Cancer vaccines targeting survivin 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 the benefit and priority of U.S. Provisional Patent Application No. 62 / 598,267, filed on December 13, 2017, the disclosure of which is hereby incorporated by reference in its entirety.

[0002] Sequence Listing This application includes a sequence listing submitted electronically in ASCII format, which is hereby incorporated by reference in its entirety. The ASCII copy was created on December 13, 2018, has the name 104409_000448_sequence_listing.txt, and is 8,797 bytes in size.

[0003] The present invention relates to survivin antigen and nucleic acid molecules encoding the same. The present invention also relates to vaccines comprising such survivin antigen and / or nucleic acid molecules. The present invention further relates to methods of using the vaccines for inducing an immune response and for preventing and / or treating a subject having cancer cells and / or tumors that express survivin.

Background Art

[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 arises from a single cell that has transformed from a normal cell into a cancerous cell. Such transformation is often a multi - step process that progresses from a pre - cancerous 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 light 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), modification of behavior (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 baculoviral apoptosis inhibitor repeat-containing protein 5 (BIRC5), is an apoptosis inhibitor that blocks caspase function and thereby prevents programmed cell death. In addition to its role in apoptosis, Survivin clearly has an evolutionarily conserved role that is essential 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, treatment 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 various 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 tested positive for Survivin expression ranged from 74% to 92%.(See Cohen, C., Lohmann, C. M., 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)). The contribution of survivin to tumorigenesis, in combination with its restricted pattern of expression and overexpression in various tumors, has made it an interesting 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. Its protein structure also lacks the 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, and survivin isoform 1 is the major transcript. Survivin is expressed during fetal development but not in fully differentiated tissues. However, it is highly expressed in many cancer cells. Therefore, survivin is a potential target antigen for cancer therapy.

Summary of the Invention

Problems to be Solved by the Invention

[0008] Vaccines for the treatment and prevention of cancer, particularly epithelial ovarian cancer (EOC), are of interest. However, existing vaccines targeting tumor cell antigens are limited by insufficient antigen expression in vivo. Therefore, there is still a need in the art for safe and effective vaccines and methods of using them for preventing and / or treating cancer and reducing the mortality rate of subjects suffering from cancer.

Means for Solving the Problems

[0009] (a) A nucleic acid sequence encoding amino acids 19 - 159 of SEQ ID NO: 2, (b) a nucleic acid sequence encoding amino acids 19 - 210 of SEQ ID NO: 4, (c) a nucleic acid sequence encoding amino acids 19 - 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 - 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 - 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 - 232 of SEQ ID NO: 8, (g) a nucleic acid sequence encoding a protein that is at least 95% identical to amino acids 19 - 159 of SEQ ID NO: 2, (h) a nucleic acid sequence encoding a protein that is at least 95% identical to amino acids 19 - 210 of SEQ ID NO: 4, (i) a nucleic acid sequence encoding a protein that is at least 95% identical to amino acids 19 - 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 - 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 - 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 - 232 of SEQ ID NO: 8. A nucleic acid molecule comprising one or more nucleic acid sequences selected from the group consisting thereof is provided herein.

[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 full length of nucleotides 55 - 423 of SEQ ID NO: 1, (d) a fragment comprising at least 90% of the full 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 comprising 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 comprising 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 full length of SEQ ID NO: 1; (d) a fragment comprising at least 90% of the full 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 full length of a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 1; and (h) a fragment comprising at least 90% of the full 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 set forth in SEQ ID NO: 1.

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

[0015] The nucleic acid molecule described herein is used as a drug.

[0016] The nucleic acid molecules described in this specification are used as agents in the treatment of cancer.

[0017] The nucleic acid molecules described in this specification are used in the preparation of agents.

[0018] The nucleic acid molecules described in this specification are used in the preparation of agents for the treatment of cancer.

[0019] The vector contains the nucleic acid molecules described in this specification.

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

[0021] The composition contains one or more nucleic acid molecules described in this specification.

[0022] The composition described in this specification contains a pharmaceutically acceptable carrier.

[0023] The composition described in this specification contains one or more vectors described in this specification.

[0024] The protein comprises an amino acid sequence 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 containing at least 90% of the full length of amino acids 19 to 159 of SEQ ID NO: 2; (e) a fragment containing at least 90% of the full length of amino acids 19 to 210 of SEQ ID NO: 4; (f) a fragment containing 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; (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 full 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 full 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 containing at least 90% of the full 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 containing at least 90% of the full length of SEQ ID NO: 2; (e) a fragment containing at least 90% of the full length of SEQ ID NO: 4; (f) a fragment containing at least 90% of the full 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 containing at least 90% of the full length of an amino acid sequence that is at least 95% identical to SEQ ID NO: 2; (k) a fragment containing at least 90% of the full length of an amino acid sequence that is at least 95% identical to SEQ ID NO: 4; and (l) a fragment containing at least 90% of the full length of an amino acid sequence that is at least 95% identical to SEQ ID NO: 8.

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

[0027] The protein contains the amino acid sequence set forth in SEQ ID NO: 4.

[0028] The protein contains the amino acid sequence set forth in SEQ ID NO: 8.

[0029] The vaccine contains the nucleic acid molecule described herein.

[0030] The vaccine contains the vector described herein.

[0031] The vaccine described herein further contains a pharmaceutically acceptable excipient.

[0032] The vaccine described herein further contains an adjuvant.

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

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

[0035] In the method described herein, the administration includes an electroporation step.

[0036] The method described herein is performed at one or more sites of the subject.

[0037] A method for vaccinating a subject against cancerous cells that express survivin includes administering an amount of the vaccine described herein effective 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 present invention, exemplary embodiments of the invention are shown in the drawings, however, the invention is not limited to the specific methods, compositions, and devices disclosed. Drawings:

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[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 cancers or cancer-based tumors that express survivin.

[0040] The synthetic consensus survivin antigen can be a consensus survivin antigen derived from the sequences 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 can be further modified by introducing one or more mutations into the consensus sequence to generate a synthetic consensus sequence. The mutations can block or modify specific functional domains of the native survivin sequence, thereby disrupting or enhancing the structure or function of the functional domains. 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 can have a furin cleavage site. The synthetic consensus survivin antigen sequence can include additional localization signals to enhance the extracellular transport of the final protein product. The additional localization signals can be IgELS or other cell transport sequences.

[0041] The synthetic consensus survivin antigen can induce antigen-specific T cells and / or high-titer antibody responses, thereby inducing or eliciting an immune response that is direct or reactive against cancers or tumors expressing the antigen. In some embodiments, the induced or elicited immune response can be a cellular, humoral, or both cellular and humoral immune response. In some embodiments, the induced or elicited cellular immune response can include the induction or secretion of interferon-gamma (IFN-γ) and / or tumor necrosis factor alpha (TNF-α) 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 tumors or cancers expressing the antigen, such as, but not limited to, factors that downregulate MHC presentation, antigen-specific regulatory T cells (Tregs), cytokines such as PD-L1, FasL, IL-10, and TGF-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 vaccine of the present invention can provide any combination of specific cancer antigens for the specific prevention or treatment of cancer in a subject in need of treatment.

[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. Introducing mutations cannot be universally applied across mammalian subjects, preferably human or canine subjects, because it does not modify the cancer antigen too much. However, the resulting amino acid sequence is sufficiently altered to break tolerance or be regarded as a foreign antigen in order to generate an immune response. Another way can be to create a consensus recombinant cancer antigen that has at least 85% to a maximum of 99% amino acid sequence identity, preferably at least 90% to a maximum of 98% sequence identity, more preferably at least 93% to a maximum of 98% sequence identity, or even more preferably at least 95% to a maximum of 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. A native cancer antigen is an antigen that is normally associated with a specific cancer or tumor. Depending on the cancer antigen, the consensus sequence of the cancer antigen can span across all mammalian species, or within a subtype of a species, or across all viral strains, or serotypes. Some cancer antigens do not differ greatly from the wild-type amino acid sequence of the cancer antigen. Some cancer antigens have diverse nucleic acid / amino acid sequences across species and cannot generate a consensus sequence. In these cases, the recombinant cancer antigen is generated by breaking tolerance and generates an immune response that has at least 85% to a maximum of 99% amino acid sequence identity, preferably at least 90% to a maximum of 98% sequence identity, more preferably at least 93% to a maximum of 98% sequence identity, or even more preferably at least 95% to a maximum of 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. By combining the aforementioned approaches, the final recombinant cancer antigen can have a percentage similarity with the native cancer antigen amino acid sequence discussed above.

[0044] The vaccine can further enhance the stimulation of both cellular and humoral immune responses when further combined with antibodies against checkpoint inhibitors such as PD-1 and PDL-1. The use of anti-PD-1 or anti-PDL-1 antibodies prevents PD-1 or PDL-1 from suppressing T cell and / or B cell responses. Overall, designing cancer antigens to be recognized by the immune system helps to overcome other forms of immunosuppression 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 bone marrow-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 the 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, as compared to the cellular immune response in the non-vaccinated subject. In some embodiments, the vaccine can enhance the cellular immune response in the 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-fold, 5500-fold, 5600-fold, 5700-fold, 5800-fold, 5900-fold, or 6000-fold, as compared to the cellular immune response in the non-vaccinated subject.

[0047] The vaccine can enhance the interferon gamma (IFN-γ) level in the 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 as compared to the IFN-γ level in the non-vaccinated subject. In some embodiments, the vaccine can enhance the IFN-γ level in the 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-fold, 5500-fold, 5600-fold, 5700-fold, 5800-fold, 5900-fold, or 6000-fold as compared to the IFN-γ level in the non-vaccinated subject.

[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 the proliferation and / or differentiation of immune cells. As described in more detail below, the vaccine can further be combined with antibodies against checkpoint inhibitors such as PD-1 and PDL-1 to enhance the stimulation of both cellular and humoral immune responses. The use of anti-PD-1 or anti-PDL-1 antibodies prevents PD-1 or PDL-1 from suppressing T cell and / or B cell responses.

[0049] Definitions Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, but methods and materials similar or equivalent to those described herein can 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 illustrative only and not intended to be limiting. The terms used herein are for the purpose of describing particular 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 phrases, terms, or words that do not preclude additional acts or structures. The singular forms "a", "an", 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 explicitly described.

[0051] For the recitation of numerical ranges herein, each intervening value having the same degree of precision as the recited minimum and maximum of the range is explicitly contemplated. For example, in the range of 6-9, the numerical values 7 and 8 are contemplated in addition to 6 and 9, and in the range of 6.0-7.0, the numerical 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 explicitly 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" means an antibody of 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 can be an antibody isolated from a mammalian serum sample, a polyclonal antibody, an affinity-purified antibody, or any mixture thereof, and exhibits sufficient binding specificity to the desired epitope or sequence from which it is derived.

