Survivin-targeted cancer vaccines and their use

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 of related applications This application claims priority and interest to U.S. Provisional Patent Application No. 62 / 598,267, filed on 13 December 2017, the disclosure of which is incorporated herein by reference in its entirety.

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

[0003] This invention relates to a survivor antigen and a nucleic acid molecule encoding it. The invention also relates to a vaccine comprising such a survivor antigen and / or nucleic acid molecule. Furthermore, the invention relates to a method of using a vaccine to induce an immune response and prevent and / or treat subjects having cancer cells and / or tumors expressing survivor. [Background technology]

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

[0005] Cancer prevention, diagnosis, and treatment can take many different forms. Prevention may include screening for predispositions (e.g., specific genetic variants), behavioral modifications (e.g., smoking, diet, and physical activity levels), and vaccination against viruses (e.g., human papillomavirus, hepatitis B virus). Treatment may include chemotherapy, radiation therapy, and surgical removal of tumors or cancerous tissue. Despite the availability of numerous preventive and therapeutic methods, these often achieve only limited success in effectively preventing and / or treating cancer.

[0006] Survivin, also known as baculovirus apoptosis inhibitor repeat sequence 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 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 apoptosis inhibitory protein (IAP) family, and IAP genes are highly expressed in various cancer cells and primary tumor biopsies. Among IAPs, survivin shows the most dramatic overexpression in tumor and fetal tissues. In several ovarian cancer studies, the number of patient samples tested positive for survivin expression ranged from 74% to 92%.(Cohen, C., Lohmann, CM, Cotsonis, G., Lawson, D. & Santoianni, R. Survivin expression in ovarian carcinoma: correlation with apoptotic markers and prognosis. Modern pathology: an official journal of the United States and Canadian Academy of Pathology, Inc. 16,574-583, doi:10.1097 / 01.MP.0000073868.31297.B0(2003), Felisiak-Golabek,A.et al.Nuclear Survivin expression is a positive prognostic factor in taxane-platinum-treated ovarian cancer patients.Journal of ovarian research 4,20,doi:10.1186 / 1757-2215-4-20 (2011)). The contribution of survivin to tumorigenesis, combined with its restricted patterns of expression and overexpression in various tumors, makes it an intriguing 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, characterized by the presence of a single BIR repeat. Its protein structure also lacks a RING finger domain at the carboxyl terminus. (Chen, X., Duan, N., Zhang, C. & Zhang, W. Survivin and Tumorigenesis: Molecular Mechanisms and Therapeutic Strategies. Journal of Cancer 7, 314-323, doi: 10.7150 / jca.13332 (2016)). Several survivin isoforms have been identified, with survivin isoform 1 being the major transcript. Survivin is expressed during fetal development but not in fully differentiated tissues. However, it is highly expressed in many cancer cells. Therefore, survivin is a potential target antigen for cancer treatment. [Overview of the project] [Problems that the invention aims to solve]

[0008] Vaccines for the treatment and prevention of cancer, particularly epithelial ovarian cancer (EOC), are of great interest. However, existing vaccines targeting tumor cell antigens are limited by insufficient antigen expression in vivo. Therefore, there is still a need in this field for safe and effective vaccines and methods of their use to prevent and / or treat cancer and reduce mortality in cancer-affected individuals. [Means for solving the problem]

[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 containing at least 90% of the total length of amino acids 19-159 of SEQ ID NO: 2, (e) a nucleic acid sequence encoding a fragment containing at least 90% of the total length of amino acids 19-210 of SEQ ID NO: 4, (f) a nucleic acid sequence encoding a fragment containing at least 90% of the total 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 protein that is at least 95% identical to amino acids 19-210 of SEQ ID NO: 4 A nucleic acid molecule is provided herein, comprising one or more nucleic acid sequences selected from the group consisting of: (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 containing at least 90% of the total 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 containing at least 90% of the total 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 containing at least 90% of the total length of a protein that is at least 95% identical to amino acids 19-232 of SEQ ID NO: 8.

[0010] The nucleic acid molecule contains 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 containing at least 90% of the full length of nucleotides 55-423 of SEQ ID NO: 1, (d) a fragment containing 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 containing 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 containing 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 includes 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 containing at least 90% of the full length of SEQ ID NO: 2, (e) a nucleic acid sequence encoding a fragment containing at least 90% of the full length of SEQ ID NO: 4, (f) a nucleic acid sequence encoding a fragment containing 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 containing 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 containing 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 containing 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 contains 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 containing at least 90% of the full length of SEQ ID NO: 1, (d) a fragment containing 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 containing 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 containing 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 contains the nucleic acid sequence shown in Sequence ID No. 1.

[0014] The nucleic acid molecule contains the nucleic acid sequence shown in Sequence ID No. 3.

