutical composition comprises about 30 ng to about 50 micrograms of the vaccine nucleic acid molecule(s).In some embodiments, the pharmaceutical composition comprises about 35 ng to about 45 micrograms of the vaccine nucleic acid molecule(s). In some preferred embodiments, the pharmaceutical composition comprises about 0.1 to about 500 micrograms of the vaccine nucleic acid molecule(s). In some preferred embodiments, the pharmaceutical composition comprises about 1 to about 350 micrograms of the vaccine nucleic acid molecule(s). In some preferred embodiments, the pharmaceutical composition comprises about 25 to about 250 micrograms of the vaccine nucleic acid molecule(s). In some preferred embodiments, the pharmaceutical composition comprises about 100 to about 200 micrograms of the vaccine nucleic acid molecule(s).

[0164] In some embodiments, pharmaceutical compositions according to the invention comprise at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 ng of a vaccine nucleic acid molecule(s). In some embodiments, the pharmaceutical composition comprises at least 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 81 0, 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, 8 In one embodiment, the vaccine may comprise 0.05, 810, 815, 820, 825, 830, 835, 840, 845, 850, 855, 860, 865, 870, 875, 880, 885, 890, 895, 900, 905, 910, 915, 920, 925, 930, 935, 940, 945, 950, 955, 960, 965, 970, 975, 980, 985, 990, 995, or 1000 micrograms of the vaccine nucleic acid molecule(s). In some embodiments, a pharmaceutical composition may comprise at least 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 mg or more of the vaccine nucleic acid molecule(s).

[0165] In other embodiments, the pharmaceutical composition comprises up to 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 ng (inclusive) of the vaccine nucleic acid molecule(s). In some embodiments, the pharmaceutical composition comprises up to 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235 , 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 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, 47 5, 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, 8 The vaccine may contain 15, 820, 825, 830, 835, 840, 845, 850, 855, 860, 865, 870, 875, 880, 885, 890, 895, 900, 905, 910, 915, 920, 925, 930, 935, 940, 945, 950, 955, 960, 965, 970, 975, 980, 985, 990, 995, or 1000 micrograms (inclusive) of the vaccine nucleic acid molecule(s). In some embodiments, the pharmaceutical composition may contain up to 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 mg (inclusive) of the vaccine nucleic acid molecule(s).

[0166] The pharmaceutical composition may further include other agents for formulation purposes depending on the mode of administration used. When the pharmaceutical composition is an injectable drug composition, they are sterile, pyrogen-free, and particulate-free. It is preferable to use an isotonic formulation. In general, additives for isotonicity may include sodium chloride, dextrose, mannitol, sorbitol, and lactose. In some cases, isotonic solutions such as phosphate buffered saline are preferred. Stabilizers include gelatin and albumin. In some embodiments, a vasoconstrictor is added to the formulation.

[0167] The pharmaceutical composition can further comprise a pharma- ceutically acceptable excipient as provided above in paragraph 2. For example, the pharma- ceutically acceptable excipient can comprise a functional molecule, a vehicle, an adjuvant, a carrier, a diluent, or a transfection-facilitating agent as provided above in paragraph 2.

[0168] Indications The vaccines and pharmaceutical compositions comprising the vaccines provided herein can be used to treat or prevent cancer cells and cancer-based tumors expressing survivin.In particular, the vaccines and pharmaceutical compositions comprising the vaccines provided herein can be used to treat or prevent ovarian cancer, more particularly epithelial ovarian cancer, and most particularly serous ovarian cancer.

[0169] Method of vaccination Methods for treating and / or preventing cancer using the pharmaceutical formulations described above are provided herein. Methods for using the pharmaceutical formulations described above for treating and / or preventing cancer in a subject are also described herein. Methods for vaccinating a subject are also described herein. Methods for administering a pharmaceutical formulation described herein to a subject in need thereof are also described herein. The methods described herein, collectively referred to as therapeutic methods using the pharmaceutical formulations described herein, can include administering one or more vaccines described herein to a subject in need thereof to induce a therapeutic and / or prophylactic immune response. The vaccine can be administered to a subject to modulate the activity of the subject's immune system and enhance the immune response. The administration of the vaccine can be the transfection of the cancer antigens disclosed herein as nucleic acid molecules that are expressed in cells and delivered to the surface of the cells, whereby the immune system recognizes and induces a cellular, humoral, or cellular and humoral response. The administration of the vaccine can be used to induce or elicit an immune response against one or more of the cancer antigens disclosed herein in a subject by administering to the subject a vaccine discussed herein.

[0170] The vaccine is administered to a subject to modulate the activity of the subject's immune system, thereby enhancing the immune response. In some embodiments, the subject is a mammal. When the vaccine is administered to a mammal, thereby introducing the vector into the mammal's cells, the transfected cells express and secrete one or more of the cancer antigens disclosed herein. These secreted proteins or synthetic antigens are recognized as foreign by the immune system, which initiates an immune response that may include antibodies directed against one or more cancer antigens, and T cell responses specifically directed against one or more cancer antigens. In some examples, a mammal vaccinated with the vaccine discussed herein has an antigen-primed immune system, and when inoculated with one or more cancer antigens disclosed herein, the antigen-primed immune system can rapidly clear the subsequent cancer antigens disclosed herein, whether by humoral, cellular, or both cellular and humoral immune responses.

[0171] Methods for administering DNA vaccines are described in US Pat. Nos. 4,945,050 and 5,036,006, both of which are incorporated herein by reference in their entireties.

[0172] The vaccine can be administered to mammals to induce immune response in mammals.The mammals can be human, non-human primates, cows, pigs, sheep, goats, antelopes, bisons, buffalo, cattle, deer, hedgehogs, elephants, llamas, alpacas, mice, rats, preferably human, cattle, or pigs.The vaccine can also be administered to non-human subjects, such as chickens, to induce immune response.

[0173] The dose of the vaccine can be from 1 microgram to 10 mg of active ingredient per kilogram (kg) body weight over time (ingredient / kg body weight / dose), and can be from 20 micrograms to 10 mg of ingredient / kg body weight / dose. The vaccine can be administered every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days. The number of vaccine doses for effective treatment can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more doses.

[0174] Methods for generating an immune response with a vaccine Vaccine can be used to generate immune response in mammalian or non-mammalian subjects, including therapeutic or preventive immune response.Immune response can generate antibodies and / or killer T cells directed to one or more cancer antigens as disclosed herein.Such antibodies and T cells can be isolated.

[0175] Some embodiments provide a method of generating an immune response against one or more of the cancer antigens disclosed herein, the embodiment of which comprises administering a vaccine to a subject. Some embodiments provide a method of prophylactically vaccinating a subject against a cancer or tumor expressing one or more of the above-mentioned cancer antigens, the embodiment of which comprises administering a vaccine. Some embodiments provide a method of therapeutically vaccinating a subject suffering from a cancer or tumor expressing one or more of the cancer antigens, the embodiment of which comprises administering a vaccine. Diagnosis of a cancer or tumor expressing one or more of the cancer antigens disclosed herein prior to administration of a vaccine can be routinely performed.

[0176] How vaccines can be used to treat cancer The vaccine can be used to generate or induce an immune response in a mammal that is reactive or directed against a cancer or tumor (e.g., ovarian cancer) that expresses survivin in the mammal or subject in need thereof. The induced immune response can prevent the growth of the cancer or tumor.

[0177] The induced immune response can prevent and / or reduce metastasis of cancerous or tumor cells. Thus, the vaccine can be used in methods to treat and / or prevent cancer or tumors in a mammal or subject to which the vaccine is administered.