[0054] "Antigen" refers to a protein having a synthetic consensus survivin antigen amino acid sequence comprising: (a) amino acids 19-159 of SEQ ID NO: 2; (b) amino acids 19-210 of SEQ ID NO: 4; (c) amino acids 19-232 of SEQ ID NO: 8; (d) a fragment comprising at least 90% of amino acids 19-159 of SEQ ID NO: 2; (e) a fragment comprising at least 90% of amino acids 19-210 of SEQ ID NO: 4; (f) a fragment comprising at least 90% of amino acids 19-232 of SEQ ID NO: 8; (g) a protein that is at least 95% identical to amino acids 19-159 of SEQ ID NO: 2; (h) a protein that is at least 95% identical to amino acids 19-210 of SEQ ID NO: 4; (i) a protein that is at least 95% identical to amino acids 19-232 of SEQ ID NO: 8; (j) a fragment comprising at least 90% of a protein that is at least 95% identical to amino acids 19-159 of SEQ ID NO: 2; (k) a fragment comprising at least 90% of a protein that is at least 95% identical to amino acids 19-210 of SEQ ID NO: 4; and (l) a fragment comprising at least 90% of a protein that is at least 95% identical to amino acids 19-232 of SEQ ID NO: 8. "Antigen" also refers to a protein having a synthetic consensus survivin antigen amino acid sequence comprising: (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. The antigen may optionally contain a signal peptide, such as from another protein.

[0055] As used herein, "coding sequence" or "coding nucleic acid" means a nucleic acid (RNA or DNA molecule) that includes a nucleotide sequence encoding a protein. The coding sequence may further include start and stop signals operably linked to control elements, including a promoter and a polyadenylation signal capable of directing expression in a cell of a subject or mammal to which the nucleic acid is administered.

[0056] As used herein with respect to nucleic acids, "complementary" or "complement" can mean Watson-Crick (e.g., A-T / U and C-G) or Hoogsteen base pairing between nucleotides or nucleotide analogs of a nucleic acid molecule.

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

[0058] As used herein, "constant current" represents the current received or experienced by a tissue or the 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 described herein. The electroporation devices provided herein preferably have a feedback element with instantaneous feedback, such that this current remains at a constant number of amperes within the tissue over the lifetime of the electrical pulse. The feedback element can measure the resistance of the tissue (or cell) over the duration of the pulse and can vary its electrical energy output (e.g., increase the voltage) to the electroporation device, such that the current in the same tissue remains constant (on the order of microseconds) throughout the electrical pulse and also 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 mean the active response of the provided electroporation device, which includes measuring the current of the tissue between the electrodes and changing the energy output supplied by the EP device accordingly to maintain the current at a certain level. This certain level is preset by the user before the start of the pulse sequence or electrotherapy. Feedback can be achieved by an electroporation component of the electroporation device, such as a controller, where the electrical circuit continuously monitors the current of the tissue between the electrodes, compares the monitored current (or the current in the tissue) with a preset current, and continuously adjusts the energy output to maintain the monitored current at the preset level. Since the feedback loop is an analog closed-loop feedback, it can be instantaneous.

[0060] As used herein, "dispersed current" may mean the pattern of currents delivered from various needle electrode arrays of the electroporation devices described herein, where the pattern minimizes, or preferably eliminates, the generation of electroporation-related thermal stress in any region of the tissue being electroporated.

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

[0062] As used herein with respect to a nucleic acid sequence, "fragment" means a nucleic acid sequence or a portion thereof that encodes a polypeptide capable of inducing an immune response in a mammal that cross-reacts with an antigen disclosed herein. The fragment can be a DNA fragment selected from at least one of the various nucleotide sequences encoding the protein fragments shown below. The fragment can comprise at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of one or more of the nucleic acid sequences shown below, excluding any added heterologous signal peptide. The fragment can 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 the percent identity. The fragment can 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 can be ligated to the fragment of the coding sequence.

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

[0064] "Fragment" or "immunogenic fragment" with respect to a polypeptide sequence means a polypeptide capable of inducing an immune response in a mammal that cross-reacts with the antigens disclosed herein. The fragment can be a polypeptide fragment selected from at least one of the following various amino acid sequences. A fragment of a consensus protein can contain at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the consensus protein, excluding any added heterologous signal peptide. The fragment can contain at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of one or more of the amino sequences shown below, and optionally further contains a heterologous signal peptide, which need not be included when calculating the percent identity. The fragment can further contain an immunoglobulin signal peptide, such as an IgE or IgG signal peptide.

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

[0066] As used herein, the term "gene construct" refers to a DNA or RNA molecule that contains a nucleotide sequence encoding a protein. The coding sequence includes initiation and termination signals operably linked to regulatory elements, including a promoter and a polyadenylation signal, capable of directing expression in the cells of the subject to which the nucleic acid molecule is administered. As used herein, the term "expressible form" refers to a gene construct that, when present in the cells of a subject, contains the necessary regulatory elements operably linked to a coding sequence that encodes a protein such that the coding sequence is expressed.

[0067] As used herein, the term "homology" refers to a degree of complementarity. There can be partial homology or complete homology (i.e., identity). A partially complementary sequence that at least partially inhibits the hybridization of a completely complementary sequence to a target nucleic acid is referred to using the functional term "substantially homologous." When used with respect 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 with respect to a single-stranded nucleic acid sequence, the term "substantially homologous" as used herein refers to a probe that can hybridize to a single-stranded nucleic acid template sequence under low stringency conditions (i.e., its complement).

[0068] As used herein in the context of two or more nucleic acid or polypeptide sequences, "identical" or "identity" means that the sequences have a specified percentage of residues that are the same over a particular region. The percentage can be calculated by optimally aligning the two sequences, comparing the two sequences over the specified region, determining the number of positions at which the 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 lengths of the two sequences differ, or if the alignment generates one or more staggered ends and the specified comparison region includes only a single sequence, the residues of the single sequence are included in the denominator 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 computer sequence algorithms such as BLAST or BLAST 2.0.

[0069] As used herein, "impedance" can be used when considering a feedback mechanism and can be converted into a current value according to Ohm's law, enabling comparison with a preset current.

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

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

[0072] A nucleic acid can be single-stranded or double-stranded, or can contain portions of both double-stranded and single-stranded sequences. A nucleic acid can be DNA, RNA, or a hybrid of both genomic and cDNA, and a nucleic acid can contain combinations of deoxyribonucleotides and ribonucleotides, and the combination of bases includes uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine. A nucleic acid 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 a promoter to which it is spatially connected. A promoter can be located 5' (upstream) or 3' (downstream) of the gene under its control. The distance between the promoter and the gene can be approximately the same as the distance between that promoter and the gene it controls in the gene from which the promoter is derived. As is known in the art, variations in this distance can be accommodated without loss of promoter function.

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

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

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

[0077] As used herein, "stringent hybridization conditions" means 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 can be selected to be about 5-10 °C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm can be the temperature (under defined ionic strength, pH, and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (since the target sequence is present in excess, at the 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 at pH 7.0-8.3, such as about 0.01-1.0 M sodium ion concentration (or other salts), and the temperature is at least about 30 °C for short probes (e.g., about 10-50 nucleotides) and at least about 60 °C for long probes (e.g., greater than about 50 nucleotides). Stringent conditions can also be achieved by the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal can be at least 2-10 times background hybridization. Exemplary stringent hybridization conditions include: 50% formamide, 5x SSC, and 1% SDS, incubate at 42 °C, or 5x SSC, 1% SDS, incubate at 65 °C, wash at 65 °C with 0.2x SSC, and 0.1% SDS.

[0078] As used herein, "subject" can mean a mammal that desires or requires immunization with the vaccines described herein. The mammal can be a human, chimpanzee, dog, cat, horse, cow, mouse, or rat.

[0079] As used herein, "substantially complementary" means that the first sequence is at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the complement of the second sequence 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, or that the two sequences hybridize under stringent hybridization conditions.

[0080] As used herein, "substantially identical" means that the first and second sequences are at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 180, 270, 360, 450, 540 or more nucleotides or amino acids, or, with respect to nucleic acids, that the first sequence is substantially complementary to the complement of the second sequence.

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

[0082] As used herein with respect to nucleic acids, "variant" 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 the reference nucleic acid or its complement, or (iv) a nucleic acid that hybridizes under stringent conditions to the reference nucleic acid, its complement, or a sequence substantially identical thereto.

[0083] As used herein with respect to a peptide or polypeptide, "variant" means a peptide or polypeptide whose amino acid sequence differs by amino acid insertions, deletions, or conservative substitutions, but which retains at least one biological activity. A variant can also mean a protein having an amino acid sequence that is substantially identical to a reference protein having an amino acid sequence that retains at least one biological activity. Conservative substitution of an amino acid, i.e., replacement of an amino acid with a different amino acid having similar properties (e.g., hydrophilicity, degree, and distribution of charged regions), is typically recognized in the art as involving minor changes. These minor changes can be identified in part by considering the hydrophobic-hydrophilic index of the amino acids, as understood in the art. Kyte et al., J. Mol. Biol. 157:105-132 (1982). The hydrophobic-hydrophilic index of an amino acid is based on considering its hydrophobicity and charge. It is known in the art that amino acids with similar hydrophobic-hydrophilic indices can be substituted and still retain protein function. In one embodiment, amino acids having a hydrophobic-hydrophilic index of ±2 are substituted. It is also possible to use the hydrophilicity of amino acids to identify substitutions that will result in a protein that retains biological function. In the context of a peptide, considering the hydrophilicity of amino acids allows for the calculation of the maximum local average hydrophilicity of the peptide, a useful means that has been reported to correlate well with antigenicity and immunogenicity. U.S. Patent No. 4,554,101, which is hereby incorporated by reference in its entirety. By substituting amino acids having similar hydrophilicity values, a peptide that retains biological activity, such as immunogenicity, can be obtained, as understood in the art. The substitutions can be made with amino acids having hydrophilicity values within ±2 of each other. Both the hydrophobic-hydrophilic index and the hydrophilicity value of an amino acid are affected by the specific side chain of that amino acid. Consistent with that observation, amino acid substitutions that are compatible with biological function are understood to depend on the relative similarity of the amino acids and in particular the side chains of those amino acids, as revealed by hydrophobicity, hydrophilicity, charge, size, and other properties.

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

[0085] As used herein, "vector" means a nucleic acid sequence containing an origin of replication. The vector can be a viral vector, bacteriophage, bacterial artificial chromosome, or yeast artificial chromosome. The vector can be a DNA or RNA vector. The vector can be a self-replicating extrachromosomal vector, preferably a DNA plasmid. The vector can contain or can include one or more heterologous nucleic acid sequences.

[0086] Vaccine Provided herein are vaccines comprising a synthetic consensus survivin antigen, a nucleic acid molecule encoding the antigen, a nucleic acid molecule encoding a fragment of the antigen, a nucleic acid molecule encoding a variant of the antigen, or a nucleic acid molecule encoding 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 prophylactic immune response. The vaccine can include 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 comprising 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 comprising 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, and 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 comprising 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, and 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 comprising 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, such as cancer or tumors that express survivin. The vaccine can be used in a subject in need thereof to prevent and / or treat tumors that express survivin. The vaccine can induce a cellular and / or antibody response against survivin and tumors that express survivin.

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

[0093] The development of the cancer vaccine described herein involves identifying cancer antigens, such as survivin, which are tumor-associated ("cancer / testis", "C / T") antigens that are not recognized by the immune system. The identified cancer antigens are altered from self-antigens to foreign antigens in order to be recognized by the immune system. Redesigning the nucleic acid and amino acid sequences of the recombinant cancer antigen from self to foreign antigen disrupts the immune system's tolerance to the antigen. To break tolerance, several redesign means can be applied to the cancer antigen as described below.