[0015] The nucleic acid molecules described herein are used as pharmaceuticals.

[0016] The nucleic acid molecule described in the present specification is used as a medicament in the treatment of cancer.

[0017] The nucleic acid molecule described in the present specification is used in the preparation of a medicament.

[0018] The nucleic acid molecule described in the present specification is used in the preparation of a medicament for the treatment of cancer.

[0019] A vector comprises the nucleic acid molecule described in the present specification.

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

[0021] A composition comprises one or more nucleic acid molecules described in the present specification.

[0022] The composition described in the present specification comprises a pharmaceutically acceptable carrier.

[0023] The composition described in the present specification comprises one or more vectors described in the present 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 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, and (l) 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 232 of SEQ ID NO: 8.

[0025] The protein comprises an amino acid sequence selected from the group consisting of: (a) SEQ ID NO: 2, (b) SEQ ID NO: 4, (c) SEQ ID NO: 8, (d) a fragment comprising at least 90% of the 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 fragment comprising 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 shown in Sequence ID No. 4.

[0028] The protein contains the amino acid sequence shown in Sequence ID No. 8.

[0029] The vaccine contains nucleic acid molecules as described herein.

[0030] The vaccine contains the vector described herein.

[0031] The vaccines described herein further include pharmaceutically acceptable excipients.

[0032] The vaccines described herein further include adjuvants.

[0033] The vaccines described herein have an adjuvant of IL-12, IL-15, IL-28, or RANTES.

[0034] A method for treating a subject having cancer cells expressing Survivin comprises administering a therapeutically effective dose of the vaccine described herein.

[0035] The method described herein includes an electroporation step in the administration.

[0036] The methods described herein are performed on one or more parts of the subject.

[0037] A method of vaccinating cancer cells expressing Survivin includes administering an amount of the vaccine described herein that is effective in inducing a humoral or cellular immune response. [Brief explanation of the drawing]