[0178] Route of administration The vaccine or pharmaceutical composition may be administered orally, parenterally, sublingually, transdermally, rectally, transmucosally, topically, by inhalation, by buccal administration, intrapleurally, intravenously, intraarterially, intraperitoneally, subcutaneously, intramuscularly, intranasally, intrathecally, and / or intraarticularly, or combinations thereof. For veterinary use, the composition may be administered in a suitably tolerated formulation in accordance with normal veterinary practice. A veterinarian can readily determine the most appropriate dosing regimen and route of administration for a particular animal. The vaccine may be administered by conventional syringes, needleless injection devices, "microprojectile bombardment gene guns," or other physical methods such as electroporation ("EP"), "hydrodynamic methods," or ultrasound.

[0179] Vaccine vectors can be administered to mammals by several well-known techniques, including in vivo electroporation, liposome-mediated transfection, nanoparticle facilitated transfection, and DNA injection (also referred to as DNA vaccination) with or without recombinant vectors such as recombinant adenovirus, recombinant adenovirus-associated virus, and recombinant vaccinia. Vaccines of one or more cancer antigens can be administered by DNA injection in conjunction with in vivo electroporation.

[0180] Vaccines or pharmaceutical compositions comprising vaccines can be administered by electroporation. Administration of vaccines by electroporation can be accomplished using an electroporation device that can be configured to deliver a pulse of energy to a desired mammalian tissue that is effective to form reversible pores in cell membranes, preferably the pulse of energy is a constant current equivalent to a preset current input by a user. The electroporation device can include an electroporation component and an electrode assembly or a handle assembly. The electroporation component can include or incorporate one or more of the various elements of an electroporation device, including a controller, a current waveform generator, an impedance tester, a waveform logger, an input element, a status reporting element, a communication port, a memory component, a power supply, and a power switch. Electroporation can be accomplished 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 the transfection of cells with vectors.

[0181] Examples of electroporation devices and electroporation methods that can facilitate administration of the DNA vaccines of the present invention include those described in U.S. Patent No. 7,245,963 by Draghia-Akli et al., and U.S. Patent Publication No. 2005 / 0052630 by Smith et al., the contents of which are incorporated herein by reference in their entireties. Other electroporation devices and electroporation methods that can be used to facilitate administration of DNA vaccines include those provided in co-pending and co-owned U.S. Patent Application No. 11 / 874,072, filed October 17, 2007, which claims the benefit under 35 U.S.C. § 119(e) to U.S. Provisional Application Nos. 60 / 852,149, filed October 17, 2006, and 60 / 978,982, filed October 10, 2007, all of which are incorporated herein in their entireties.

[0182] U.S. Patent No. 7,245,963 by Draghia-Akli et al. describes modular electrode systems and their use to facilitate the introduction of biomolecules into cells of selected tissues of a body or plant. The modular electrode system may include multiple needle electrodes, a hypodermic needle, an electrical connector providing a conductive connection from a programmable constant current pulse controller to the multiple needle electrodes, and a power source. An operator can grasp the multiple needle electrodes mounted on a support structure and firmly insert them into selected tissues of a body or plant. The biomolecules are then administered into the selected tissues by the hypodermic needle. The programmable constant current pulse controller is activated to apply a constant current electrical pulse to the multiple needle electrodes. The applied constant current electrical pulse facilitates the introduction of the biomolecules into cells between the multiple electrodes. The entire contents of U.S. Patent No. 7,245,963 are incorporated herein by reference in their entirety.

[0183] US Patent Publication No. 2005 / 0052630, filed by Smith et al., describes an electroporation device that can be used to effectively facilitate the introduction of biomolecules into cells of selected tissues of the body or plants. The electroporation device comprises an electrokinetic device ("EKD device") whose operation is specified by software or firmware. The EKD device creates a series of programmable constant current pulse patterns between a row of electrodes based on user control and input of pulse parameters, allowing for the storage and retrieval of current waveform data. The electroporation device also comprises a replaceable electrode disk with a series of needle electrodes, a central injection channel for an injection needle, and a removable guide disk. The entire contents of US Patent Publication No. 2005 / 0052630 are incorporated herein by reference in their entirety.

[0184] The electrode arrays and methods described in U.S. Patent No. 7,245,963 and U.S. Patent Publication No. 2005 / 0052630 may be suitable for deep penetration of tissues such as muscle as well as other tissues or organs. The configuration of the electrode array allows the injection needle to be fully inserted into the target organ and the injection to be administered perpendicular to the target tissue in the area pre-delineated by the electrodes. The electrodes described in U.S. Patent No. 7,245,963 and U.S. Patent Publication No. 2005 / 005263 are preferably 20 mm long and 21 gauge.

[0185] Additionally, the following patents are contemplated in some embodiments incorporating electroporation devices and their use: U.S. Patent No. 5,273,525 issued December 28, 1993, U.S. Patent No. 6,110,161 issued August 29, 2000, U.S. Patent No. 6,261,281 issued July 17, 2001, and U.S. Patent No. 6,958,060 issued October 25, 2005, as well as electroporation devices as described in U.S. Patent No. 6,939,862 issued September 6, 2005. Additionally, patents covering subject matter provided in U.S. Patent No. 6,697,669 issued February 24, 2004, which relates to administration of DNA using any of a variety of devices, and U.S. Patent No. 7,328,064 issued February 5, 2008, which focuses on methods of injecting DNA, are contemplated herein. The above patents are incorporated by reference in their entirety.

[0186] Provided herein are methods for preparing vectors containing a nucleic acid molecule(s) encoding the synthetic consensus survivin antigen discussed herein. The vectors, after a final subcloning step into a mammalian expression plasmid, can be used to inoculate cell cultures in large-scale fermentation tanks using methods known in the art.

[0187] The DNA vectors for use in the EP device of the present invention can be formulated or manufactured using a combination of known devices and techniques, but are preferably manufactured using the optimized manufacturing techniques described in U.S. Published Application No. 2009 / 0004716, filed May 23, 2007. In some examples, the DNA vectors used in these studies can be formulated at a concentration of 10 mg / mL or higher. The manufacturing techniques also include or incorporate a variety of devices and protocols commonly known to those skilled in the art, in addition to those described in U.S. Patent No. 60 / 939,792, including those described in U.S. Patent No. 7,238,522, issued July 3, 2007. The above-referenced applications and patents, U.S. Patent No. 60 / 939,792 and U.S. Patent No. 7,238,522, are each incorporated herein in their entirety. EXAMPLES

[0188] The present invention is further illustrated in the following examples. It should be understood that these embodiments, although showing preferred embodiments of the present invention, are given by way of illustration only. From the above discussion and these examples, those skilled in the art can ascertain the essential features of the present invention, and can make various changes and modifications of the present invention to suit various uses and conditions without departing from its spirit and scope. Thus, in addition to those shown and described herein, various modifications of the present invention will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

[0189] Example 1 - Generation of consensus survivin isoform 1 Twenty-nine survivin isoform 1 sequences were collected from GenBank (www.ncbi.nlm.nih.gov / genbank / ). The GenBank accession numbers of the selected survivin isoform 1 sequences are NP_001125727.1, 3UIG,1F3H, BAC22748.2, NP_001159.2, CAG46540.1, BAD97148.1, XP_002748841.1, XP_003818322.1, NP_001253110.1, XP_011810718.1, XP_011832690.1, XP_001083183.1, XP_003786134.1, and XP_01253110.1. 516801.1, XP_007957800.1, XP_001915435.1, XP_008523102.1, AAT37504.1, XP_004432883.1, XP_003417277.1, XP_004374167.1, NP_999306.1, XP_002918421.1, NP_001003348.1, NP_001009280.1, XP_004748859.1, NP_001001855.2, and AAU89275.1.