[0094] Since the recombinant cancer antigen of the vaccine is not recognized as self, tolerance is broken. Breaking tolerance induces an antigen-specific T cell and / or high-titer antibody response, thereby inducing or eliciting an immune response that is direct or reactive against cancer or tumors expressing the antigen. In some embodiments, the induced or elicited immune response can be a cellular, humoral, or both cellular and humoral immune response. In some embodiments, the induced or elicited cellular immune response can include the induction or secretion of interferon-gamma (IFN-γ) and / or tumor necrosis factor alpha (TNF-α) and / or interleukin 2 (IL-2). In other embodiments, the induced or elicited immune response reduces or inhibits one or more immunosuppressive factors that promote the growth of tumors or cancers expressing the antigen, such as, but not limited to, factors that downregulate MHC presentation, antigen-specific regulatory T cells (Tregs), cytokines such as PD-L1, FasL, IL-10, and TGF-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 certain embodiments, the vaccine can mediate clearance or prevent the growth of tumor cells by (1) increasing cytotoxic T lymphocytes such as CD8 + and / or CD107a + (CTL) to attack and kill tumor cells, (2) increasing the T helper cell response, and / or (3) increasing the inflammatory response via IFN-γ, IL-2, and TFN-α, or preferably all of the foregoing.

[0096] The vaccine can be a DNA vaccine. DNA vaccines are disclosed in U.S. Patent Nos. 5,593,972, 5,739,118, 5,817,637, 5,830,876, 5,962,428, 5,981,505, 5,580,859, 5,703,055, and 5,676,594, which are hereby incorporated by reference in their entirety. The DNA vaccine can further contain an element or reagent that inhibits its integration into the chromosome.

[0097] The vaccine can contain RNA encoding a cancer antigen. The RNA vaccine can be introduced into cells.

[0098] The vaccine can be an attenuated live vaccine, a vaccine that uses a recombinant vector to deliver an antigen, a subunit vaccine, and a glycoprotein vaccine. For example, but not limited to these, the vaccine is described in U.S. Patent Nos. 4,510,245, 4,797,368, 4,722,848, 4,790,987, 4,920,209, 5,017,487, 5,077,044, 5,110,587, 5,112,749, 5,174,993, 5,223,424, 5,225,336, 5,240,703, 5,242,829, 5,294,441, 5,294,548, 5,310,668, 5,387,744, 5,389,368, 5,424,065, 5,451,499, 5,453,364, 5,462,734, 5,470,734, 5,474,935, 5,482,713, 5,591,439, 5,643,579, 5,650,309, 5,698,202, 5,955,088, 6,034,298, 6,042,836, 6,156,319, and 6,589,529, each of which is hereby incorporated by reference in its entirety.

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

[0100] The vaccines of the present invention can have the characteristics required of 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 a protective T cell response, ease of administration, few side effects, biological stability, and low cost per dose. 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] Vaccine combined with an immune checkpoint inhibitor The vaccine can further comprise one or more inhibitors of one or more immune checkpoint molecules (i.e., immune checkpoint inhibitors). Immune checkpoint molecules are described in more detail below. An immune checkpoint inhibitor is any nucleic acid or protein that prevents the suppression of any 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 the proliferation and / or differentiation of immune cells.

[0102] Such inhibitors can be nucleic acid sequences, amino acid sequences, small molecules, or combinations thereof. The nucleic acid sequence can be DNA, RNA, cDNA, variants thereof, fragments thereof, or combinations thereof. The nucleic acid can also include additional sequences encoding 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 having a low molecular weight, e.g., less than 800 daltons, an enzyme substrate, a ligand (or an 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, a variant thereof, a fragment thereof, or combinations thereof.

[0103] In some embodiments, the immune checkpoint inhibitor can be one or more nucleic acid sequences encoding an antibody, a variant thereof, a fragment thereof, or combinations thereof. In other embodiments, the immune checkpoint inhibitor can be an antibody, a variant thereof, a fragment thereof, or combinations thereof.

[0104] Immune checkpoint molecule The immune checkpoint molecule can be a nucleic acid sequence, an amino acid sequence, a small molecule, or combinations thereof. The nucleic acid sequence can be DNA, RNA, cDNA, variants thereof, fragments thereof, or combinations thereof. The nucleic acid can also include additional sequences encoding 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 having a low molecular weight, e.g., less than 800 daltons, an enzyme substrate, a ligand (or an 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, a variant thereof, a fragment thereof, or combinations 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 in T cells and pro-B cells, thus contributing to the fate and / or differentiation of these cells. In particular, PD-1 is a type I membrane protein of the CD28 / CTLA-4 family of T cell regulators, negatively regulating T cell receptor (TCR) signals and thereby negatively regulating the immune response. Since PD-1 can negatively regulate CD8+ T cell responses, it inhibits CD8-mediated cytotoxicity and promotes tumor growth.

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

[0107] Anti-immune checkpoint molecule antibody As described above, the immune checkpoint inhibitor can be an antibody. The antibody can bind or react with an antigen, i.e., the immune checkpoint molecule described above. Thus, the antibody can be considered an anti-immune checkpoint molecule antibody or an immune checkpoint molecule antibody. The antibody can be encoded by the nucleic acid sequence contained therein.

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

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

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

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

[0112] An antibody can include a set of heavy and light chain complementarity determining regions (the "CDRs"), and is inserted between a set of heavy and light chain frameworks (the "FRs") that provide support for the CDRs and mutually define the spatial relationship of the CDRs. The CDR set can include three hypervariable regions of the heavy or light chain V region. In order from the N-terminus of the heavy or light chain, these regions are designated "CDR1", "CDR2", "CDR3", respectively. Thus, the antigen binding site can include six CDRs, including a CDR set from each of the heavy and light chain V regions.

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

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

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

[0116] PD-1 antibody An anti-immune checkpoint molecule antibody can be an anti-PD-1 antibody (also referred to herein as a “PD-1 antibody”), a variant thereof, a fragment thereof, or a combination thereof. The PD-1 antibody can be nivolumab. An anti-PD-1 antibody can inhibit PD-1 activity and thereby induce, elicit, or increase an immune response against a tumor or cancer and decrease tumor growth.

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

[0118] Antigen As described above, the vaccine can contain an antigen or a nucleic acid encoding the antigen. The antigen can be survivin, its fragment, its variant, or a combination of the fragment and its variant.

[0119] Therefore, the vaccine can be used to treat a subject suffering from a cancer or tumor that expresses 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 a subject having a cancer or tumor that expresses survivin to prevent the occurrence of such tumors in the subject. The synthetic consensus survivin antigen can be different from the native survivin gene, and thus provides therapy or prophylaxis against tumors expressing the synthetic consensus survivin antigen. Accordingly, synthetic consensus survivin antigen sequences that are different from the native survivin gene (i.e., mutant or synthetic survivin genes or sequences) are provided herein.

[0120] The transcripts of the native survivin gene are processed into various mRNAs. Specific survivin mRNA isoforms can be selected, for example, based on their expression in cancer cells. In certain embodiments, the survivin isoform is selected based on its expression in ovarian cancer cells. The synthetic consensus survivin antigen sequences described herein avoid alternative processing, generate one full-length transcript, and result in a stronger induction of effector T and B cell responses.

[0121] Isolated nucleic acid molecules comprising the heterologous sequences described above are provided. Isolated nucleic acid molecules consisting of the heterologous sequences described above are provided. The isolated nucleic acid molecules comprising the heterologous sequences described above can be incorporated into vectors such as plasmids, viral vectors, and other forms of nucleic acid molecules as described below. Nucleic acid sequences encoding a synthetic consensus survivin antigen are provided herein. The coding sequences encoding the synthetic consensus survivin antigen have sequences as described above.

[0122] Protein molecules comprising the heterologous amino acid sequences described above are provided. Protein molecules consisting of the heterologous amino acid sequences described above are provided. 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 inducing an immune response against tumors expressing survivin.

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

[0124] A 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, where the portion of the second isoform is, for example, non-homologous to any portion of the first isoform. The synthetic consensus survivin antigen can include consensus sequences and / or modifications for improved expression. Modifications include codon optimization, RNA optimization, addition of a Kozak sequence (e.g., GCC ACC) to enhance translation initiation, and / or addition of an immunoglobulin leader sequence to enhance the immunogenicity of the synthetic consensus survivin antigen. The synthetic consensus survivin antigen can include an immunoglobulin signal peptide, such as, but not limited to, an immunoglobulin E (IgE) or immunoglobulin G (IgG) signal peptide. In some embodiments, the synthetic consensus survivin antigen can include a localization signal sequence, such as a mutation or deletion to a nuclear localization signal to disrupt nuclear localization during translation. In some embodiments, the survivin consensus antigen can include a hemagglutinin (HA) tag. The survivin consensus antigen can be designed to induce a more potent and broad 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 specific structures and / or functions of native survivin to generate synthetic consensus survivin antigen sequences. In one embodiment, mutations are introduced to ablate the anti-apoptotic activity of survivin. In certain embodiments, the T34A, T48A, and C84A mutations are introduced into the consensus survivin isoform 1 sequence to ablate the anti-apoptotic function. (See Muchmore, S.W. et al. Crystal structure and mutagenic analysis of the inhibitor-of-apoptosis protein Survivin. Molecular cell 6, 173-182 (2000); O’Connor, D.S. et 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, R.M., Colnaghi, R. & Wheatley, S.P. Threonine 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, e.g., the dominant survivin isoform, or the consensus sequence can include a combination of a portion of the first isoform and a portion of the second isoform, or a truncated portion of the 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 preferred embodiments, the synthetic consensus survivin antigen sequence shares at least 95.0% identity with SEQ ID NO: 1 or SEQ ID NO: 3. In this embodiment, the nucleic acid sequences of SEQ ID NO: 1 or SEQ ID NO: 3 encode the amino acid sequences of SEQ ID NO: 2 or SEQ ID NO: 8, respectively. In other embodiments, the synthetic consensus survivin antigen sequence shares at least 95.0% identity, at least 95.2% identity, at least 95.4% identity, at least 95.6% identity, at least 95.8% identity, at least 96.0% identity, at least 96.2% identity, at least 96.4% identity, at least 96.6% identity, at least 96.8% identity, at least 97.0% identity, at least 97.2% identity, at least 97.4% identity, at least 97.6% identity, at least 97.8% identity, at least 98.0% identity, at least 98.2% identity, at least 98.4% identity, or at least 98.6% identity, at least 98.8% identity, at least 99.0% identity, at least 99.2% identity, at least 99.4% identity, at least 99.6% identity, at least 99.8% identity, or 100% identity with SEQ ID NO: 1 or SEQ ID NO: 3.

[0128] Vector The vaccine can comprise one or more vectors comprising a heterologous nucleic acid encoding 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 comprise a heterologous nucleic acid encoding the antigen. The vector can have a nucleic acid sequence comprising an origin of replication. The vector can be a plasmid, bacteriophage, bacterial artificial chromosome, or yeast artificial chromosome. The vector can be either a self-replicating episomal vector or a vector integrated into the host genome.

[0129] The one or more vectors can be expression constructs, which are generally plasmids used to introduce a specific gene into a target cell. When the expression vector enters the cell, the protein encoded by the gene is produced by the cell transcription and translation machinery ribosome complex. Plasmids often are designed to contain regulatory sequences that function as enhancer and promoter regions and lead 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 protein.