[0038] The summary and the following detailed description will be better understood when read in conjunction with the accompanying drawings. For illustrative purposes, 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: [Figure 1] A schematic diagram of synthetic consensus survivor antigen isoform 1 is shown. Asterisks indicate mutations essential for the loss of anti-apoptotic activity. [Figure 2] The overall structures of synthetic consensus survivorbin antigen isoform 1 monomer A (left) and monomer B (right) are shown. Changes from the natural survivorbin are shown by spheres. [Figure 3] A schematic diagram of synthetic consensus survivorbin antigen isoform 1T3 is shown. Asterisks indicate mutations essential for the loss of anti-apoptotic activity. The dark gray region on the right (3') represents the cleavage-type survivorbin isoform 3 (T3) region. [Figure 4] The structure of pGX1428 containing synthetic consensus survivor antigen isoform 1 is shown. [Figure 5] The structure of pGX1429 containing the synthetic consensus survivor antigen isoform 1T3 is shown. [Figure 6] Immunoblotting revealed the expression of synthetic consensus survivorbin antigen 1 and synthetic consensus survivorbin antigen 1 T3 proteins in human rhabdomyosarcoma (RD) cells transfected with pGX1428 and pGX1429, respectively. Protein bands of the expected molecular weights were detected for pGX1428 (17.5 kD) and pGX1429 (25.3 kD). β-actin was used as a loading control. [Figure 7A-C]The immunogenicity of synthetic consensus Survivin antigen 1 and synthetic consensus Survivin antigen 1T3 is demonstrated. Female CB6F1 tadpoles were immunized three times at 3-week intervals with the indicated dose of Survivin 1 (pGX1428) (Figures 7A-7D), Survivin 1T3 pGX1429 (E-H) (n=8 / group), or pGX0001 (empty vector) (n=4). Figure 7A: Survivin 1-specific IFNγ response by ELISpot at the indicated dose of pGX1428. Figure 7B: Survivin 1-specific CD4+ T cell response. Figure 7C: Survivin 1-specific CD8+ T cell response. [Figure 7D-F] Figure 7D: Survivin 1T3-specific IFNγ response mediated by ELISpot at the prescribed dose of pGX1429. Figure 7E: Survivin 1T3-specific CD4+ T cell response. Figure 7F: Survivin 1T3-specific CD8+ T cell response. [Figure 7G] Figure 7G: Cytokine profiles of Survivin 1-specific CD4+ T cell and CD8+ T cell responses. [Figure 7H] Figure 7H: Cytokine profiles of Survivin 1 T3-specific CD4+ T cell and CD8+ T cell responses. [Figure 8] This document presents a gating strategy for flow cytometry. [Figure 9] This shows the relative frequencies of CD4+ and CD8+ T cells. The cellular immune response induced by pGX1428 and pGX1429 was primarily in the CD4+ T cell compartment compared to the CD8+ T cell compartment. [Figure 10A] This shows the cytolytic activity of Survivin-specific T cells. The cytolytic activity of antigen-specific T cells induced by Survivin 1 and Survivin 1T3. Figure 10A: Frequency of Survivin 1-specific CD4+CD107a+ T cells. [Figure 10B] Figure 10B: Frequency of Survivin 1T3-specific CD4+CD107a+ T cells. [Figure 10C] Figure 10C: Cytokine profile of CD4+CD107a+ T cells. [Figure 10D] Figure 10D: Frequency of Survivin 1-specific CD8+CD107a+ T cells. [Figure 10E] Figure 10E: Frequency of Survivin 1T3-specific CD8+CD107a+ T cells. [Figure 10F] Figure 10F: Cytokine profile of CD4+CD107a+ T cells. [Figure 11A] This shows the epitope mapping of the Survivin IFN-γ response. The width of the IFNγ response induced by Survivin 1 and Survivin 1T3. Figure 11A: Survivin 1 peptide pool after treatment with 50 μg of pGX1428, pGX1429, or pGX0001. [Figure 11B] Figure 11B: Survivin T3 peptide pool after treatment with 50 μg of pGX1429 or pGX0001. Matrix pool location indicated by dashed boxes and sequence comparison with pGX1428 and pGX1429. [Figure 11C] Figure 11C: Sequence comparison of pGX1428 and pGX1429. The amino acids in red text indicate epitopes identified by matrix mapping. The underlined text represents the sequence portion added to the synthetic consensus survivor antigen: the N-terminus shows the IgELS and RGRKRRS fulin cleavage sites. [Figure 11D] Figure 11D: Design of Survivin 1 and Survivin T3 matrices. Peptides that induced the response are indicated by dashed boxes. [Figure 12] This document shows survivalbin-specific IFNγ SFU / 106 PBMCs from individual non-human primates immunized using embodiments of the present disclosure, including synthetic consensus survivalbin 1T3. [Figure 13] This document shows survivorbin-specific IFNγ SFU / 106 PBMCs from individual non-human primates immunized using embodiments of the present disclosure containing synthetic consensus survivorbin 1T3 with a low dose of IL-12 (0.04 mg). [Figure 14] This document shows survivorbin-specific IFNγ SFU / 106 PBMCs from individual non-human primates immunized using embodiments of the present disclosure containing synthetic consensus survivorbin 1T3 with a high dose of IL-12 (0.20 mg). [Figure 15A] The results for individual animals in the NHP study are shown, and a comparison of immunization with the synthetic consensus survivorbin 1 construct and the synthetic consensus survivorbin 1 T3 construct is further presented. Figure 15A shows the results for group 1 animals. [Figure 15B] Figure 15B shows the results for animals in group 2. [Figure 15C] Figure 15C shows the results for animals in group 3. [Modes for carrying out the invention]

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

[0040] Synthetic consensus survivin antigens (SSUs) can be derived from survivin sequences from different species or different isoforms within a species, and are therefore non-natural. SSUss can be further modified by introducing one or more mutations into the consensus sequence to generate a synthetic consensus sequence. Mutations can block or alter specific functional domains of the natural survivin sequence, thereby disrupting or enhancing the structure or function of the functional domain. In some embodiments, additional sequences are added to the synthetic consensus survivin antigen sequence to introduce a new structure or function. For example, the synthetic consensus survivin antigen sequence may have a furin cleavage site. The synthetic consensus survivin antigen sequence may include additional localization signals to enhance the extracellular transport of the final protein product. These additional localization signals may be IgELS or other cell transport sequences.

[0041] Synthetic consensus survivor antigens can induce antigen-specific T cell and / or high-titer antibody responses, thereby inducing or triggering an immune response that is direct or reactive to cancer or tumors expressing the antigen. In some embodiments, the induced or triggered immune response may be cellular, humoral, or both. In some embodiments, the induced or triggered cellular immune response may 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 induced immune response may reduce or inhibit one or more immunosuppressive factors that promote the growth of antigen-expressing tumors or cancers, 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 TFG-β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by immunosuppressive cells, CTLA-4, PD-1, MDSC, MCP-1, and factors that upregulate immune checkpoint molecules.

[0042] The vaccine of the present invention can provide any combination of specific cancer antigens for the specific prevention or treatment of target cancers requiring treatment.