[0190] A consensus sequence was generated using the DNASTAR® Lasergene software package (version 13.0.0.357). The 29 sequences listed above were imported into MegAlign and aligned using the ClustalW multiple sequence alignment program. The resulting consensus survivin isoform 1 sequence shares 97.2-97.9% homology with native human survivin isoform 1. To eliminate potential biological functions of the resulting consensus survivin isoform 1 protein, three mutations were introduced, eliminating the anti-apoptotic activity of survivin. The three mutations are T34A, T48A, and C84A. In addition, an upstream Kozak sequence and an IgE leader sequence were added to the N-terminus to obtain a higher level of expression. Furthermore, the codon usage of this gene was adapted to the codon bias of Homo sapiens genes. (Andre, S. et al. Increased immune response elicited by DNA vaccination with a synthetic gp120 sequence with optimized codon usage. Journal of virology 72, 1497-1503 (1998); Deml, L. et al. Multiple effects of codon usage optimization on expression and immunogenicity of DNA candidate vaccines encoding the human immunodeficiency virus type 1 Gag protein. Journal of virology 75, 10991-11001, doi:10.1128 / JVI.75.22.10991-11001.2001 (2001)). In addition, RNA optimization was performed to avoid regions with very high (>80%) or very low (<30%) GC content, as well as cis-acting sequence motifs such as internal TATA boxes, chi-sites, and ribosome entry sites.As a result, the synthetic consensus survivin antigen isoform 1 protein shares 95.1-95.8% identity with the human native survivin isoform 1 protein. The nucleotide sequence of the synthetic consensus survivin antigen isoform 1 is shown in SEQ ID NO: 1. The amino acid sequence of the synthetic consensus survivin antigen isoform 1T3 is shown in SEQ ID NO: 2.

[0191] The synthetic consensus survivin antigen isoform 1 was digested with BamHI and XhoI and cloned into a proprietary expression vector pGX0001 with an expression cassette placed under the transcriptional control of the cytomegalovirus immediate early promoter. The resulting plasmid was named pGX1428. Full-length sequencing was performed and then analyzed to confirm correctness. A schematic diagram of the synthetic consensus survivin antigen isoform 1 construct is shown in Figure 1. The overall structure of the synthetic consensus survivin antigen isoform 1 is shown in Figure 2. [Table 1] [Table 2]

[0192] Example 2 - Generation of consensus survivin isoform 1T3 Eight survivin isoform 3 sequences were collected from GenBank (www.ncbi.nlm.nih.gov / genbank / ) to generate the human consensus survivin isoform 3. The GenBank accession numbers of the selected survivin isoform 3 sequences are NP_001012270.1, XP_008969790.1, XP_008011275.1, XP_009189614.1, XP_011897653.1, XP_011810642.1, XP_011844315.1, and XP_011718355.1.

[0193] The consensus sequence was generated using the DNASTAR® Lasergene software package (version 13.0.0.357). The eight sequences listed above were imported into MegAlign and aligned using the ClustalW multiple sequence alignment program. Six of these sequences from lower animals contained 11 extra amino acid residues at their C-terminus that are not present in the human sequence. These extra residues were removed in generating the consensus human survivin isoform 3 to prevent the induction of off-target immune responses in humans. After generating the human consensus survivin isoform 3, the identical amino acid sequence between survivin isoforms 1 and 3 was removed from the consensus survivin isoform 3. The truncated consensus survivin isoform 3 sequence (survivin antigen T3) shares 96.8% sequence homology with the native human survivin isoform T3 sequence. The survivin antigen isoform 1T3 immunogen was generated by adding a furin cleavage site between survivin antigen 1 (described above) and survivin antigen T3.

[0194] Once the consensus synthetic consensus survivin antigen isoform 1T3 DNA sequence was obtained, an upstream Kozak sequence and an IgE leader sequence were added to the N-terminus to obtain higher levels of expression. (Yang. genes(Andre,S.et al.Increased immune response elicited by DNA vaccination with a synthetic gp120 sequence with optimized codon usage.Journal of virology 72,1497-1503(1998), Deml,L.et al.Multiple effects of codon usage optimization on expression and immunogenicity of DNA candidate vaccines encoding the human immunodeficiency virus type 1 Gag protein.Journal of virology 75, 10991-11001, doi:10.1128 / JVI.75.22.10991-11001.2001 (2001)). In addition, RNA optimization was performed to avoid regions with very high (>80%) or very low (<30%) GC content, as well as cis-acting sequence motifs such as internal TATA boxes, chi-sites, and ribosome entry sites.The synthetic consensus survivin antigen isoform 1T3 was digested with BamHI and XhoI and cloned into the expression vector pGX0001 with an expression cassette placed under the transcriptional control of the cytomegalovirus immediate early promoter. The resulting plasmid was named pGX1429. Full-length sequencing was performed and then analyzed to confirm correctness. As shown in Table 1, the nucleotide sequence of the synthetic consensus survivin antigen isoform 1T3 is shown in SEQ ID NO: 3. As shown in Table 1, the amino acid sequence of the synthetic consensus survivin antigen isoform 1T3 is shown in SEQ ID NO: 8. A schematic diagram of the synthetic consensus survivin antigen isoform 1T3 construct is shown in FIG. 3. The characteristics of the synthetic consensus survivin antigen isoform 1T3 construct are shown in Table 3. [Table 3]

[0195] Example 3 - Construction of pGX Survivin Expression Vector pGX0001 (modified pVAX1 expression vector) under the control of the human cytomegalovirus immediate early promoter (hCMV promoter) was used as the backbone vector. The original pVAX1 was obtained from Thermo Fisher Scientific.

[0196] Modifications were introduced into pVAX1 to create pGX0001 and were identified based on the reported sequence of pVAX1 available from Thermo Fisher Scientific. These modifications are listed below and no issues were detected with plasmid amplification and antigen transcription and translation. Thus far, no changes have been observed in the sequence of pGX0001 in any of the data for plasmid products in platforms that use pGX0001 as a backbone. [Table 4-1] Base pairs 2, 3, and 4 are changed from ACT to CTG in the backbone upstream of the CMV promoter.

[0197] pGX1428 is a DNA plasmid encoding synthetic consensus survivin antigen isoform 1 (survivin antigen 1) protein. The associated mRNA production is driven by the human CMV promoter (hCMV promoter) and terminated by the bovine growth hormone 3' end poly-adenylation signal (bGH polyA). The pGX0001 backbone contains a kanamycin resistance gene (KanR) and a plasmid replication origin (pUC ori) for production purposes. These elements do not function in eukaryotic cells. pGX1428 was generated by cloning the synthetic consensus survivin antigen isoform 1 (survivin antigen 1) DNA sequence into pGX0001 at the BamHI and XhoI sites as shown in Figure 4.

[0198] pGX1429 is a DNA plasmid encoding the synthetic consensus survivin antigen isoform 1T3 (survivin antigen 1T3) protein. The associated mRNA production is driven by the human CMV promoter (hCMV promoter) and terminated by the bovine growth hormone 3' end poly-adenylation signal (bGH polyA). The pGX0001 backbone contains a kanamycin resistance gene (KanR) and a plasmid replication origin (pUC ori) for production purposes. These elements do not function in eukaryotic cells. pGX1429 was generated by cloning the synthetic consensus survivin antigen isoform 1T3 (survivin antigen 1T3) DNA sequence at the BamHI and XhoI sites into pGX0001 as shown in Figure 5.

[0199] Example 4 - Immunogenicity of synthetic consensus survivin antigen constructs The immunogenicity of vaccine constructs designed to target human survivin, synthetic consensus survivin antigen 1 (pGX1428), and synthetic consensus survivin antigen 1T3 (pGX1429) was evaluated in mice. Expression of antigen protein by each construct was also assessed in vitro by Western blotting.

[0200] material and method Plasmids Synthetic consensus survivin antigen 1 (pGX1428) and synthetic consensus survivin antigen 1T3 (pGX1429) were designed as described herein. For in vitro and in vivo studies, plasmids (10 mg) were ordered from GenScript, both pGX1428 (lot #786114S-1 / G52238) and pGX1429 (lot #786114S-2 / G52239). Antigen sequences of the 10 mg plasmid stocks were confirmed by Sanger sequencing.