[0130] The vector can 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 can direct the expression of a specific nucleotide sequence in a suitable target cell. The vector can have a promoter operably linked to a nucleotide sequence encoding an antigen and can be operably linked to a termination signal. The vector can also contain sequences necessary for proper translation of the nucleotide sequence, as well as sequences for cloning and subcloning the vector and its fragments. A vector containing a nucleotide sequence of interest can be chimeric, which means that at least one of its components is heterologous with respect to at least one of the other components. Expression of the nucleotide sequence in the expression cassette can be under the control of a constitutive promoter or an inducible promoter, where transcription is initiated only when the host cell is exposed to certain specific external stimuli. In the case of multicellular organisms, the promoter can also be specific to a particular tissue or organ or developmental stage. In a preferred embodiment, the plasmid vector is pGX1428 described herein and further contains the nucleic acid sequence of SEQ ID NO: 1, or pGX1429 described herein and further contains the nucleic acid sequence of SEQ ID NO: 3.

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

[0133] The plasmid can contain a nucleic acid sequence encoding one or more of the various antigens disclosed above, including a consensus antigen capable of inducing an immune response against a synthetic antigen, a fragment of such a protein, a variant of such a protein, a fragment of a variant or fusion protein, which are composed of a combination of a consensus protein and / or a fragment of a consensus protein and / or a variant of a consensus protein and / or a fragment of a variant of a consensus protein.

[0134] A single plasmid can contain the coding sequence of a single antigen, the coding sequences of two antigens, the coding sequences of three antigens, or the coding sequences of four antigens.

[0135] In some embodiments, the plasmid can further comprise CCR20 alone or a coding sequence encoding it as part of these plasmids. Similarly, the plasmid can further comprise the coding sequences of IL-12, IL-15, and / or IL-28.

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

[0137] The plasmid can also include a promoter operably linked to the coding sequence. The promoter operably linked to the coding sequence can be a promoter from a human immunodeficiency virus (HIV) promoter such as the simian virus 40 (SV40), mouse mammary tumor virus (MMTV) promoter, bovine immunodeficiency virus (BIV) long terminal repeat (LTR) promoter, Moloney virus promoter, avian leukemia virus (ALV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter, Epstein - Barr virus (EBV) promoter, or Rous sarcoma virus (RSV) promoter. The promoter can also be a promoter from a human gene such as human actin, human myosin, human hemoglobin, human muscle creatine, or human metallothionein. The promoter can also be a tissue - specific promoter, such as a natural or synthetic, muscle - or skin - specific promoter. Examples of such promoters are described in U.S. Patent Application Publication No. US2004 / 0175727, the content of which is incorporated herein by reference in its entirety.

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

[0139] The plasmid may also contain an enhancer upstream of the coding sequence. The enhancer can be a viral enhancer such as human actin, human myosin, human hemoglobin, human muscle creatine, or those from CMV, FMDV, RSV, or EBV. Polynucleotide functional enhancers are described in U.S. Patent Nos. 5,593,972, 5,962,428, and W094 / 016737, the contents of each of which are incorporated by reference in their entirety.

[0140] The plasmid may also contain a mammalian origin of replication to maintain the plasmid episomally and produce multiple copies of the plasmid in the cell. The plasmid can 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 can generate high-copy episomal replication without integration. The backbone of the plasmid can be pA V0242. The plasmid can 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 the cells to which the plasmid is administered. The coding sequence can contain codons that may allow for more efficient transcription of the coding sequence in the host cell.

[0142] The encoded array may also include an Ig leader array. The leader array may be 5' of the coding array. The consensus antigen encoded by this array 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 can be pSE420 (Invitrogen, San Diego, Calif.), which can be used for protein production in Escherichia coli (E. coli). The plasmid can also be YES2 (Invitrogen, San Diego, Calif.), which can be used for protein production in the Saccharomyces cerevisiae strain of yeast. The plasmid can also be of the MAXBAC™ complete baculovirus expression system (Invitrogen, San Diego, Calif.), which can be used for protein production in insect cells. The plasmid can also be pcDNA I or pcDNA3 (Invitrogen, San Diego, Calif.), which can be used for protein production in mammalian cells such as Chinese hamster ovary (CHO) cells.

[0144] The vector can transform target cells by integrating into the cell genome or can be a circular plasmid that exists extrachromosomally (e.g., a self-replicating plasmid having an origin of replication).

[0145] The vector can be pVAX, pcDNA3.0, or provax, or any other expression vector capable of expressing the DNA encoding the antigen and enabling the cell to translate the sequence into an antigen recognized by the immune system.

[0146] Also provided herein are linear nucleic acid vaccines or linear expression cassettes ("LECs") that can be efficiently delivered to a subject by electroporation and express one or more desired antigens. The LEC can be any linear DNA lacking 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. Expression of the antigen can be controlled by the promoter. The LEC may not contain any antibiotic resistance gene and / or phosphate backbone. The LEC may not contain other nucleic acid sequences unrelated to the expression of the desired antigen gene.

[0147] The LEC can be derived from any plasmid that can be localized. The plasmid can be capable of expressing an 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 enable the cell to translate the sequence into an antigen recognized by the immune system.

[0148] The LEC can be pcrM2. The LEC can be pcrNP. 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 can be any promoter capable of driving gene expression and controlling the expression of the isolated nucleic acid. Such a promoter is a cis-acting sequence element necessary for transcription via DNA-dependent RNA polymerase that transcribes the antigen sequence described herein. The choice of promoter used to direct the expression of the heterologous nucleic acid depends on the particular application. The promoter can be placed at approximately the same distance from the transcription start within the vector, which is from the transcription start site in its natural setting. However, differences in this distance can be adjusted without loss of promoter function.

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

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

[0152] The vector can include enhancers and introns having functional splice donor and acceptor sites. The vector can contain a transcription termination region downstream of the structural gene to provide efficient termination. The termination region can be obtained from the same gene as the promoter sequence or from a different gene.

[0153] Method for preparing a vector A method for preparing a vector comprising a nucleic acid molecule encoding the synthetic consensus survivin antigen discussed herein is provided herein. The vector can be used to seed cell cultures in large-scale fermentation tanks using methods known in the art after the final subcloning step.

[0154] The vector is used with an EP device and can be formulated or manufactured using combinations of known devices and techniques, as described in more detail below, but is preferably manufactured using the optimized plasmid manufacturing techniques described in U.S. Provisional Patent Application No. 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 a concentration of 10 mg / mL or greater. The manufacturing techniques also include, in addition to those described in U.S. Patent Application No. 60 / 939,792, which includes those described in U.S. Patent No. 7,238,522, issued July 3, 2007, various devices and protocols generally known to those of skill in the art, or incorporate them. The applications and patents referenced above, U.S. Patent Application No. 60 / 939,792 and U.S. Patent No. 7,238,522, are hereby incorporated by reference in their entirety.

[0155] Vaccine excipients and other components The vaccine may further comprise a pharmaceutically acceptable excipient. Pharmaceutically acceptable excipients can be functional molecules such as vehicles, carriers, or diluents. Pharmaceutically acceptable excipients can be transfection promoters and can include surfactants such as immunostimulating 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 promoters.

[0156] The transfection promoter is a polyanion, a polycation (including poly-L-glutamate (LGS)), or a lipid. The transfection promoter is poly-L-glutamate, and the poly-L-glutamate can be present in the vaccine at a concentration of less than 6 mg / ml. The transfection promoter can also include immunostimulatory complexes (ISCOMs), Freund's incomplete adjuvant, monophosphoryl lipid A containing LPS analogs, muramyl peptides, quinone analogs, and surfactants such as vesicles such as squalene and squalene, and hyaluronic acid administered together with the gene construct can also be used. DNA vector vaccines can also include transfection promoters such as lipids, liposomes, and can be in the form of a DNA-liposome mixture, lecithin liposomes or other liposomes known in the art (see, for example, W09324640), calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other known transfection promoters. The transfection promoter is a polyanion, a polycation (including poly-L-glutamate (LGS)), or a lipid. The concentration of the transfection agent in the vaccine is less than 4 mg / ml, less than 2 mg / ml, less than 1 mg / ml, 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] A pharmaceutically acceptable excipient can be one or more adjuvants. The adjuvant can be another gene that is expressed in an alternative vector or delivered as a protein in combination with the above vector in a vaccine.One or more adjuvants can be selected from the group consisting of CCL20, alpha-interferon (IFN-α), beta-interferon (IFN-β), gamma-interferon, platelet-derived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermal growth factor (EGF), cutaneous T cell-attracting chemokine (CTACK), 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-1α, MIP-1β, IL-8, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, p150.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, a variant of IL-18, CD40, CD40L, a vascular growth factor, a fibroblast growth factor, IL-7, a nerve growth factor, a vascular endothelial growth factor, Fas, a TNF receptor, Flt, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, inactive NIK, SAP K, SAP-1, JNK, an interferon-responsive gene, 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, a signal sequence or coding sequence encoding a deleted signal sequence, optionally a different signal peptide such as from IgE, or a coding sequence encoding a different signal peptide such as from IgE, IL-15, 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 can 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 US Application No. 08 / 956,865, and US Provisional Application No. 61 / 569600 filed on 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, and 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 and 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 and 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 immunomodulatory agents are disclosed in U.S. Application No. 10 / 560,653, which is incorporated herein by reference. Examples of DR5 and other immunomodulatory agents 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 those encoding MCP-1, MIP-1a, MIP-1b, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, p150.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, a variant of IL-18, CD40, CD40L, a vascular growth factor, a fibroblast growth factor, IL-7, IL-22, a nerve growth factor, a vascular endothelial growth factor, Fas, a TNF receptor, Flt, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, an inactive NIK, SAP K, SAP-1, JNK, an interferon response gene, 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 include a gene vaccine adjuvant as described in U.S. No. 021,579, filed April 1, 1994, which is incorporated herein in its entirety by reference.

[0161] The vaccine may contain the plasmid in an amount of about 1 nanogram to 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 micrograms of DNA. In some preferred embodiments, the vaccine may contain about 10 nanograms to about 800 micrograms of DNA. In some preferred embodiments, the vaccine may contain about 0.1 to about 500 micrograms of DNA. In some preferred embodiments, the vaccine may contain about 1 to about 350 micrograms 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 microgram 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, about 100 to about 200 micrograms of an antigen or a plasmid encoding the same.

[0162] The vaccine can be formulated according to the mode of administration used. Injectable vaccine pharmaceutical compositions can be sterile, pyrogen-free, and free of microparticles. Isotonic formulations or solutions can be used. Additives for isotonicity can include sodium chloride, dextrose, mannitol, sorbitol, and lactose. The vaccine can contain a vasoconstrictor. Isotonic solutions can include phosphate buffered saline. The vaccine can further contain a stabilizer including gelatin and albumin. Stabilizers including LGS or polycations or polyanions can stabilize the formulation for a long time at room temperature or ambient temperature.

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

[0164] In some embodiments, the pharmaceutical composition according to the present invention comprises at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 ng of the nucleic acid molecule(s) of the vaccine. In some embodiments, the pharmaceutical composition may comprise at least 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 605, 610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695, 700, 705, 710, 715, 720, 725, 730, 735, 740, 745, 750, 755, 760, 765, 770, 775, 780, 785, 790, 795, 800, 805, 810, 815, 820, 825, 830, 835, 840, 845, 850, 855, 860, 865, 870, 875, 880, 885, 890, 895, 900, 905, 910, 915, 920, 925, 930, 935, 940, 945, 950, 955, 960, 965, 970, 975, 980, 985, 990, 995, or 1000 micrograms of the nucleic acid molecule(s) of the vaccine. In some embodiments, the pharmaceutical composition may comprise at least 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 mg or more of the nucleic acid molecule(s) of the vaccine.