[0043] One approach to designing the nucleic acids of recombinant cancer antigens and the amino acid sequences that encode them is to introduce mutations that alter specific amino acids in the overall amino acid sequence of a natural cancer antigen. While the introduction of mutations does not significantly alter the cancer antigen and therefore cannot be universally applied across mammalian subjects, preferably humans or dogs, the resulting amino acid sequence is altered sufficiently to disrupt tolerance or be recognized as an exogenous antigen in order to generate an immune response. Another approach may involve creating a consensus recombinant cancer antigen having at least 85% to a maximum of 99% amino acid sequence identity, preferably at least 90% to a maximum of 98%, more preferably at least 93% to a maximum of 98%, or even more preferably at least 95% to a maximum of 98% sequence identity compared to its corresponding natural cancer antigen. In some cases, recombinant cancer antigens have 95%, 96%, 97%, 98%, or 99% amino acid sequence identity compared to their corresponding natural cancer antigens. Natural cancer antigens are antigens typically associated with specific cancers or cancerous tumors. Depending on the cancer antigen, the consensus sequence of the cancer antigen can span across mammalian species, within species subtypes, across virus strains, or across serotypes. Some cancer antigens do not differ significantly from the wild-type amino acid sequence of the cancer antigen. Some cancer antigens have a wide variety of nucleic acid / amino acid sequences across species and cannot generate a consensus sequence. In these cases, recombinant cancer antigens are generated by disrupting resistance and produce an immune response with 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 natural cancer antigen. In some cases, recombinant cancer antigens have 95%, 96%, 97%, 98%, or 99% amino acid sequence identity compared to the corresponding natural cancer antigen. By combining the approaches described above, the final recombinant cancer antigen can have the percentage of similarity to the natural cancer antigen amino acid sequence discussed above.

[0044] Vaccines can be further combined with antibodies against checkpoint inhibitors such as PD-1 and PDL-1 to enhance stimulation of both cellular and humoral immune responses. Using anti-PD-1 or anti-PDL-1 antibodies prevents PD-1 or PDL-1 from suppressing T-cell and / or B-cell responses. Overall, designing cancer antigens to be recognized by the immune system helps 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%. After immunization, 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%. The vaccine can prevent and block the increase of monocyte chemotactic protein 1 (MCP-1), a cytokine secreted from 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] Vaccines can enhance the cellular immune response in vaccinated subjects by approximately 50 to 6000 times, 50 to 5500 times, 50 to 5000 times, 50 to 4500 times, 100 to 6000 times, 150 to 6000 times, 200 to 6000 times, 250 to 6000 times, or 300 to 6000 times compared to the cellular immune response in unvaccinated subjects. In some embodiments, the vaccine elevates the cellular immune response in vaccinated subjects by approximately 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, and 220 times compared to the cellular immune response in unvaccinated subjects. It can be augmented by 0x, 2300x, 2400x, 2500x, 2600x, 2700x, 2800x, 2900x, 3000x, 3100x, 3200x, 3300x, 3400x, 3500x, 3600x, 3700x, 3800x, 3900x, 4000x, 4100x, 4200x, 4300x, 4400x, 4500x, 4600x, 4700x, 4800x, 4900x, 5000x, 5100x, 5200x, 5300x, 5400x, 5500x, 5600x, 5700x, 5800x, 5900x, or 6000x.

[0047] Vaccines can enhance interferon-gamma (IFN-γ) levels in vaccinated subjects by approximately 50 to 6000 times, 50 to 5500 times, 50 to 5000 times, 50 to 4500 times, 100 to 6000 times, 150 to 6000 times, 200 to 6000 times, 250 to 6000 times, or 300 to 6000 times compared to IFN-γ levels in unvaccinated subjects. In some embodiments, the vaccine raises IFN-γ levels in vaccinated subjects by approximately 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, and 22 times compared to IFN-γ levels in unvaccinated subjects. It can be augmented by 00x, 2300x, 2400x, 2500x, 2600x, 2700x, 2800x, 2900x, 3000x, 3100x, 3200x, 3300x, 3400x, 3500x, 3600x, 3700x, 3800x, 3900x, 4000x, 4100x, 4200x, 4300x, 4400x, 4500x, 4600x, 4700x, 4800x, 4900x, 5000x, 5100x, 5200x, 5300x, 5400x, 5500x, 5600x, 5700x, 5800x, 5900x, or 6000x.

[0048] As described in more detail below, vaccines may further contain 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. Immune checkpoint inhibitors are any nucleic acids or proteins that prevent 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 cytokines, chemokines, or signaling for the proliferation and / or differentiation of immune cells. As described in more detail below, vaccines can be further combined with antibodies against checkpoint inhibitors such as PD-1 and PDL-1 to enhance the stimulation of both cellular and humoral immune responses. Using anti-PD-1 or anti-PDL-1 antibodies prevents PD-1 or PDL-1 from suppressing the T cell and / or B cell response.

[0049] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. In case of any conflict, the definitions in this document shall prevail. Preferred methods and materials are described below, but similar or equivalent methods and materials may be used in carrying out or testing the 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 and not intended to limit. The terms used herein are intended solely to describe specific embodiments and not to limit them.