[0201] In vitro antigen expression The expression of antigen proteins by pGX1428 and pGX1429 was confirmed by Western blotting. As shown in Figure 6, human rhabdomyosarcoma (RD) cells (ATCC, CCL-136) maintained in DMEM medium containing 10% FBS (ThermoFisher) were transfected with pGX1428, pGX1429, or pGX0001 (6 μg / 10 cm) using Turbofectin 8 (Origene). 2 Dish) were transfected. 48 h after transfection, cells were lysed using RIPA cell lysis buffer (ThermoFisher) and cell lysates were collected. Following a BCA assay (ThermoFisher) to determine total protein concentration, 15 μg of cell lysate was electrophoresed on a 4–12% SDS-PAGE gel (ThermoFisher). Detection was performed with a monoclonal antibody against amino acids 1–142 of human survivin (Santa Cruz Biotech, clone D8, sc-17779) and then visualized with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Santa Cruz Biotech, sc-2005) using an ECL Western blot analysis system (GE Amersham). As a loading control, blots were reprobed for actin expression using an anti-β-actin monoclonal antibody (Santa Cruz Biotech, clone, C4, sc-47778 HRP).

[0202] Animals and immunization Female 8-week-old CB6F1 mice were purchased from Jackson Laboratories. All animals were housed in a temperature-controlled light cycle facility at BTS Research (San Diego, CA). Animal care was performed in accordance with the National Institutes of Health and Animal Care and Use Proposal (ACUP) guidelines (BTS ACUP #15-091). Mice were divided into nine groups as shown in Table 4. [Table 4-2]

[0203] Mice in the immunized groups were vaccinated with the indicated doses of pGX0001, pGX1428, or pGX1429 according to SOP R20-003147 CELLECTRA® 3P Mouse Treatment. Briefly, plasmids were formulated in sterile water for injection (VetOne) and the indicated doses were delivered by intramuscular injection into the tibialis anterior muscle with an injection volume of 30 μL. Each intramuscular injection was immediately followed by electroporation (EP) using a CELLECTRA® 2000 Adaptive Constant Current Electroporation Device (Inovio Pharmaceuticals) with a 3P array. The device was configured to deliver two 0.1 Amp pulses with a 52 ms pulse width, separated by a 1 second delay. Mice received three immunizations at 3-week intervals. Mice were sacrificed 1 week after the last immunization and spleens were harvested for cellular immunity readings. No other tissues were collected.

[0204] Splenic lymphocyte isolation Splenocytes were aseptically isolated and placed in 5 mL of R10 medium (Rosewell Park Memorial Institute medium 1640 supplemented with 10% fetal bovine serum and 1% antibiotic-antimycotic). Splenocytes were isolated by mechanically disrupting the spleen using a Stomacher machine (Seward Laboratory Systems Inc.), and the resulting product was filtered using a 40 μm cell strainer (BD Falcon). The resulting product was centrifuged, and the pellet was treated with ACK lysis buffer (Lonza) for 5 min for lysis of RBCs. Splenocytes were then centrifuged, washed with PBS, resuspended in R10 medium, and immediately used for further analysis.

[0205] IFNγ ELISpot Mouse IFNγ ELISpot assay was performed using MabTech's kit (MabTech, #3321-4APW-10) to evaluate antigen-specific cellular responses. Briefly, 96-well plates pre-coated with anti-mouse IFNγ antibody (mAb AN18) were washed in PBS and blocked with complete culture medium (RPMI 1640 supplemented with 10% FBS and antibiotics) for 2 hours at room temperature. Splenic lymphocytes were resuspended in R10 medium (then added in triplicate, with an input cell number of 2 × 105 cells per well). A set of peptides was synthesized (GenScript), each containing 15 amino acid residues with 11 amino acid overlaps representing the entire synthetic consensus survivin antigen 1 and synthetic consensus survivin antigen 1 T3 protein sequences. These sets of peptides were resuspended in DMSO (Sigma) and pooled into two peptide pools at a concentration of approximately 2 μg / ml of peptide. The first peptide pool contains peptides corresponding to the synthetic consensus survivin antigen 1 antigen protein, and the second peptide pool contains peptides corresponding to the synthetic consensus survivin antigen 1 T3 antigen protein. Concavalin A (Sigma) at 5 μg / ml was used as a positive control, and complete culture medium was used as a negative control. Plates were incubated at 37°C in an incubator with a 5% CO2 atmosphere for 18 hours. Then, a biotinylated anti-mouse IFNγ detection antibody (MabTech mAb R4-6A2) was added and the plate was incubated for 2 h at room temperature. The plate was washed, streptavidin-ALP (MabTech) was added and the plate was incubated for 1 h at room temperature. Spot detection was completed using BCIP / NBT substrate according to the kit manufacturer's instructions (MabTech). Spots on the plate were counted using an automated ELISPOT reader (Cellular Technology). The average number of spot-forming units (SFU) was calculated as 1 × 10 for data presentation. 6 The splenocytes were adjusted.

[0206] In Figures 7A-7H, antigen-specific responses by IFNγ ELISpot were measured using 1 x 10 6Reported as number of IFNγ spot-forming units (SFU) per splenocyte.

[0207] Flow cytometry The cellular immune responses induced by synthetic consensus survivin antigen 1 and synthetic consensus survivin antigen 1T3 were further characterized by flow cytometry. Briefly, 2 × 10 6Splenocytes were stimulated immediately after isolation with synthetic consensus survivin antigen 1 and survivin 1T3 peptides according to each group for 6 h in the presence of Brefeldin A (BD Biosciences), monensin (BD Biosciences), and FITC anti-mouse CD107a antibody (BD Biosciences, clone 1D4B). After peptide stimulation, splenocytes were spun down and resuspended in 20 μL per well of mouse BD Fc Block (BD Biosciences) solution. Fc Block was used at an initial dilution of 1:40 in PBS and incubated for 5 min at 4 °C. After incubation, add 30 μL per well of the remaining extracellular antibody (in PBS) and incubate for 30 min at 4 °C. Upon addition of the extracellular staining solution, the final volume in each well is 50 μL, consisting of Fc Block at a final dilution of 1:100 and extracellular antibody at the appropriate working dilution. Cells were then stained with a viability dye (Vivid V450, Thermo-Fisher) and the following antibodies: PerCP-Cy5.5 anti-mouse CD4 (BD Biosciences, clone RM4-5) and APC anti-mouse CD8a (BD Biosciences, clone 63-6.7). Cells were fixed and permeabilized for 20 minutes at 4°C (BD Biosciences, #554714). Intracellular staining was then completed with the following antibodies: APC-Cy7 anti-mouse CD3e (BD Biosciences, clone 145-2C11), BV605 anti-mouse IFNγ (BD Biosciences, clone XMG1.2), APC-R700 anti-mouse IL-2 (BD Biosciences, clone JEs6-5H4), and PE anti-mouse TNF-α (BD Biosciences, clone MP6-XT22). ICS data was collected on a 10-color FACS CANTO (BD Biosciences) and analysis was completed using FlowJo software. The flow cytometry gating strategy is shown in Figure 8.

[0208] For cells to be called antigen-specific by flow cytometry, the frequency of the reported parameters must exceed that of the media-only control. For cells to be identified as producing antigen-specific CD107a, they must be identified as positive for antigen-specific production of IFNγ and / or IL-2 and / or TNFα as identified by Boolean gating.

[0209] statistical analysis Statistical analyses were completed using IBM SPSS Statistics 22 (IBM Corporation). Between-group analyses were performed using ANOVA with post-hoc Tukey's test (HSD) to adjust for multiple comparisons. Homogeneity of variance was checked using F statistics prior to multiple comparisons. For all statistical analyses, a p-value of 0.050 was considered significant.