[0165] In other embodiments, the pharmaceutical composition comprises nucleic acid molecule(s) of the vaccine at up to 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 ng (including these values). In some embodiments, the pharmaceutical composition may comprise nucleic acid molecule(s) of the vaccine at 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, 475, 480, 485, 490, 495, 500, 605, 610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695, 700, 705, 710, 715, 720, 725, 730, 735, 740, 745, 750, 755, 760, 765, 770, 775, 780, 785, 790, 795, 800, 805, 810, 815, 820, 825, 830, 835, 840, 845, 850, 855, 860, 865, 870, 875, 880, 885, 890, 895, 900, 905, 910, 915, 920, 925, 930, 935, 940, 945, 950, 955, 960, 965, 970, 975, 980, 985, 990, 995, or 1000 micrograms (including these values). In some embodiments, the pharmaceutical composition may comprise nucleic acid molecule(s) of the vaccine at 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 (including these values).

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

[0167] The pharmaceutical composition can further contain pharmaceutically acceptable excipients as provided in paragraph 2 above. For example, pharmaceutically acceptable excipients can include functional molecules, vehicles, adjuvants, carriers, diluents, or transfection promoters as provided in paragraph 2.

[0168] Indications The vaccines and pharmaceutical compositions containing the vaccines provided herein can be used for the treatment or prevention of cancer cells expressing survivin and cancer-based tumors. In particular, the vaccines and pharmaceutical compositions containing the vaccines provided herein can be used for the treatment or prevention of ovarian cancer, more particularly epithelial ovarian cancer, and most particularly serous ovarian cancer.

[0169] Vaccination method Methods for treating and / or preventing cancer using the above pharmaceutical formulations are provided herein. Also described herein are methods of using the above pharmaceutical formulations for treating and / or preventing cancer in a subject. Also described herein are methods of vaccinating a subject. Also described herein are methods of administering the pharmaceutical formulations described herein to a subject in need thereof. The methods described herein, collectively referred to as the treatment methods using the pharmaceutical formulations described herein, may include administering to a subject in need thereof one or more of the vaccines described herein to induce a therapeutic and / or prophylactic immune response. The vaccine may be administered to a subject to modulate the activity of the subject's immune system and enhance the immune response. Administration of the vaccine may be transfection of a cancer antigen disclosed herein as a nucleic acid molecule that, when expressed in a cell and delivered to the cell surface, causes the immune system to recognize and induce a cellular, humoral, or cellular and humoral response. Administration of the vaccine may be used to induce or elicit an immune response in a subject against one or more of the cancer antigens disclosed herein by administering the vaccine discussed herein to the subject.

[0170] The vaccine can be 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 a vaccine is administered to a mammal, thereby introducing a vector into the cells of the mammal, 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 can include antibodies made against one or more of the cancer antigens and a T cell response specific to one or more of the cancer antigens. In some instances, a mammal vaccinated with the vaccine discussed herein has an antigen-stimulated immune system, and when challenged with one or more of the cancer antigens disclosed herein, the antigen-stimulated immune system can rapidly eliminate the subsequent cancer antigens disclosed herein, whether by a humoral, cellular, or both cellular and humoral immune response.

[0171] Methods of administering DNA vaccines are described in U.S. Patent Nos. 4,945,050 and 5,036,006, both of which are incorporated herein by reference in their entirety.

[0172] A vaccine can be administered to a mammal to induce an immune response in the mammal. The mammal can be a human, non-human primate, cow, pig, sheep, goat, antelope, bison, buffalo, ox, deer, hamster, elephant, llama, alpaca, mouse, rat, preferably a human, cow, or pig. Similarly, a vaccine can be administered to a non-human subject, such as a chicken, to induce an immune response.

[0173] The dosage of the vaccine can be from 1 microgram to 10 mg (active ingredient / kg body weight / dose) per kilogram (kg) of body weight over time, and can be from 20 micrograms to 10 mg of ingredient / kg body weight / dose. The vaccine can be administered once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, eleven times, twelve times, thirteen times, fourteen times, fifteen times, sixteen times, seventeen times, eighteen times, nineteen times, twenty times, twenty-one times, twenty-two times, twenty-three times, twenty-four times, twenty-five times, twenty-six times, twenty-seven times, twenty-eight times, twenty-nine times, thirty times, or thirty-one times per day. The number of vaccine administrations for effective treatment can be one, two, three, four, five, six, seven, eight, nine, ten or more administrations.

[0174] Method for generating an immune response with a vaccine A vaccine can be used to generate an immune response in a mammalian or non-mammalian subject, including a therapeutic or prophylactic immune response. The immune response can generate antibodies and / or killer T cells directed against one or more cancer antigens as disclosed herein. Such antibodies and T cells can be isolated.

[0175] Some embodiments provide methods of generating an immune response against one or more of the cancer antigens disclosed herein, and such embodiments include administering a vaccine to a subject. Some embodiments provide methods of prophylactically vaccinating a subject against a cancer or tumor that expresses one or more of the aforementioned cancer antigens, and such embodiments include administering a vaccine. Some embodiments provide methods of therapeutically vaccinating a subject afflicted with a cancer or tumor that expresses one or more of the cancer antigens, and such embodiments include administering a vaccine. Diagnosis of a cancer or tumor that expresses one or more of the cancer antigens disclosed herein prior to administration of the vaccine can be routinely performed.

[0176] Methods of cancer treatment with a vaccine 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 the metastasis of cancerous or tumor cells. Thus, the vaccine can be used in a method of treating and / or preventing a cancer or tumor in a mammal or subject to which the vaccine has been administered.

[0178] Route of administration The vaccine or pharmaceutical composition can 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 by combinations thereof. For veterinary use, the composition can be administered as a formulation that is suitably acceptable according to normal veterinary practice. A veterinarian can readily determine the most appropriate dosing regimen and route of administration for a particular animal. The vaccine can 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] Vectors for vaccines 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 adeno-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] A vaccine or pharmaceutical composition containing a vaccine can be administered by electroporation. Administration of the vaccine by electroporation can be achieved using an electroporation device configured to deliver a pulse of energy effective to form reversible pores in the cell membrane to a desired tissue of a mammal, preferably, the pulse of energy is a constant current equivalent to a pre-set current input by a user. The electroporation device can comprise an electroporation component and an electrode assembly or a handle assembly. The electroporation component can include one or more of the various elements of the 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 source, and a power switch, and can incorporate them. Electroporation can be achieved using an in vivo electroporation device, for example, the CELLECTRA® EP System (Inovio Pharmaceuticals, Inc., Blue Bell, PA) or the Elgen electroporator (Inovio Pharmaceuticals, Inc.) to facilitate transfection of cells by a vector.

[0181] Examples of electroporation devices and electroporation methods that can facilitate the administration of the DNA vaccine 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 filed by Smith et al., the contents of which are hereby incorporated by reference in their entirety. Other electroporation devices and electroporation methods that can be used to facilitate the administration of the DNA vaccine include those provided in co-pending and co-owned U.S. Patent Application No. 11 / 874,072, filed on October 17, 2007, claiming the benefit of U.S. Provisional Application No. 60 / 852,149, filed on October 17, 2006, and U.S. Provisional Application No. 60 / 978,982, filed on October 10, 2007, under 35 U.S.C. § 119(e), all of which are hereby incorporated by reference in their entirety.

[0182] U.S. Patent No. 7,245,963 by Draghia-Akli et al. describes a modular electrode system and its use for facilitating the introduction of biomolecules into cells of selected tissues of the body or a plant. The modular electrode system can comprise a plurality of needle electrodes, a subcutaneous needle, an electrical connector providing electrical connection from a programmable constant current pulse controller to the plurality of needle electrodes, and a power source. The operator can grip the plurality of needle electrodes mounted on a support structure and firmly insert them into a selected tissue of the body or plant. Next, the biomolecule is administered into the selected tissue by the subcutaneous needle. The programmable constant current pulse controller is activated to apply a constant current electrical pulse to the plurality of needle electrodes. The applied constant current electrical pulse facilitates the introduction of the biomolecule into the cells between the plurality of electrodes. The entire content of U.S. Patent No. 7,245,963 is hereby incorporated by reference in its entirety.

[0183] U.S. 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 (an "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 the input of pulse parameters, and enables the storage and acquisition of current waveform data. The electroporation device also comprises a replaceable electrode disk having a series of needle electrodes, a central injection channel for an injection needle, and a removable guide disk. The entire disclosure of U.S. Patent Publication No. 2005 / 0052630 is hereby incorporated by reference in its entirety into this specification.

[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 into not only tissues such as muscle but also other tissues or organs. Due to the configuration of the electrode array, the injection needle can be fully inserted into the target organ, and the injection is administered perpendicular to the target tissue in the region 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] In addition, the following patents are contemplated in some embodiments incorporating an electroporation device and its 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, and electroporation devices described in U.S. Patent No. 6,939,862, issued September 6, 2005. Further, patents encompassing the subject matter provided in U.S. Patent No. 6,697,669, issued February 24, 2004, relating to the administration of DNA using any of a variety of devices, and U.S. Patent No. 7,328,064, issued February 5, 2008, which noted a method of injecting DNA, are contemplated herein. The above patents are hereby incorporated by reference in their entirety.

[0186] Methods are provided herein for preparing vectors comprising nucleic acid molecule(s) encoding the synthetic consensus survivin antigen discussed herein. The vectors can be used to seed cell cultures in large scale fermentation tanks using methods known in the art after the final subcloning step into mammalian expression plasmids.

[0187] The DNA vectors for use in the EP devices of the present invention can be formulated or manufactured using combinations 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 instances, the DNA vectors used in these studies can be formulated at a concentration of 10 mg / mL or greater. The manufacturing techniques also include, in addition to those described in U.S. Patent Application No. 60 / 939,792, which includes those described in U.S. Patent No. 7,238,522, issued July 3, 2007, various devices and protocols generally known to those of ordinary skill in the art, or incorporate them. The applications and patents referenced above, U.S. Patent Application No. 60 / 939,792 and U.S. Patent No. 7,238,522, are hereby incorporated by reference in their entirety.

Examples

[0188] The present invention is further illustrated in the following examples. These embodiments represent preferred embodiments of the invention, but are shown by way of example only. From the foregoing discussion and these examples, one of ordinary skill in the art can ascertain the essential characteristics of the invention and adapt various changes and modifications of the invention to various uses and conditions without departing from the spirit and scope thereof. Accordingly, various modifications of the invention in addition to those shown and described herein will be apparent to those of ordinary skill 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, XP_012516801.1, 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] The 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 ablate the potential biological functions of the resulting consensus survivin isoform 1 protein, three mutations were introduced, abolishing the anti-apoptotic activity of survivin. The three mutations are T34A, T48A, and C84A. Furthermore, to obtain higher levels of expression, the upstream Kozak sequence and IgE leader sequence were added to the N-terminus. Additionally, 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 also 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 1 T3 is shown in SEQ ID NO: 2.