[0050] As used herein, the terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and their variations are intended to be unrestrictive transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “and,” and “the” include multiple references unless explicitly indicated by the context. This disclosure also contemplates other embodiments that “comprise,” “consist of,” and “essentially consist of,” the embodiments or elements presented herein, whether expressly described or not.

[0051] In the enumeration of numerical ranges in this specification, each intervening value is explicitly intended to have the same degree of precision as the minimum and maximum values ​​of the enumerated range. For example, in the range 6–9, the numbers 7 and 8 are intended in addition to 6 and 9, and in the range 6.0–7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly intended.

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

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

[0054] The "antigen" is a fragment containing (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) at least 90% of amino acids 19-159 of SEQ ID NO: 2, (e) at least 90% of amino acids 19-210 of SEQ ID NO: 4, (f) 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, and (h) at least 95% identical to amino acids 19-210 of SEQ ID NO: 4. This refers to a protein having a synthetic consensus survivin antigen amino acid sequence, comprising: (i) a protein that is at least 95% identical to amino acids 19-232 of SEQ ID NO: 8; (j) a fragment containing 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 containing 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 containing 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 that includes (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) 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 containing at least 90% of the full length of a protein that is at least 95% identical to SEQ ID NO: 2, (k) a fragment containing 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 containing at least 90% of the full length of a protein that is at least 95% identical to SEQ ID NO: 8. Antigens may optionally contain signal peptides, such as those from other proteins.

[0055] As used herein, “encoding sequence” or “encoding nucleic acid” means a nucleic acid (RNA or DNA molecule) containing a nucleotide sequence that codes for a protein. The encoding sequence may further include start and terminate signals operably linked to a regulatory element, including a promoter and a polyadenylation signal capable of directing expression in the cells of the target or mammal to which the nucleic acid is administered.

[0056] As used herein with respect to nucleic acids, “complement” or “complementary” may mean Watson-Crick (e.g., AT / U and CG) or Hoogsteen base pairs between nucleotides or nucleotide analogs of nucleic acid molecules.

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

[0058] As used herein, “constant current” refers to the current received or experienced by a tissue or the cells that make up that 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 so that the current remains constant at an ampere within the tissue over the lifetime of the electrical pulse. The feedback element measures the resistance of the tissue (or cells) over the duration of the pulse and can cause the electroporation device to change its electrical energy output (e.g., increase the voltage), so that the current in the same tissue remains constant throughout the electrical pulse (on the order of microseconds) and between pulses. In some embodiments, the feedback element includes a controller.

[0059] As used herein, “current feedback” or “feedback” may be used interchangeably and may mean the active response of a provided electroporation device, which includes measuring the tissue current between electrodes and, accordingly, modifying the energy output supplied by the EP device to maintain the current at a constant level. This constant level is preset by the user before the start of a pulse sequence or electrotherapy. Feedback can be achieved by the electroporation components of the electroporation device, such as a controller, so that the electrical circuit continuously monitors the tissue current between electrodes, compares the monitored current (or current in the tissue) to a preset current, and continuously adjusts the energy output to maintain the monitored current at the preset level. The feedback loop may be instantaneous because it is analog closed-loop feedback.

[0060] As used herein, “dispersed current” may mean a pattern of current 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 area of ​​the tissue being electroporated.

[0061] As used interchangeably herein, “electroporation,” “electrical permeability,” or “electrokinetic enhancement” (“EP”) refers to the use of transmembrane electric field pulses to induce microscopic pathways (pores) in biological membranes, which in their presence allow biomolecules such as plasmids and vectors, oligonucleotides, siRNAs, drugs, ions, and water to pass from one side of the cell membrane to the other.

[0062] As used herein with respect to nucleic acid sequences, “fragment” means a nucleic acid sequence or portion thereof that encodes a polypeptide capable of inducing an immune response in mammals that cross-react with the antigens disclosed herein. A fragment may be a DNA fragment selected from at least one of the various nucleotide sequences encoding the protein fragments shown below. A fragment may contain at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of one or more nucleic acid sequences shown below, excluding any added heterologous signal peptides. A fragment may contain at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of one or more nucleic acid sequences shown below, and optionally further include a sequence encoding a heterologous signal peptide, which does not need to be included when calculating the identity percentage. A fragment may further include a sequence encoding a signal peptide, such as an immunoglobulin signal peptide, e.g., an IgE or IgG signal peptide. A sequence encoding an N-terminal methionine and / or a signal peptide may be ligated to the fragment of the coding sequence.