[0210] result Expression of synthetic consensus survivin antigen protein Two constructs were designed to target human survivin, synthetic consensus survivin antigen 1 (pGX1428), and synthetic consensus survivin antigen 1T3 (pGX1429). Expression of synthetic consensus survivin antigen 1 and synthetic consensus survivin antigen 1T3 antigen proteins by pGX1428 and pGX1429, respectively, was confirmed by Western blotting. Briefly, human rhabdomyosarcoma (RD) cells were transfected with pGX1428, pGX1429, or pGX0001 (empty vector, negative control) plasmids. Cell lysates were probed for expression of synthetic consensus survivin antigen protein and anti-human survivin antibody (BIRC5). Protein bands of the expected molecular weights of synthetic consensus survivin antigen 1 (17.5 kD) and synthetic consensus survivin antigen 1T3 (25.3 kD) were detected (Figure 6). A faint band was detected in the negative control (pGX0001), most likely due to low levels of endogenous survivin protein expression in RD cell lines. Anti-β-actin bands were detected with similar intensity, indicating that equal amounts of protein were loaded in each lane. In summary, pGX1428 and pGX1429 were found to express their respective antigen proteins.

[0211] Immunogenicity of a synthetic consensus survivin antigen vaccine construct IFNγ ELISpot The immunogenicity of the two synthetic consensus survivin antigen constructs was evaluated at four doses (10 μg, 20 μg, 30 μg, and 50 μg) by IFNγ ELISpot and flow cytometry (n=8 / group). Mice were immunized with an empty vector backbone (pGX0001) as a negative control (n=4 / group). Vaccination with the synthetic consensus survivin antigen resulted in a significant IFNγ response compared to negative control vaccinated mice. However, there was minimal evidence for a dose-dependent increase in IFNγ production induced by synthetic consensus survivin antigen 1 (FIG. 10A), suggesting that the maximal response was achieved at the lowest dose. In detail, synthetic consensus survivin antigen 1 IFNγ SFU were 1,082 ± 574, 1,186 ± 747, 1,135 ± 647, and 848 ± 350 at 10 μg, 20 μg, 30 μg, and 50 μg, respectively. Synthetic consensus survivin antigen 1 IFNγ responses were significantly greater with pGX1428 at 10 μg (p = 0.031), 20 μg (p = 0.015), and 30 μg (p = 0.021) doses than untreated (2 ± 3), but not at the 50 μg (p = 0.134) dose. Vaccination with synthetic consensus survivin antigen 1T3 resulted in significant IFNγ responses, with some evidence for a dose-dependent increase with increasing dose levels (Figure 10D). Survivin 1T3 IFNγ SFU were 516 ± 156, 812 ± 534, 1,016 ± 654, and 818 ± 339 at 10 μg, 20 μg, 30 μg, and 50 μg, respectively. Synthetic consensus survivin antigen 1T3 IFNγ responses were significantly greater with the 20 μg (p = 0.039), 30 μg (p = 0.006), and 50 μg (p = 0.037) doses of pGX1429 than untreated (5 ± 6), but not with the 10 μg (p = 0.337) dose. IFNγ responses are summarized in Table 5. [Table 5]

[0212] Flow cytometry Both synthetic consensus survivin antigen 1 and synthetic consensus survivin antigen 1T3 induced stronger responses in the CD4+ T cell compartment compared to responses in the CD8+ T cell compartment (Figure 9). Synthetic consensus survivin antigen 1 induced a frequency of antigen-specific CD4+ T cell responses that were significantly more robust than untreated (0.03% ± 0.05%) in the 20μg (1.08% ± 0.65%) (p = 0.024) and 50μg (1.21% ± 0.73%) (p = 0.009) dose groups, but not in the 10μg (0.86% ± 0.31%) (p = 0.105) or 30μg (0.82% ± 0.46%) (p = 0.134) dose groups (Figure 7B). The synthetic consensus survivin antigen 1T3 induced frequencies of antigen-specific CD4+ T cell responses that were significantly more robust than untreated (0.05% ± 0.05%) in the 20 μg (1.02% ± 0.59%) (p = 0.010), 30 μg (1.08% ± 0.47%) (p = 0.006), and 50 μg (1.13% ± 0.44%) (p = 0.004) dose groups, but not in the 10 μg (0.62% ± 0.35%) (p = 0.248) dose group (Figure 7E). Cytokine profiles of synthetic consensus survivin antigen-specific CD4+ T cells were similar across dose groups for both constructs and consisted primarily of IFNγ+IL-2+TNFα+, IFNγ+IL-2-TNFα+, or IFNγ+IL-2-TNFα- cells (Figure 7G,H). The frequencies of antigen-specific CD4+ T cells are detailed in Table 6. [Table 6]

[0213] There was no significant difference in the frequency of antigen-specific CD8+ T cells induced by synthetic consensus survivin antigen 1 (p=0.117) (Figure 7C). Survivin 1T3 induced a significantly higher frequency of antigen-specific CD8+ T cells between dose groups (p=0.043). The frequency of antigen-specific CD8+ T responses in groups immunized with 10 μg (0.15% ± 0.09%) (p=0.919), 20 μg (0.27% ± 0.21%) (p=0.274), or 30 μg (0.25% ± 0.14%) (p=0.377) of pGX1429 did not approach statistical significance compared to untreated (0.07% ± 0.01%). The frequency of survivin 1T3-specific CD8+ T cells approached statistical significance (0.36% ± 0.21%) (p = 0.051) in the group immunized with 50 μg of pGX1429 (Figure 7F). Both synthetic consensus survivin antigen 1 and synthetic consensus survivin antigen 1T3 induced CD8+ T cell responses similar in magnitude and phenotype. Antigen-specific CD8+ T cells were predominantly IFNγ+IL-2-TNFα-, IFNγ+IL-2-TNFα+ (Figure 7G, Figure 7H). The frequency of antigen-specific CD8+ T cells is described in more detail in Table 7. [Table 7]

[0214] Approximately 25% of the cytokine-positive CD4+ T cells induced by synthetic consensus survivin antigen 1 (FIG. 10A) and synthetic consensus survivin antigen 1T3 (FIG. 10B) were also positive for CD107a, indicating a possible CD4+ T cell-mediated cytolytic function. All doses of synthetic consensus survivin antigen 1 induced significantly higher frequencies of CD4+CD107a+ T cells than untreated (0.01%±0.01%). In detail, the frequency of antigen-specific CD4+CD107a+ T cells was 0.25% ± 0.08%, 0.36% ± 0.11%, 0.21% ± 0.15%, and 0.38% ± 0.13% in the 10 μg (p = 0.013), 20 μg (p < 0.001), 30 μg (p = 0.050), and 50 μg (p < 0.001) dose groups, respectively (Figure 10A). The synthetic consensus survivin antigen 1T3 induced significantly higher frequencies of CD4+CD107a+ T cells than untreated (0.01 ± 0.01) in all groups except the 10 μg dose group (0.18% ± 0.09%) (p = 0.147). Antigen-specific CD4 + CD107a + The frequencies of T cells were 0.24% ± 0.12%, 0.27% ± 0.12%, and 0.29% ± 0.16% in the 20 μg (p = 0.030), 30 μg (p = 0.010), and 50 μg (p = 0.004) dose groups, respectively (Figure 10B). + T cell frequencies are detailed in further Table 8. [Table 8]

[0215] Similar to the magnitude of antigen-specific CD8+ T cells, synthetic consensus survivin antigen 1 did not induce significant changes in the frequency of CD8+CD107a+ T cells among all groups (p=0.101) (Figure 10C). Synthetic consensus survivin antigen 1T3 induced significant changes in the frequency of CD8+CD107a+ T cells among all dose groups (p=0.034) (Figure 10D). The frequency of antigen-specific CD8+CD107a+ T cells was significantly increased (p=0.026) in the group immunized with 50 μg of pGX1429 (0.28%±0.22%) compared with the untreated group (0.03±0.01), but not in the groups immunized with 10 μg (0.11%±0.08%) (p=0.813), 20 μg (0.16%±0.11%) (p=0.450), or 30 μg (0.16%±0.07%) (p=0.424) of the synthetic consensus survivin antigen 1T3. The cytokine profile of synthetic consensus survivin antigen-specific CD8+CD107a+ T cells was similar for both constructs across dose groups and consisted primarily of IFNγ+IL-2-TNFα+ and IFNγ+IL-2-TNFα- cells (Figures 10E and 10F). + T cell frequencies are detailed in Table 9. [Table 9] Breadth of IFN-.GAMMA. responses induced by synthetic consensus survivin antigen constructs

[0216] The breadth of IFNγ responses to survivin induced by pGX1428 and pGX1429 was investigated by epitope mapping using a peptide matrix pooling approach (Figures 11A-11D). Pooled splenic lymphocytes from mice immunized with the highest dose of pGX1428 (n=8) (Figure 11A) or pGX1429 (n=8) (Figure 11B) were examined.