[0191] The synthetic consensus survivin antigen isoform 1 was digested with BamHI and XhoI and cloned into the unique 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 its 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 1 T3 To generate human consensus survivin isoform 3, eight survivin isoform 3 sequences were collected from GenBank (www.ncbi.nlm.nih.gov / genbank / ). 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 an extra 11 amino acid residues at their C-terminus that were not present in the human sequence. These extra residues were removed in generating the consensus human survivin isoform 3 to prevent 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 the survivin antigen 1 (described above) and survivin antigen T3.

[0194] Once the consensus synthetic consensus survival antigen isoform 1 T3 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, J.S. et al. Induction of potent Th1-type immune responses from a novel DNA vaccine for West Nile virus New York isolate (WNV-NY1999). The Journal of infectious diseases 184, 809-816, doi:10.1086 / 323395 (2001)). 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 also performed to avoid cis-acting sequence motifs such as regions with very high (>80%) or very low (<30%) GC content, as well as internal TATA boxes, chi-sites, ribosome entry sites, etc.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 its correctness. As shown in Table 1, the nucleotide sequence of synthetic consensus survivin antigen isoform 1T3 is shown in SEQ ID NO: 3. As shown in Table 1, the amino acid sequence of 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 Figure 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 generate pGX0001 and identified based on the reported sequence of pVAX1 available from Thermo Fisher Scientific. These modifications are listed below and no problems have been detected with plasmid amplification and antigen transcription and translation. To date, no changes have been observed in the sequence of pGX0001 in any data of plasmid products within the platform using pGX0001 as the backbone.

Table 4-1

[0197] pGX1428 is a DNA plasmid encoding a synthetic consensus survivin antigen isoform 1 (survivin antigen 1) protein. The related 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) for production purposes and a plasmid replication origin (pUC ori). These elements do not function in eukaryotic cells. pGX1428 was prepared by cloning the synthetic consensus survivin antigen isoform 1 (survivin antigen 1) DNA sequence at the BamHI and XhoI sites into pGX0001, as shown in Figure 4.

[0198] pGX1429 is a DNA plasmid encoding a synthetic consensus survivin antigen isoform 1T3 (survivin antigen 1T3) protein. The related 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) for production purposes and a plasmid replication origin (pUC ori). These elements do not function in eukaryotic cells. pGX1429 was prepared 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. The expression of the antigen proteins by each construct was also evaluated in vitro by Western blotting.

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

[0201] In Vitro Antigen Expression 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 2 dish) using Turbofectin 8 (Origene). Forty-eight hours after transfection, the cells were lysed using RIPA cell lysis buffer (ThermoFisher), and the cell lysates were collected. Following a BCA assay (ThermoFisher) to determine the total protein concentration, 15 μg of cell lysate was electrophoresed on a 4–12% SDS-PAGE gel (ThermoFisher). Detection was performed using a monoclonal antibody against amino acids 1–142 of human survivin (Santa Cruz Biotech, clone D8, sc-17779), and then visualized using Western blotting analysis system (GE Amersham) with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Santa Cruz Biotech, sc-2005). As a loading control, the actin expression of the blot was reprobed 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 conducted in accordance with the guidelines of the National Institutes of Health and the Animal Care and Use Proposal (ACUP) (BTS ACUP #15-091). The 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 Therapy. Briefly, the plasmid was formulated in sterile water for injection (VetOne) and delivered by intramuscular injection into the anterior tibialis muscle at an injection volume of 30 μL of the indicated dose. Immediately following each intramuscular injection, electroporation (EP) using a CELLECTRA® 2000 Adaptive Constant Current Electroporation Device (Inovio Pharmaceuticals) with a 3P array was performed. This device was configured to deliver two 0.1 Amp pulses with a 52 ms pulse width separated by a 1 second delay. The mice received three immunizations at 3-week intervals. One week after the last immunization, the mice were sacrificed and spleens were harvested for cellular immune readouts. No other tissues were collected.

[0204] Isolation of Spleen Lymphocytes Spleen cells were isolated aseptically and placed in 5 mL of R10 medium (Rosewell Park Memorial Institute medium 1640 supplemented with 10% fetal bovine serum and 1% antibiotic - antifungal agent). Spleen cells 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 minutes for RBC lysis. Next, the spleen cells were centrifuged, washed with PBS, resuspended in R10 medium, and used immediately for further analysis.

[0205] IFNγ ELISpot The mouse IFNγ ELISpot assay was performed using a kit from 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 and then added in triplicate at 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 acids overlapping, representing the entire synthetic consensus survivin antigen 1 and the 11 - amino - acid sequence of the synthetic consensus survivin antigen 1 T3 protein. These sets of peptides were resuspended in DMSO (Sigma) and pooled into two peptide pools at a peptide concentration of approximately 2 μg / ml. The first peptide pool contained peptides corresponding to the synthetic consensus survivin antigen 1 antigen protein, and the second peptide pool contained peptides corresponding to the synthetic consensus survivin antigen 1 T3 antigen protein. 5 μg / ml of concanavalin A (Sigma) was used as a positive control, and complete culture medium was used as a negative control. The plates were incubated at 37°C for 18 hours in an incubator with a 5% CO2 atmosphere. Next, biotinylated anti - mouse IFNγ detection antibody (MabTech mAb R4 - 6A2) was added, and the plates were incubated for 2 hours at room temperature. The plates were washed, and streptavidin - ALP (MabTech) was added, and the plates were incubated for 1 hour at room temperature. Spot detection was completed using BCIP / NBT substrate according to the manufacturer's instructions (MabTech). Spots on the plates were counted using an automated ELISPOT reader (Cellular Technology). The average number of spot - forming units (SFU) was adjusted to 1×10 6 per spleen cell.

[0206] In Figures 7A - 7H, the antigen - specific responses by IFNγ ELISpot were greater than 1×10 in the medium - only control in terms of SFU 6It was reported as the 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 1 T3 were further characterized by flow cytometry. Briefly, 2 × 10 from vaccinated and untreated mice 6Spleen cells were stimulated for 6 hours in the presence of Brefeldin A (BD Biosciences), monensin (BD Biosciences), and FITC anti-mouse CD107a antibody (BD Biosciences, clone 1D4B) immediately after isolation with synthetic consensus survivin antigen 1 and survivin 1T3 peptide according to each group. After stimulation with the peptide, the spleen cells were centrifuged 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 at 4°C for 5 minutes. After incubation, the remaining extracellular antibody (in PBS) was added at 30 μL per well and incubated at 4°C for 30 minutes. When the extracellular staining solution was added, the final volume per well was 50 μL, composed of Fc Block at a final dilution of 1:100 and the appropriate working dilution of the extracellular antibody. The 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). The cells were fixed and permeabilized at 4°C for 20 minutes (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 were collected on a 10-color FACS CANTO (BD Biosciences), and analysis was completed using FlowJo software. The gating strategy for flow cytometry is shown in Figure 8.

[0208] For a cell to be termed antigen-specific by flow cytometry, the frequency of the reported parameter needs to exceed the frequency of the medium-only control. For a cell to be identified as producing antigen-specific CD107a, the antigen-specific production of IFNγ and / or IL-2 and / or TNFα identified by Boolean gating must be identified as positive.

[0209] Statistical analysis Statistical analysis was completed using IBM SPSS Statistics 22 (IBM Corporation). Analysis between groups was performed using ANOVA and post hoc Tukey's test (HSD) to adjust for multiple comparisons. Homogeneity of variance was confirmed using the F statistic prior to multiple comparisons. For all statistical analyses, a p-value of 0.050 was considered significant.

[0210] Results 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 1 T3 (pGX1429). Expression of synthetic consensus survivin antigen 1 and synthetic consensus survivin antigen 1 T3 antigen proteins by pGX1428 and pGX1429 was confirmed by Western blotting, respectively. Briefly, human rhabdomyosarcoma (RD) cells were transfected with pGX1428, pGX1429, or pGX0001 (empty vector, negative control) plasmid. Cell lysates were probed for the expression of synthetic consensus survivin antigen proteins and anti-human survivin antibody (BIRC5). Protein bands of the predicted molecular weights of synthetic consensus survivin antigen 1 (17.5 kD) and synthetic consensus survivin antigen 1 T3 (25.3 kD) were detected (Figure 6). A faint band was detected in the negative control (pGX0001), most likely due to low-level endogenous survivin protein expression in the RD cell line. Bands for anti-β-actin were detected at similar intensities, 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 the synthetic consensus survivin antigen vaccine construct IFNγ ELISpot The immunogenicity of 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). As a negative control (n = 4 / group), the empty vector backbone (pGX0001) was immunized into mice. Vaccination with the synthetic consensus survivin antigen resulted in a significant IFNγ response compared to mice vaccinated with the negative control vaccine. However, there was minimal evidence for a dose-dependent increase in IFNγ production induced by synthetic consensus survivin antigen 1 (Figure 10A), suggesting that the maximum response was achieved at the lowest dose. Specifically, the synthetic consensus survivin antigen 1 IFNγ SFU was 1,082 ± 574, 1,186 ± 747, 1,135 ± 647, and 848 ± 350 at 10 μg, 20 μg, 30 μg, and 50 μg, respectively. The synthetic consensus survivin antigen 1 IFNγ response was significantly greater than that of untreated (2 ± 3) at the 10 μg (p = 0.031), 20 μg (p = 0.015), and 30 μg (p = 0.021) doses of pGX1428, but not at the 50 μg (p = 0.134) dose. Vaccination with synthetic consensus survivin antigen 1T3 resulted in a significant IFNγ response and had some evidence of a dose-dependent increase with increasing dose levels (Figure 10D). The survivin 1T3 IFNγ SFU was 516 ± 156, 812 ± 534, 1,016 ± 654, and 818 ± 339 at 10 μg, 20 μg, 30 μg, and 50 μg, respectively. The synthetic consensus survivin antigen 1T3 IFNγ response was significantly greater than that of untreated (5 ± 6) at the 20 μg (p = 0.039), 30 μg (p = 0.006), and 50 μg (p = 0.037) doses of pGX1429, but not at the 10 μg (p = 0.337) dose. The IFNγ responses are summarized in Table 5.

Table 5

[0212] Flow cytometry Both synthetic consensus survivin antigen 1 and synthetic consensus survivin antigen 1 T3 induced a stronger response in the CD4+ T cell compartment compared to the response in the CD8+ T cell compartment (Figure 9). Synthetic consensus survivin antigen 1 induced a significantly stronger frequency of antigen-specific CD4+ T cell responses than untreated (0.03% ± 0.05%) in the 20 μg (1.08% ± 0.65%) (p = 0.024) and 50 μg (1.21% ± 0.73%) (p = 0.009) dose groups, but not in the 10 μg (0.86% ± 0.31%) (p = 0.105) or 30 μg (0.82% ± 0.46%) (p = 0.134) dose groups (Figure 7B). Synthetic consensus survivin antigen 1 T3 induced a significantly stronger frequency of antigen-specific CD4+ T cell responses 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). The cytokine profile of synthetic consensus survivin antigen-specific CD4+ T cells was similar across all dose groups for both constructs and consisted mainly of IFNγ+IL-2+TNFα+, IFNγ+IL-2-TNFα+, or IFNγ+IL-2-TNFα- cells (Figure 7G, Figure 7H). The frequency of antigen-specific CD4+ T cells is described in more detail 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 among the dose groups (p = 0.043). The frequencies of antigen-specific CD8+ T responses in the 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%) 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 similar CD8+ T cell responses in terms of magnitude and phenotype. Antigen-specific CD8+ T cells were mainly IFNγ+IL-2-TNFα- and IFNγ+IL-2-TNFα+ (Figures 7G, 7H). The frequencies of antigen-specific CD8+ T cells are described in more detail in Table 7.