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

[0064] With respect to polypeptide sequences, “fragments” or “immunogenic fragments” means polypeptides capable of inducing an immune response in mammals that cross-react with the antigens disclosed herein. Fragments can be polypeptide fragments selected from at least one of the following diverse amino acid sequences. A consensus protein fragment may contain at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the consensus protein, excluding any added heterologous signal peptides. A fragment may contain at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of one or more amino sequences shown below, and optionally further contain heterologous signal peptides, which do not need to be included when calculating identity percentages. A fragment may further contain signal peptides such as immunoglobulin signal peptides, e.g., IgE or IgG signal peptides.

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

[0066] As used herein, the term “gene construct” refers to a DNA or RNA molecule containing a nucleotide sequence that codes for a protein. The coding sequence includes start and terminate signals operably linked to regulatory elements, including a promoter and a polyadenylation signal capable of directing the expression of the nucleic acid molecule in the target cell to which it is administered. As used herein, the term “expressible form” refers to a gene construct containing necessary regulatory elements functionally linked to the coding sequence that codes for a protein, so that the coding sequence may be expressed if present in the target cell.

[0067] As used herein, the term “homology” refers to some degree of complementarity. There may be partial homology or complete homology (i.e., identity). A partially complementary sequence that at least partially inhibits a fully complementary sequence from hybridizing to a target nucleic acid is referred to using the functional term “substantially homologous.” When used in relation to double-stranded nucleic acid sequences such as cDNA or genomic clones, the term “substantially homologous” as used herein refers to a probe that can hybridize to a strand of the double-stranded nucleic acid sequence under low stringency conditions. When used in relation to single-stranded nucleic acid sequences, the term “substantially homologous” as used herein refers to a probe that can hybridize to a single-stranded nucleic acid template sequence (i.e., its complement) under low stringency conditions.

[0068] As used herein in the context of two or more nucleic acid or polypeptide sequences, “identical” or “sequence” means that the sequences have a certain proportion of residues that are the same across a given region. The proportion can be calculated by optimally aligning the two sequences, comparing them across a given region, determining the number of positions where identical residues occur in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the given region, and multiplying the result by 100 to obtain the proportion of sequence identity. If the two sequences are of different lengths, or if the alignment produces one or more staggered ends and the given comparison region contains 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 sequencing algorithms such as BLAST or BLAST 2.0.

[0069] The term "impedance" as used herein may be used when considering feedback mechanisms, and since it can be converted to a current value according to Ohm's law, it becomes possible to compare it with a pre-set current.

[0070] As used herein, “immune response” means the activation of the host’s immune system, for example, the mammalian immune system, in response to the introduction of an antigen. The immune response may take the form of a cellular response, a humoral response, or both.

[0071] As used herein, “nucleic acid,” “oligonucleotide,” or “polynucleotide” means at least two nucleotides covalently linked to one another. A single-stranded description also defines the sequence of the complementary strand. Thus, nucleic acid also encompasses the complementary strand of the described single-stranded nucleic acid. Many variants of nucleic acid can be used for the same purposes as a given nucleic acid. Thus, 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, nucleic acid also encompasses probes that hybridize under stringent hybridization conditions.

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

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

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

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

[0076] "Signal peptide" and "leader sequence" are used interchangeably herein and refer to amino acid sequences that can be ligated at the amino terminus of proteins described herein. Signal peptides / leader sequences are typically directed toward protein localization. The signal peptides / leader sequences used herein are preferably designed to facilitate the secretion of the protein from the cell from which it is produced. During secretion from the cell, signal peptides / leader sequences are often cleaved from the rest of the protein, often referred to as the mature protein. Signal peptides / leader sequences are ligated at the amino terminus (i.e., the N terminus) of the protein.

[0077] As used herein, “stringent hybridization conditions” refer to conditions under which a first nucleic acid sequence (e.g., a probe) hybridizes to a second nucleic acid sequence (e.g., a target) in complex mixtures of nucleic acids. Stringent conditions are sequence-dependent and vary in different contexts. Stringent conditions may be selected to be approximately 5–10°C lower than the thermal melting point (Tm) of the specific sequence at a defined ionic strength pH. Tm may be the temperature at which 50% of the probe complementary to the target hybridizes to the target sequence at equilibrium (under defined ionic strength, pH, and nucleic acid concentration) (since the target sequence is present in excess, at Tm, 50% of the probe is occupied at equilibrium). Stringent conditions may be a salt concentration of less than approximately 1.0 M sodium ions at pH 7.0–8.3, e.g., approximately 0.01–1.0 M sodium ion concentration (or other salts), and a temperature of at least approximately 30°C for short probes (e.g., approximately 10–50 nucleotides) and at least approximately 60°C for long probes (e.g., more than approximately 50 nucleotides). Stringent conditions can also be achieved by adding an stabilizer such as formamide. With respect to selective or specific hybridization, a positive signal may be at least 2–10 times greater than background hybridization. Exemplary stringent hybridization conditions include: 50% formamide, 5x SSC, and 1% SDS, incubated at 42°C, or 5x SSC, 1% SDS, incubated at 65°C, washed with 0.2x SSC and 0.1% SDS at 65°C.