[0217] The following synthetic consensus survivin antigen 1 epitope was present in both pGX1428 and pGX1429: LPPAWQLFLKDHRISTFKN (SEQ ID NO:5) (Matrix pools 1, 2, 3, 4, and 7) LKLDRERAKNKIAKETNNK (SEQ ID NO: 6) (Matrix pools 1, 2, 3, 4, and 11) Synthetic consensus survivin antigen T3 epitope present in pGX1429: EWLHHFQGLFP (SEQ ID NO: 7) (Matrix pools 3, 4, and 7)

[0218] The total magnitude of the cellular immune responses induced by synthetic consensus survivin antigen 1 and synthetic consensus survivin antigen 1T3 were similar, but the response elicited by the synthetic consensus survivin antigen 1T3 (pGX1429) region against the synthetic consensus survivin antigen 1 (pGX1428) region of synthetic consensus survivin antigen 1T3 was approximately half the magnitude of the response induced by the synthetic consensus survivin antigen 1 construct (pGX1428). Epitope mapping by IFNγ ELISpot using a matrix approach revealed that both synthetic consensus survivin antigen constructs generated responses against the same epitopes of the synthetic consensus survivin antigen 1 antigen, but the responses driven by synthetic consensus survivin antigen 1T3 against these epitopes were lower in magnitude. We also confirmed the presence of unique epitopes in the T3 region of the synthetic consensus survivin antigen 1T3 antigen.

[0219] The total magnitude of the cellular immune responses induced by the synthetic consensus survivin antigen 1 (pGX1428) and synthetic consensus survivin antigen 1T3 (pGX1429) were similar, but the responses elicited by the synthetic consensus survivin antigen 1T3 (pGX1429) region against the synthetic consensus survivin antigen 1 region of the synthetic consensus survivin antigen 1T3 antigen were approximately half the magnitude of the responses induced by the synthetic consensus survivin antigen 1 construct (pGX1428). Epitope mapping by IFNγ ELISpot using a matrix approach revealed that both synthetic consensus survivin antigen constructs generated responses against the same epitopes as the synthetic consensus survivin antigen 1 antigen, but the responses driven by the synthetic consensus survivin antigen 1T3 against these epitopes were lower in magnitude. Using this approach, we also confirmed the presence of unique epitopes in the T3 region of the synthetic consensus survivin antigen 1T3 antigen. Synthetic consensus survivin antigen 1T3 also significantly increased the frequency of antigen-specific CD8+ and CD8+CD107a+ T cells compared to untreated, whereas synthetic consensus survivin antigen 1 did not significantly increase antigen-specific CD8+ T cells in mice. Synthetic consensus survivin antigen 1T3 (pGX1429) was selected to move into monovalent non-human primate studies based on its potential to further expand the breadth of the cellular immune response to survivin compared to pGX1428 in mice.

[0220] Example 5 - Non-human primate studies using synthetic consensus survivin antigen 1T3 (pGX1429) To investigate the potential of synthetic consensus survivin 1T3 alone and in combination with low and high doses of IL-12, 18 adult rhesus macaques, identified by their unique NHP ID number, were divided into three groups of six and immunized with pGX1429 as follows: six animals were immunized with 3.0 mg pGX1429, six with 3.0 mg pGX1429 plus 0.04 mg pGX6006 (opt rh IL-12) as an adjuvant, and six with 3.0 mg pGX1429 plus 0.20 mg pGX6006 (opt rh IL-12) as an adjuvant, formulated in SSC in a 1.0 mL injection volume. Immunization injections were administered at weeks 0, 4, 8, and 12. All immunizations were performed intramuscularly on alternating contralateral limbs with a CELLECTRA® 2000 5P-IM EP device, with an injection volume of 1 ml formulated in sterile WFI. EP conditions were as follows: OpBlock 0070-IM, 0.5 Amp, 3 pulses, 52 ms, 0.2 seconds between pulses. Survivin immunogenicity was evaluated at weeks 2, 6, 10, and 14. Survivin-specific IFN-gamma responses are shown in Figures 12 and 13. The homology between native rhesus survivin and pGX1429 is shown in Table 10. As shown in Figures 13 and 14, an increase in survivin-specific responses was observed in animals immunized with synthetic consensus survivin antigen 1T3 plus 0.20 mg IL-12 as an adjuvant.

[0221] PBMC isolation Non-human primate whole blood was collected in sodium citrate cell preparation tubes (BD Biosciences in CPT CPT) containing anticoagulant and gel barrier. Whole blood is centrifuged immediately (within 2 hours) after collection to separate and enrich for PMBCs before overnight transport. Red blood cells and neutrophils settle to the bottom of the tube and are held in place by the gel barrier. Plasma and lymphocytes remain above the gel barrier. Each CPT can hold approximately 8 mL of blood and is transported at room temperature. Spinned CPT tubes were processed for PBMC isolation. After lysis of red blood cells with ammonium chloride-potassium (ACK) buffer, viable cells were counted using an Invitrogen Countess™ Automated Cell Counter and resuspended in complete culture medium (RPMI 1640 supplemented with 10% FBS, antibiotics, and β-mercaptoethanol). Upon completion of the assays described herein, the remaining PBMCs were frozen in freezing medium (10% DMSO from Sigma in 90% FBS from Seradigm) in cryovials and stored long term in liquid nitrogen.

[0222] IFNγ ELISpot To evaluate vaccine-induced antigen-specific cellular responses, monkey IFNγ ELISpot assays were performed on isolated PMBCs at each time point using a kit (MabTech IFNγ ELISpotPro, #3421M-2APW-10). Briefly, 96-well plates pre-coated with anti-monkey IFNγ antibody (mAb MT126L) were washed with PBS and blocked with complete culture medium (RPMI 1640 supplemented with 10% FBS, antibiotics, and β-mercaptoethanol) for 2 hours at room temperature. NHP PBMCs were resuspended in R10 medium (then 2 × 10 per well). 5(The input cell number of cells was added in triplicate). A set of peptides was synthesized (GenScript), each containing 15 amino acid residues with 11 amino acid overlaps representing the entire synthetic consensus protein sequence. These sets of peptides were resuspended in DMSO (Sigma) and pooled into pools at a concentration of approximately 2 μg / mL of each respective peptide. All antigen-specific pooled peptides were used at a 1:100 dilution and combined with PBMCs for a final dilution of 1:200 in each well. Variation in the size of each antigen protein resulted in two peptide pools of survivin.

[0223] Anti-CD3 (mAb CD-2 Mabtech) and / or PMA (Sigma) with ionomycin (Sigma) were used as positive controls. Complete R10 culture medium was used as a negative control. Plates were incubated for approximately 18 hours at 37°C in an incubator with a 5% CO2 atmosphere. After removing the cells and adding an ALP-conjugated anti-monkey IFNγ detection antibody (MabTech Ab 7-B6-1-ALP), plates were incubated for 2 hours at room temperature. The sandwich immunoenzymatic assay was then developed using BCIP / NBT substrate solution according to the kit manufacturer's instructions (MabTech). Blue-black precipitates form as spots, revealing individual IFNγ-producing cells. Spots are then scanned and counted by a CTL ImmunoSpot® analyzer and software (Cellular Technology) and quality controlled by a trained operator. IFNγ responses were assessed as 1x10 SFU greater than the SFU in the medium-only control. 6 Reported as spot forming units (SFU) for PBMC. [Table 10] [Table 11]

[0224] Groups 1, 2, and 3 received the following: Group 1 - 3.0 mg of pGX1429 (synthetic consensus survivin 1T3), formulated IM in an SSC with an injection volume of 1.0 mL. Group 2 - 3.0 mg pGX1429 (synthetic consensus survivin 1T3) + 0.04 pGX6006 (opt. rIL-12). Formulated IM, 1.0 mL injection volume in SSC. Group 3 - 3.0 mg pGX1429 (synthetic consensus survivin 1T3) + 0.20 pGX6006 (opt. rIL-12). Formulated IM, 1.0 mL injection volume in SSC.