Table 7

[0214] Approximately 25% of cytokine-positive CD4+ T cells induced by synthetic consensus survivin antigen 1 (Figure 10A) and synthetic consensus survivin antigen 1 T3 (Figure 10B) were also positive for CD107a, indicating the potential for cell lysis function via CD4+ T cells. All doses of synthetic consensus survivin antigen 1 induced a significantly higher frequency of CD4+CD107a+ T cells than untreated (0.01% ± 0.01%). Specifically, the frequencies of antigen-specific CD4+CD107a+ T cells were 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). Synthetic consensus survivin antigen 1 T3 induced a significantly higher frequency 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). The frequencies of antigen-specific CD4 + CD107a + + 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). The frequencies of antigen-specific CD4 + + T cells with cytolytic ability are described in more detail in Table 8.

Table 8

[0215] Similar to the size of antigen-specific CD8+ T cells, synthetic consensus survivin antigen 1 did not induce a significant change in the frequency of CD8+CD107a+ T cells among all groups (p = 0.101) (Figure 10C). Synthetic consensus survivin antigen 1T3 induced a significant change 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 in the group immunized with 50 μg of pGX1429 (0.28% ± 0.22%) compared to untreated (0.03 ± 0.01) (p = 0.026), 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 synthetic consensus survivin antigen 1T3. The cytokine profiles of synthetic consensus survivin antigen-specific CD8+CD107a+ T cells were similar between both constructs across dose groups and consisted mainly of IFNγ+IL-2-TNFα+ and IFNγ+IL-2-TNFα- cells (Figure 10E, Figure 10F). The frequency of antigen-specific CD8 + T cells with cytolytic ability is described in more detail in Table 9.

Table 9

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

[0217] The following synthetic consensus survivin antigen 1 epitopes were 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 was similar, but the response induced by the synthetic consensus survivin antigen 1T3 (pGX1429) region to the synthetic consensus survivin antigen 1 (pGX1428) region of synthetic consensus survivin antigen 1 was approximately half the magnitude of the response induced by the synthetic consensus survivin antigen 1 construct (pGX1428). Epitope mapping by IFNγ ELISpot using the matrix approach revealed that both synthetic consensus survivin antigen constructs generated responses to the same epitopes of the synthetic consensus survivin antigen 1 antigen, but the responses driven by synthetic consensus survivin antigen 1T3 to these epitopes were of lower magnitude. It was also confirmed that there were epitopes unique to the T3 region of the synthetic consensus survivin antigen 1T3 antigen.

[0219] The total magnitude of the cellular immune responses induced by synthetic consensus survivin antigen 1 (pGX1428) and synthetic consensus survivin antigen 1 T3 (pGX1429) was similar, but the response induced by the synthetic consensus survivin antigen 1 T3 (pGX1429) region against the synthetic consensus survivin antigen 1 region of the synthetic consensus survivin antigen 1 T3 antigen was approximately half the magnitude of the response induced by the synthetic consensus survivin antigen 1 construct (pGX1428). Epitope mapping by IFNγ ELISpot using the 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 synthetic consensus survivin antigen 1 T3 against these epitopes were of lower magnitude. Using this approach, it was also confirmed that there were epitopes specific to the T3 region of the synthetic consensus survivin antigen 1 T3 antigen. Synthetic consensus survivin antigen 1 T3 also significantly increased the frequencies of antigen-specific CD8+ and CD8+CD107a+ T cells compared to untreated, while synthetic consensus survivin antigen 1 did not significantly increase antigen-specific CD8+ T cells in mice. Synthetic consensus survivin antigen 1 T3 (pGX1429) was selected to proceed to a monovalent non-human primate study based on its potential to further expand the breadth of the cellular immune response against survivin compared to pGX1428 in mice.

[0220] Example 5 - Non-Human Primate Study Using Synthetic Consensus Survivin Antigen 1 T3 (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 monkeys, identified by their unique NHP ID numbers, were divided into three groups of six and immunized with pGX1429 as follows. Six animals were immunized with 3.0 mg of pGX1429, six were immunized with 3.0 mg of pGX1429 plus 0.04 mg of pGX6006 (opt rh IL-12) as an adjuvant, and six were immunized with 3.0 mg of pGX1429 plus 0.20 mg of pGX6006 (opt rh IL-12) as an adjuvant, formulated at an injection volume of 1.0 mL in SSC. Immunization injections were administered at weeks 0, 4, 8, and 12. All immunizations were performed intramuscularly alternately in the contralateral limbs at an injection volume of 1 ml formulated in sterile WFI using the CELLECTRA® 2000 5P-IM EP device. The EP conditions were as follows: OpBlock 0070-IM, 0.5 Amp, 3 pulses, 52 milliseconds, 0.2 seconds between pulses. Survivin immunogenicity was evaluated at weeks 2, 6, 10, and 14. The survivin-specific IFN-gamma responses are shown in Figures 12 and 13. The homology between native rhesus monkey survivin and pGX1429 is shown in Table 10. As shown in Figures 13 and 14, an increase in the survivin-specific response was observed in animals immunized with 0.20 mg of IL-12 as an adjuvant in addition to the synthetic consensus survivin antigen 1T3.

[0221] PBMC isolation Non-human primate whole blood was collected into sodium citrate cell preparation tubes (CPT, BD Biosciences) containing an anticoagulant and a gel barrier. Immediately after collection (within 2 hours), the whole blood was centrifuged to separate and enrich PMBCs prior to overnight shipment. Red blood cells and neutrophils were precipitated to the bottom of the tube and held in place by the gel barrier. Plasma and lymphocytes remained above the gel barrier. Each CPT can hold approximately 8 mL of blood and is shipped at room temperature. The spun CPT tubes were processed for PBMC isolation. After lysing 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). When the assays described herein were complete, the remaining PBMCs were frozen in cryovials in freezing medium (10% DMSO from Sigma in 90% FBS from Seradigm) and stored long-term in liquid nitrogen.

[0222] IFNγ ELISpot To evaluate vaccine-induced antigen-specific cellular responses, a monkey IFNγ ELISpot assay was performed on PMBCs isolated 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 for 2 hours at room temperature with complete culture medium (RPMI 1640 supplemented with 10% FBS, antibiotics, and β-mercaptoethanol). NHP PBMCs were resuspended in R10 medium (then 2 × 10 5The 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 acids overlapping, representing the entire synthetic consensus protein sequence. This set of peptides was resuspended in DMSO (Sigma) and pooled at a concentration of approximately 2 μg / mL for each respective peptide. All antigen-specific pooled peptides were used at a 1:100 dilution and combined with PBMCs, resulting in a final dilution of 1:200 in each well. Due to the variation in the size of each antigen protein, two peptide pools of survivin were obtained.

[0223] Anti-CD3 (mAb CD-2 Mabtech) and / or PMA (Sigma) and ionomycin (Sigma) were used as positive controls. Complete R10 culture medium was used as a negative control. The plates were incubated at 37 °C for approximately 18 hours in an incubator with a 5% CO2 atmosphere. After removing the cells and adding the ALP-conjugated anti-sal IFNγ detection antibody (MabTech Ab 7-B6-1-ALP), the plates were incubated at room temperature for 2 hours. Next, a sandwich immunoenzymatic assay was developed using the BCIP / NBT substrate solution according to the manufacturer's instructions (MabTech). A blue-black precipitate was formed as spots, revealing individual IFNγ-producing cells. The spots were then scanned and counted using a CTLImmunoSpot® analyzer and software (Cellular Technology) and quality-controlled by a trained operator. The IFNγ response was reported as spot-forming units (SFU) per million PBMCs. 6 reported as spot-forming units (SFU) per million PBMCs.

Table 10

Table 11

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

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

[0226] Results The survivin-specific IFNγ responses for each of Groups 1 - 3 are shown in Figures 12 - 14. The results show the responses at each time point 2 weeks after dosing. Overall, all groups and individual animals had an increase in response by the end of the study, 2 weeks after Dose 4, compared to the pre-bled baseline. Addition of the higher dose of IL-12 (0.2 mg) resulted in a more consistent response at each time point compared to survivin alone or survivin + 0.04 mg of IL-12. A higher response early in PD2 was also observed with survivin + 0.2 mg of IL-12.

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

Table 12

Table 13

Table 14

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

[0229] The overall results indicate that synthetic consensus survivin administered alone can induce an immune response in 100% of NHPs. The addition of the IL-12 adjuvant improved the responses observed with synthetic consensus survivin, resulting in earlier and greater response PD2, but only when using higher doses of IL-12.

[0230] It is understood that the foregoing detailed description and accompanying examples are merely illustrative and are not to be construed as limitations on the scope of the invention, which is defined only 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 structure, substituents, derivatives, intermediates, synthesis, compositions, formulations, or methods of use of the invention, can be made without departing from the spirit and scope thereof. [1] (a) A nucleic acid sequence encoding amino acids 19 - 159 of SEQ ID NO: 2, (b) A nucleic acid sequence encoding amino acids 19 - 210 of SEQ ID NO: 4, (c) A nucleic acid sequence encoding amino acids 19 - 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 - 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 molecule comprising one or more nucleic acid sequences selected from the group consisting of nucleic acid sequences encoding fragments 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 full length of nucleotides 55 to 423 of SEQ ID NO: 1, (d) A fragment comprising at least 90% of the full 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 A nucleic acid molecule comprising one or more nucleic acid sequences selected from the group consisting of fragments 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 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 molecule comprising one or more nucleic acid sequences selected from the group consisting of nucleic acid sequences encoding fragments comprising at least 90% of the full 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 full length of SEQ ID NO: 1, (d) A fragment comprising at least 90% of the full 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 full length of a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 1, and A nucleic acid molecule comprising one or more nucleic acid sequences selected from the group consisting of fragments comprising at least 90% of the full 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 set forth in SEQ ID NO: 1. [6] A nucleic acid molecule comprising the nucleic acid sequence set forth in SEQ ID NO: 3. [7] The nucleic acid molecule according to any one of items 1 to 6 for use as a medicament. [8] The nucleic acid molecule according to any one of items 1 to 7 for use as a medicament in the treatment of cancer. [9] The nucleic acid molecule according to any one of items 1 to 6 for use in the preparation of a medicament.

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

[11] A vector comprising the nucleic acid molecule according to any one of items 1 to 10.

[12] The vector according to item 11, comprising a plasmid or a viral vector.

[13] A composition comprising one or more nucleic acid molecules according to any one of items 1 to 10.

[14] The composition according to item 13, comprising a pharmaceutically acceptable carrier.

[15] A composition comprising one or more vectors according to item 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 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, (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 full 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 full 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 full 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 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) 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 full 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 full length of an amino acid 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 fragments comprising at least 90% of the full 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 the nucleic acid molecule according to any one of items 1 to 10. A vaccine comprising the vector according to item 11 or 12.

[23] The vaccine according to any one of items 18 to 20, further comprising a pharmaceutically acceptable excipient.

[24] The vaccine according to any one of items 21 to 23, further comprising an adjuvant.