[0078] As used herein, “subject” may mean a mammal that desires or requires to be immunized with the vaccine described herein. Mammals may be humans, chimpanzees, dogs, cats, horses, cattle, mice, or rats.

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

[0080] As used herein, “substantially identical” means that the first and second sequences contain 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. This means that, across the acid region, the sequences are identical by 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%, or, with respect to nucleic acids, the first sequence is substantially complementary to the complement of the second sequence.

[0081] As used herein, “to treat,” “to cure,” or “to treat” may mean protecting an animal from disease by preventing, suppressing, inhibiting, or completely eliminating the disease. Prevention of disease involves administering the vaccine of the present invention to an animal before the onset of the disease. Suppression of disease involves administering the vaccine of the present invention to an animal after the induction of the disease but before its clinical manifestation. Inhibition of disease involves administering the vaccine of the present invention to an animal after the clinical manifestation 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) a complement of a reference nucleotide sequence or a portion thereof, (iii) a nucleic acid substantially identical to a reference nucleic acid or its complement, or (iv) a nucleic acid that, under stringent conditions, hybridizes to a reference nucleic acid, its complement, or a sequence substantially identical thereto.

[0083] As used herein with respect to peptides or polypeptides, “variant” means a peptide or polypeptide having a different amino acid sequence due to an amino acid insertion, deletion, or conservative substitution, but retaining at least one biological activity. A variant may 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 amino acids, i.e., replacing an amino acid with an amino acid having a similar property (e.g., hydrophilicity, degree, and distribution of the charged region), is recognized in the art as typically involving only minor changes. These minor changes can be identified, in part, by considering the hydrophobicity-hydrophilicity index of an amino acid, as understood in the art. Kyte et al., J.Mol.Biol.157:105-132 (1982). The hydrophobicity-hydrophilicity index of an amino acid is based on considering its hydrophobicity and charge. It is known in the art that amino acids with similar hydrophobicity-hydrophilicity indices can be substituted and still retain protein function. In one embodiment, amino acids having a hydrophobicity-hydrophilicity index of ±2 are substituted. The hydrophilicity of amino acids can also be used to identify substitutions that would result in proteins retaining biological function. In the context of peptides, considering the hydrophilicity of amino acids allows for the calculation of the peptide's maximum local mean hydrophilicity, a useful means that has been reported to correlate well with antigenicity and immunogenicity. U.S. Patent No. 4,554,101, fully incorporated herein by reference. By substituting amino acids with similar hydrophilicity values, peptides retaining biological activity, such as immunogenicity, can be obtained, as understood in the art. Substitutions can be made with amino acids having hydrophilicity values ​​within ±2 of each other. Both the hydrophobicity-hydrophilicity index and hydrophilicity value of an amino acid are influenced by the specific side chain of that amino acid. Consistent with that observation, it is understood that amino acid substitutions compatible with biological function depend on the relative similarity of amino acids and, in particular, on their side chains, as revealed by their hydrophobicity, hydrophilicity, charge, size, and other properties.

[0084] A variant can be a nucleic acid sequence that is substantially identical across the full length or a fragment of the complete gene sequence. A 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 across the full length or a fragment of the gene sequence. A variant can be an amino acid sequence that is substantially identical across the full length or a fragment of the amino acid sequence. An 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 across 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. A vector may be a viral vector, a bacteriophage, a bacterial artificial chromosome, or a yeast artificial chromosome. A vector may be a DNA or RNA vector. A vector may be a self-replicating extrachromosomal vector, preferably a DNA plasmid. A vector may contain or may contain one or more heterologous nucleic acid sequences.

[0086] vaccine Vaccines comprising a synthetic consensus survivor antigen, a nucleic acid molecule encoding an antigen, a nucleic acid molecule encoding a fragment of an antigen, a nucleic acid molecule encoding a variant of an antigen, or a nucleic acid molecule encoding a combination thereof are provided herein. The vaccine may be capable of inducing an immune response to the antigen in a subject. The immune response may be a therapeutic or prophylactic immune response. The vaccine may comprise one or more 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 survivor 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 containing 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 containing 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 containing 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 containing 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 includes a nucleic acid sequence encoding SEQ ID NO: 4, a nucleic acid sequence encoding a fragment containing 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 containing 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 includes SEQ ID NO: 2, a fragment containing 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 containing 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 survivor antigen, the antigen comprising 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 survivor antigen, the antigen comprising 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] Vaccines can be used to protect against cancer, for example, cancer or tumors that express survivin. Vaccines can be used in subjects that need them to prevent and / or treat tumors that express survivin. Vaccines can induce cellular and / or antibody responses to survivin and tumors that express survivin.