[0225] All groups were immunized according to the following schedule: ·Immunization 1 (week 0) ·Immunization 2 (4th week) ·Immunization 3 (8th week) ·Immunization 4 (12th week) ·Immunization 5 (optional)

[0226] result Survivin-specific IFNγ responses are shown in Figures 12-14 for each of groups 1-3. Results show responses at each time point over the two week period following dose. Overall, all groups and individual animals had an increased response by the end of the study two weeks after dose 4 compared to pre-bleed baseline. Adding a higher dose of IL-12 (0.2 mg) resulted in more consistent responses at each time point compared to survivin alone or survivin + 0.04 mg IL-12. Higher responses early in PD2 were also seen with survivin + 0.2 mg IL-12.

[0227] As shown in Tables 12-14, there were no differences in any of the physiological parameters measured with immunization. No significant differences were noted in RBC, HCT, neutrophils, lymphocytes, monocytes, or eosinophils (results not shown). These values ​​were within the expected range for animals of this species, sex, and age undergoing similar experimental procedures. Any variations from the normal ranges described were sporadic in nature, present in only one sex, and not related to dose level or timing. [Table 12] [Table 13] [Table 14]

[0228] There were no significant changes in body weight over the course of the study for all groups (data not shown).

[0229] Overall results show that synthetic consensus survivin administered alone is capable of inducing an immune response in 100% of NHPs. The addition of IL-12 adjuvant improved the responses seen with synthetic consensus survivin, resulting in earlier and greater PD2 responses, but only with higher doses of IL-12.

[0230] It is understood that the foregoing detailed description and the accompanying examples are merely illustrative and are not to be construed as limitations on the scope of the invention, which is defined solely by the appended claims and their equivalents.

[0231] Various changes and modifications to the embodiments of the present disclosure will be apparent to those skilled in the art. Such changes and modifications to the embodiments of the present disclosure, including but not limited to those related to the chemical structures, substituents, derivatives, intermediates, syntheses, compositions, formulations, or methods of use of the present invention, can be made without departing from the spirit and scope thereof. [1] (a) a nucleic acid sequence encoding amino acids 19 to 159 of SEQ ID NO: 2; (b) a nucleic acid sequence encoding amino acids 19 to 210 of SEQ ID NO:4; (c) a nucleic acid sequence encoding amino acids 19 to 232 of SEQ ID NO:8; (d) a nucleic acid sequence encoding a fragment comprising at least 90% of the entire length of amino acids 19 to 159 of SEQ ID NO:2; (e) a nucleic acid sequence encoding a fragment comprising at least 90% of the entire length of amino acids 19 to 210 of SEQ ID NO:4; (f) a nucleic acid sequence encoding a fragment comprising at least 90% of the entire length of amino acids 19 to 232 of SEQ ID NO:8; (g) a nucleic acid sequence encoding a protein that is at least 95% identical to amino acids 19 to 159 of SEQ ID NO:2; (h) a nucleic acid sequence encoding a protein that is at least 95% identical to amino acids 19 to 210 of SEQ ID NO:4; (i) a nucleic acid sequence encoding a protein that is at least 95% identical to amino acids 19 to 232 of SEQ ID NO:8; (j) a nucleic acid sequence encoding a fragment comprising at least 90% of the entire length of a protein that is at least 95% identical to amino acids 19 to 159 of SEQ ID NO:2; (k) a nucleic acid sequence encoding a fragment comprising at least 90% of the entire length of a protein that is at least 95% identical to amino acids 19 to 210 of SEQ ID NO:4; and (l) a nucleic acid molecule comprising one or more nucleic acid sequences selected from the group consisting of nucleic acid sequences encoding a fragment comprising at least 90% of the full length of a protein that is at least 95% identical to amino acids 19 to 232 of SEQ ID NO:8. [2] (a) nucleotides 55 to 423 of SEQ ID NO:1; (b) nucleotides 55 to 636 of SEQ ID NO:3; (c) a fragment comprising at least 90% of the entire length of nucleotides 55 to 423 of SEQ ID NO:1; (d) a fragment comprising at least 90% of the entire length of nucleotides 55 to 636 of SEQ ID NO:3; (e) a fragment that is at least 95% identical to nucleotides 55 to 423 of SEQ ID NO:1; (f) a fragment that is at least 95% identical to nucleotides 55 to 636 of SEQ ID NO:3; (g) a fragment comprising at least 90% of a nucleic acid sequence that is at least 95% identical to nucleotides 55 to 423 of SEQ ID NO:1; and (h) a nucleic acid molecule comprising one or more nucleic acid sequences selected from the group consisting of a fragment comprising at least 90% of a nucleic acid sequence that is at least 95% identical to nucleotides 55 to 636 of SEQ ID NO:3. [3] (a) a nucleic acid sequence encoding SEQ ID NO:2; (b) a nucleic acid sequence encoding SEQ ID NO:4; (c) a nucleic acid sequence encoding SEQ ID NO:8; (d) a nucleic acid sequence encoding a fragment comprising at least 90% of the entire length of SEQ ID NO:2; (e) a nucleic acid sequence encoding a fragment comprising at least 90% of the entire length of SEQ ID NO:4; (f) a nucleic acid sequence encoding a fragment comprising at least 90% of the entire length of SEQ ID NO:8; (g) a nucleic acid sequence encoding a protein that is at least 95% identical to SEQ ID NO:2; (h) a nucleic acid sequence encoding a protein that is at least 95% identical to SEQ ID NO:4; (i) a nucleic acid sequence encoding a protein that is at least 95% identical to SEQ ID NO:8; (j) a nucleic acid sequence encoding a fragment comprising at least 90% of the entire length of a protein that is at least 95% identical to SEQ ID NO:2; (k) a nucleic acid sequence encoding a fragment comprising at least 90% of the entire length of a protein that is at least 95% identical to SEQ ID NO:4; and (l) a nucleic acid molecule comprising one or more nucleic acid sequences selected from the group consisting of nucleic acid sequences encoding a fragment comprising at least 90% of the entire length of a protein that is at least 95% identical to SEQ ID NO:8. [4] (a) SEQ ID NO:1, (b) SEQ ID NO:3, (c) a fragment comprising at least 90% of the entire length of SEQ ID NO:1; (d) a fragment comprising at least 90% of the entire length of SEQ ID NO:3; (e) a fragment that is at least 95% identical to SEQ ID NO:1; (f) a fragment that is at least 95% identical to SEQ ID NO:3; (g) a fragment comprising at least 90% of the entire length of a nucleic acid sequence that is at least 95% identical to SEQ ID NO:1; and (h) a nucleic acid molecule comprising one or more nucleic acid sequences selected from the group consisting of a fragment comprising at least 90% of the entire length of a nucleic acid sequence that is at least 95% identical to SEQ ID NO:3. [5] A nucleic acid molecule comprising the nucleic acid sequence shown in SEQ ID NO:1. [6] A nucleic acid molecule comprising the nucleic acid sequence shown in SEQ ID NO:3. [7] A nucleic acid molecule described in any one of claims 1 to 6 for use as a pharmaceutical. [8] A nucleic acid molecule described in any one of claims 1 to 7 for use as a drug in the treatment of cancer. [9] A nucleic acid molecule described in any one of claims 1 to 6 for use in preparing a pharmaceutical agent.