[25] The vaccine according to item 24, wherein the adjuvant is IL-12, IL-15, IL-28, or RANTES.

[26] A method for treating a subject having cancerous cells expressing survivin, the method comprising administering a therapeutically effective amount of the vaccine according to any one of items 20 to 25.

[27] The method according to item 26, wherein the administration comprises an electroporation step.

[28] The method according to item 26 or 27, wherein the administration is performed at one or more sites of the subject.

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

Claims

1. A promoter and: (a) a nucleic acid sequence encoding amino acids 19-159 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 19-232 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 and comprising SEQ ID NO:5; (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 and comprising SEQ ID NO:5; (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, including SEQ ID NO:5, and comprising SEQ ID NO:5; (g) a nucleic acid sequence encoding a protein that is at least 99% identical to amino acids 19 to 159 of SEQ ID NO:2, the protein comprising SEQ ID NO:5, which comprises alanines at positions 51, 65, and 101, and a cysteine ​​at position 150, relative to SEQ ID NO:2; (h) a nucleic acid sequence encoding a protein that is at least 99% identical to amino acids 19 to 210 of SEQ ID NO:4, the protein comprising SEQ ID NO:5, the nucleic acid sequence comprising alanines at positions 51, 65, and 101, and a cysteine ​​at position 150 relative to SEQ ID NO:4; and (i) a nucleic acid sequence encoding a protein that is at least 99% identical to amino acids 19 to 232 of SEQ ID NO:8, the protein comprising SEQ ID NO:5 and including alanines at positions 51, 65 and 101, and a cysteine ​​at position 150 relative to SEQ ID NO:8; and a nucleic acid molecule comprising one or more nucleic acid sequences selected from the group consisting of:

2. A promoter and: (a) nucleotides 55 to 423 of SEQ ID NO:1; (b) nucleotides 55 to 636 of SEQ ID NO:3; (c) a fragment that contains at least 90% of the entire length of nucleotides 55 to 423 of SEQ ID NO:1 and encodes SEQ ID NO:

5. (d) a fragment that contains at least 90% of the entire length of nucleotides 55 to 636 of SEQ ID NO:3 and encodes SEQ ID NO:

5. (e) a fragment that is at least 99% identical to nucleotides 55 to 423 of SEQ ID NO:1, which fragment encodes alanine at amino acid positions 51, 65, and 101, and a cysteine ​​at position 150 relative to SEQ ID NO:5, SEQ ID NO:2; and (f) a fragment that is at least 99% identical to nucleotides 55 to 636 of SEQ ID NO:3, which encodes alanines at amino acid positions 51, 65, and 101 of SEQ ID NO:5, and a cysteine ​​at position 150 relative to SEQ ID NO:

8. and a nucleic acid molecule comprising one or more nucleic acid sequences selected from the group consisting of:

3. A promoter and: (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, the fragment comprising SEQ ID NO:5; (e) a nucleic acid sequence encoding a fragment comprising at least 90% of the entire length of SEQ ID NO:4, comprising SEQ ID NO:5; (f) a nucleic acid sequence encoding a fragment comprising at least 90% of the entire length of SEQ ID NO:8, comprising SEQ ID NO:5; (g) a nucleic acid sequence encoding a protein that is at least 99% identical to SEQ ID NO:2, the protein comprising SEQ ID NO:5, which comprises alanines at positions 51, 65, and 101, and a cysteine ​​at position 150, relative to SEQ ID NO:2; (h) a nucleic acid sequence encoding a protein that is at least 99% identical to SEQ ID NO:4, the protein comprising SEQ ID NO:5, which comprises alanines at positions 51, 65, and 101, and a cysteine ​​at position 150 relative to SEQ ID NO:4; and (i) a nucleic acid sequence encoding a protein that is at least 99% identical to SEQ ID NO:8, the protein comprising SEQ ID NO:5 and containing alanines at positions 51, 65, and 101, and a cysteine ​​at position 150 relative to SEQ ID NO:8; and a nucleic acid molecule comprising one or more nucleic acid sequences selected from the group consisting of:

4. A promoter and: (a) SEQ ID NO:1; (b) SEQ ID NO:3; (c) a fragment that contains at least 90% of the entire length of SEQ ID NO:1 and encodes SEQ ID NO:5 (d) a fragment that contains at least 90% of the entire length of SEQ ID NO:3 and encodes SEQ ID NO:5; (e) a fragment that is at least 99% identical to SEQ ID NO:1, which encodes alanine at amino acid positions 51, 65, and 101, and cysteine ​​at position 150, relative to SEQ ID NO:5 and SEQ ID NO:2; and (f) a fragment that is at least 99% identical to SEQ ID NO:3, which encodes alanine at amino acid positions 51, 65, and 101, and a cysteine ​​at position 150 relative to SEQ ID NO:5 and SEQ ID NO:

8. and a nucleic acid molecule comprising one or more nucleic acid sequences selected from the group consisting of:

5. A promoter and the following amino acid sequence: WPFLEGCACAPERMAEAGFIHCPAENEPDLAQCFFCFKELEGWEPDDDPIEEHKKHSSGAAFLSVKKQFEELTLSE and 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:

6. A vector comprising a promoter and 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 correspond to the following amino acid sequence: WPFLEGCACAPERMAEAGFIHCPAENEPDLAQCFFCFKELEGWEPDDDPIEEHKKHSSGAAFLSVKKQFEELTLSEF and separated by a polypeptide consisting of SEQ ID NO:6 and SEQ ID NO:7 correspond to the following amino acid sequences: EFEETAKKVRCAIEQLAAMDRGRKRRSMQRKPTIRRKNLRKLRRKCAVPSSSWLPWTEASGWSCLVP is separated by a polypeptide consisting of The vector.

7. A vector comprising a promoter and a nucleic acid molecule comprising the nucleic acid sequence set forth in SEQ ID NO:

1.

8. A vector comprising a promoter and a nucleic acid molecule comprising the nucleic acid sequence set forth in SEQ ID NO:

3.

9. The vector according to any one of claims 1 to 8, wherein the promoter is upstream of the nucleic acid molecule.

10. The vector of claim 9, wherein the nucleic acid molecule is operably linked to a regulatory element comprising a polyadenylation signal.

11. 10. The vector of claim 9, wherein the promoter is selected from the group consisting of human cytomegalovirus (hCMV) promoter, simian virus 40 (SV40), mouse mammary tumor virus (MMTV) promoter, human immunodeficiency virus (HIV) promoter, bovine immunodeficiency virus (BIV) long terminal repeat (LTR) promoter, Moloney virus promoter, avian leukosis virus (ALV) promoter, Epstein-Barr virus (EBV) promoter, Ras sarcoma virus (RSV) promoter, human actin gene promoter, human myosin gene promoter, human hemoglobin gene promoter, human muscle creatine gene promoter, and human metallothionein gene promoter.

12. 10. The vector of claim 9, wherein the promoter is a modified human cytomegalovirus (hCMV) promoter.

13. The vector of claim 10, wherein the polyadenylation signal is the bovine growth hormone polyadenylation signal (bGH polyA).

14. The vector according to any one of claims 1 to 13, wherein the vector is a plasmid or a viral vector.

15. A composition comprising one or more vectors according to any one of claims 1 to 14.

16. 16. The composition of claim 15, further comprising a pharma- ceutically acceptable carrier.

17. below: (a) amino acids 19-159 of SEQ ID NO:2; (b) amino acids 19-210 of SEQ ID NO:4; (c) amino acids 19-232 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, comprising SEQ ID NO:5; (e) a fragment comprising at least 90% of the entire length of amino acids 19 to 210 of SEQ ID NO:4, comprising SEQ ID NO:5; (f) a fragment comprising at least 90% of the entire length of amino acids 19 to 232 of SEQ ID NO:8 and comprising SEQ ID NO:

5. (g) an amino acid sequence that is at least 99% identical to amino acids 19 to 159 of SEQ ID NO:2, the amino acid sequence comprising alanines at positions 51, 65, and 101, and a cysteine ​​at position 150, relative to SEQ ID NO:5, SEQ ID NO:2; (h) an amino acid sequence that is at least 99% identical to amino acids 19 to 210 of SEQ ID NO:4, the amino acid sequence comprising alanines at positions 51, 65, and 101, and a cysteine ​​at position 150, relative to SEQ ID NO:5, SEQ ID NO:4; and (i) an amino acid sequence that is at least 99% identical to amino acids 19 to 232 of SEQ ID NO:8, the amino acid sequence comprising alanines at positions 51, 65 and 101, and a cysteine ​​at position 150 relative to SEQ ID NO:5 and SEQ ID NO:8; The vector according to any one of claims 1 to 14, which encodes a protein comprising an amino acid sequence selected from the group consisting of:

18. below: (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 and including SEQ ID NO:5; (e) a fragment comprising at least 90% of the entire length of SEQ ID NO:4 and including SEQ ID NO:5; (f) a fragment comprising at least 90% of the entire length of SEQ ID NO:8 and including SEQ ID NO:5; (g) an amino acid sequence that is at least 99% identical to SEQ ID NO:2, the amino acid sequence comprising SEQ ID NO:5, alanines at positions 51, 65, and 101, and a cysteine ​​at position 150, relative to SEQ ID NO:2; (h) an amino acid sequence that is at least 99% identical to SEQ ID NO:4, the amino acid sequence comprising SEQ ID NO:5, alanines at positions 51, 65 and 101, and a cysteine ​​at position 150 relative to SEQ ID NO:4; and (i) an amino acid sequence that is at least 99% identical to SEQ ID NO:8, the amino acid sequence comprising alanines at positions 51, 65 and 101, and a cysteine ​​at position 150, relative to SEQ ID NO:5 and SEQ ID NO:8; The vector according to any one of claims 1 to 14, which encodes a protein comprising an amino acid sequence selected from the group consisting of:

19. A vaccine comprising the vector of any one of claims 1 to 14, wherein the nucleic acid molecule encodes an antigen comprising the amino acid sequence set forth in SEQ ID NO:

2.

20. A vaccine comprising the vector of any one of claims 1 to 14, wherein the nucleic acid molecule encodes an antigen comprising the amino acid sequence set forth in SEQ ID NO:

4.

21. A vaccine comprising the vector of any one of claims 1 to 14, wherein the nucleic acid molecule encodes an antigen comprising the amino acid sequence set forth in SEQ ID NO:

8.

22. 20. The vaccine of claim 19, wherein the nucleic acid molecule comprises the sequence of SEQ ID NO:

1.

23. 22. The vaccine of claim 21 , wherein the nucleic acid molecule comprises the sequence of SEQ ID NO:

3.

24. 24. The vaccine of any one of claims 19 to 23, further comprising a pharma- ceutically acceptable excipient.

25. 24. The vaccine of any one of claims 19 to 23, further comprising an adjuvant.

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

27. A vaccine according to any one of claims 19 to 26 for use in the treatment of a survivin-expressing cancer.

28. The vaccine according to any one of claims 19 to 26 for use in vaccination against survivin-expressing cancer.

29. A vector according to any one of claims 1 to 14 for use as a medicament.

30. A vector according to any one of claims 1 to 14 for use as a medicament in the treatment of cancer.

31. A vector according to any one of claims 1 to 14 for use in the manufacture of a medicament.

32. A vector according to any one of claims 1 to 14 for use in the manufacture of a medicament for the treatment of cancer.

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