[10] A nucleic acid molecule described in any one of claims 1 to 6 and 9 for use in the preparation of a medicament for the treatment of cancer.

[11] A vector comprising a nucleic acid molecule described in any one of claims 1 to 10.

[12] The vector of claim 11, comprising a plasmid or a viral vector.

[13] A composition comprising one or more nucleic acid molecules described in any one of claims 1 to 10.

[14] The composition of claim 13, comprising a pharma- ceutically acceptable carrier.

[15] A composition comprising one or more vectors as described in claim 11 or 12.

[16] (a) amino acids 19 to 159 of SEQ ID NO: 2; (b) amino acids 19 to 210 of SEQ ID NO:4; (c) amino acids 19 to 232 of SEQ ID NO:8; (d) a fragment comprising at least 90% of the entire length of amino acids 19 to 159 of SEQ ID NO: 2; (e) a fragment comprising at least 90% of the entire length of amino acids 19 to 210 of SEQ ID NO: 4; (f) a fragment comprising at least 90% of the entire length of amino acids 19 to 232 of SEQ ID NO:8; (g) an amino acid sequence that is at least 95% identical to amino acids 19 to 159 of SEQ ID NO:2; (h) an amino acid sequence that is at least 95% identical to amino acids 19 to 210 of SEQ ID NO:4; (i) an amino acid sequence that is at least 95% identical to amino acids 19 to 232 of SEQ ID NO:8; (j) a fragment containing at least 90% of the entire length of an amino acid sequence that is at least 95% identical to amino acids 19 to 159 of SEQ ID NO: 2; (k) a fragment containing at least 90% of the entire length of an amino acid sequence that is at least 95% identical to amino acids 19 to 210 of SEQ ID NO: 4; (l) a protein comprising an amino acid sequence selected from the group consisting of fragments comprising at least 90% of the entire length of an amino acid sequence that is at least 95% identical to amino acids 19 to 232 of SEQ ID NO:8.

[17] (a) SEQ ID NO: 2; (b) SEQ ID NO:4; (c) SEQ ID NO:8; (d) a fragment comprising at least 90% of the entire length of SEQ ID NO:2; (e) a fragment comprising at least 90% of the entire length of SEQ ID NO:4; (f) a fragment comprising at least 90% of the entire length of SEQ ID NO:8; (g) an amino acid sequence that is at least 95% identical to SEQ ID NO:2; (h) an amino acid sequence that is at least 95% identical to SEQ ID NO:4; (i) an amino acid sequence that is at least 95% identical to SEQ ID NO:8; (j) a fragment comprising at least 90% of the entire length of an amino acid sequence that is at least 95% identical to SEQ ID NO:2; (k) a fragment comprising at least 90% of the entire length of an amino sequence that is at least 95% identical to SEQ ID NO:4; and (l) a protein comprising an amino acid sequence selected from the group consisting of a fragment comprising at least 90% of the entire length of an amino acid sequence that is at least 95% identical to SEQ ID NO:8.

[18] A protein comprising the amino acid sequence shown in SEQ ID NO:2.

[19] A protein comprising the amino acid sequence shown in SEQ ID NO:4.

[20] A protein comprising the amino acid sequence shown in SEQ ID NO:8.

[21] A vaccine comprising a nucleic acid molecule described in any one of claims 1 to 10.

[22] A vaccine comprising the vector described in claim 11 or 12.

[23] A vaccine described in any one of claims 18 to 20, further comprising a pharma- ceutically acceptable excipient.

[24] A vaccine described in any one of claims 21 to 23, further comprising an adjuvant.

[25] The vaccine of claim 24, wherein the adjuvant is IL-12, IL-15, IL-28, or RANTES.

[26] A method for treating a subject having cancerous cells that express survivin, comprising administering a therapeutically effective amount of a vaccine described in any one of claims 20 to 25.

[27] The method of claim 26, wherein the administration includes an electroporation step.

[28] The method of claim 26 or 27, wherein administration is at one or more sites in the subject.

[29] A method for vaccinating a subject against cancerous cells expressing survivin, comprising administering a vaccine described in any one of claims 20 to 25 in an amount effective to induce a humoral or cellular immune response.

Claims

1. WPFLEGCACAPERMAEAGFIHCPAENEPDLAQCFFCFKELEGWEPDDDPIEEHKKHSSGAAFLSVKKQFEELTLSEF A nucleic acid molecule encoding a synthetic consensus survivin antigen comprising the amino acid sequences of SEQ ID NO:5 and SEQ ID NO:6 separated by a polypeptide consisting of the amino acid sequence of SEQ ID NO:

6.

2. A nucleic acid molecule encoding a synthetic consensus survivin antigen comprising the amino acid sequences of SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, SEQ ID NO:5 and SEQ ID NO:6 are as follows: WPFLEGCACAPERMAEAGFIHCPAENEPDLAQCFFCFKELEGWEPDDDPIEEHKKHSSGAAFLSVKKQFEELTLSEF and separated by a polypeptide consisting of the amino acid sequence SEQ ID NO:6 and SEQ ID NO:7 are as follows: EFEETAKKVRCAIEQLAAMDRGRKRRSMQRKPTIRRKNLRKLRRKCAVPSSSWLPWTEASGWSCLVP The nucleic acid molecule is separated by a polypeptide consisting of the amino acid sequence of

3. WPFLEGCACAPERMAEAGFIHCPAENEPDLAQCFFCFKELEGWEPDDDPIEEHKKHSSGAAFLSVKKQFEELTLSEF A synthetic consensus survivin antigen comprising the amino acid sequences of SEQ ID NO:5 and SEQ ID NO:6 separated by a polypeptide consisting of the amino acid sequence of SEQ ID NO:

6.

4. A synthetic consensus survivin antigen comprising the amino acid sequences of SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, SEQ ID NO:5 and SEQ ID NO:6 are as follows: WPFLEGCACAPERMAEAGFIHCPAENEPDLAQCFFCFKELEGWEPDDDPIEEHKKHSSGAAFLSVKKQFEELTLSEF and a polypeptide comprising the amino acid sequence of SEQ ID NO:6 and SEQ ID NO:7 are as follows: EFEETAKKVRCAEQLAMDRGKRSMQRKPTIRRKNRRKRKCAVPSWLPWTEASGWSCLVP A synthetic consensus survivin antigen separated by a polypeptide consisting of the amino acid sequence of

5. 3. A nucleic acid molecule according to claim 1 or 2 for use as a medicament.

6. 3. A nucleic acid molecule according to claim 1 or 2 for use as a medicament in the treatment of cancer.

7. 3. A nucleic acid molecule according to claim 1 or 2 for use in the manufacture of a medicament.

8. 3. A nucleic acid molecule according to claim 1 or 2 for use in the preparation of a medicament for the treatment of cancer.

9. A vector comprising the nucleic acid molecule of claim 1 or 2.

10. The vector according to claim 9, which comprises a plasmid or a viral vector.

11. A composition comprising one or more nucleic acid molecules according to claim 1 or 2.

12. The composition of claim 11 comprising a pharma- ceutically acceptable carrier.

13. A composition comprising one or more vectors according to claim 9 or 10.

14. A vaccine comprising the nucleic acid molecule of claim 1 or 2.

15. A vaccine comprising the vector of claim 9 or 10.

16. 16. The vaccine of claim 14 or 15, further comprising a pharma- ceutically acceptable excipient.

17. The vaccine of any one of claims 14 to 16, further comprising an adjuvant.

18. 18. The vaccine of claim 17, wherein the adjuvant is IL-12, IL-15, IL-28, or RANTES.

19. A vaccine according to any one of claims 14 to 18 for use in the treatment of a survivin-expressing cancer.

20. A vaccine according to any one of claims 14 to 18 for use in vaccination against survivin-expressing cancer.

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