[0092] In one embodiment, the vaccine can be used to protect, prevent and / or treat, or induce cellular and / or antibody responses against ovarian cancer cells expressing survivin, more specifically epithelial ovarian cancer cells expressing survivin, more specifically serous ovarian cancer cells expressing survivin.

[0093] The development of cancer vaccines described herein involves identifying cancer antigens, such as survivors, that are not recognized by the immune system and are tumor-associated ("cancer / testis", "C / T") antigens. The identified cancer antigens are modified from autoantigens to exogenous antigens in order to be recognized by the immune system. Redesigning the nucleic acid and amino acid sequences of recombinant cancer antigens from autologous to exogenous antigens disrupts the immune system's resistance to the antigen. Several redesignation methods may be applied to cancer antigens to disrupt tolerance, as described below.

[0094] Recombinant cancer antigens in vaccines are not recognized as self, thus disrupting tolerance. Disrupting tolerance induces antigen-specific T cell and / or high-titer antibody responses, thereby inducing or triggering an immune response that is direct or reactive against cancer or tumors expressing the antigen. In some embodiments, the induced or triggered immune response may be cellular, humoral, or both. In some embodiments, the induced or triggered cellular immune response may 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 induced immune response may reduce or inhibit one or more immunosuppressive factors that promote the growth of antigen-expressing tumors or cancers, 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 TFG-β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by immunosuppressive cells, CTLA-4, PD-1, MDSC, MCP-1, and factors that upregulate immune checkpoint molecules.

[0095] In certain embodiments, the vaccine is (1) CD8 + and / or CD107a + (1) Increase cytotoxic T lymphocytes such as (CTLs) to attack and kill tumor cells; (2) Increase the T helper cell response; and / or (3) Increase the inflammatory response via IFN-γ, IL-2, and TFN-α, or preferably all of the foregoing, thereby mediating clearance or preventing tumor cell proliferation.

[0096] The vaccine may be a DNA vaccine. DNA vaccines are disclosed in U.S. Patents 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 fully incorporated herein by reference. The DNA vaccine may further include elements or reagents that inhibit its integration into chromosomes.

[0097] Vaccines may contain RNA that codes for cancer antigens. RNA vaccines can be introduced into cells.

[0098] Vaccines may be live attenuated vaccines, vaccines that deliver antigens using recombinant vectors, subunit vaccines, and glycoprotein vaccines, for example, but are not limited to these, U.S. vaccines No. 4,510,245, No. 4,797,368, No. 4,722,848, No. 4,790,987, No. 4,920,209, No. 5,017,487, No. 5,077,044, No. 5,110,587, No. 5,112,749, No. 5,174,993, No. 5,223,424, No. 5,225,336, No. 5,240,703, No. 5,242,829, No. 5,294,441, and No. 5,294,548, No. 5,310,668, No. 5,387,744, No. 5,389,368, No. 5,424,065, No. 5,451,4 No. 99, No. 5,453,364, No. 5,462,734, No. 5,470,734, No. 5,474,935, No. 5,482,713, No. 5, These are described in Nos. 591,439, 5,643,579, 5,650,309, 5,698,202, 5,955,088, 6,034,298, 6,042,836, 6,156,319, and 6,589,529, each of which is incorporated herein by reference.

[0099] In some embodiments, the nucleic acid vaccine may further include coding sequences for molecular adjuvants, in some cases the molecular adjuvants may be IL-12, IL-15, IL-28, IL-31, IL-33, and / or RANTES, and in some cases the molecular adjuvants may be checkpoint inhibitors, including anticytotoxic T lymphocyte antigen 4 (CTLA-4), anti-programmed death receptor 1 (PD-1), and anti-lymphocyte activator gene (LAG-3). The coding sequences for IL-12, IL-15, IL-28, IL-31, IL-33, and / or RANTES may be contained in one or more nucleic acid molecules containing coding sequences for one or more antigens. The coding sequences for IL-12, IL-15, IL-28, IL-31, IL-33, and / or RANTES may be contained in one or more separate nucleic acid molecules, such as one or more separate plasmids or vectors, which are administered in combination with the nucleic acid vaccine.

[0100] The vaccine of the present invention may possess the characteristics required of an effective vaccine, such as being safe so as not to cause disease or death itself, protecting against disease, inducing neutralizing antibodies, inducing a protective T-cell response, being easy to administer, having few side effects, being biologically stable, and having a low cost per dose. The vaccine may achieve some or all of these characteristics by containing nucleic acid molecules (may include) encoding cancer antigens, as discussed below.

[0101] Vaccines combined with immune checkpoint inhibitors The vaccine may further contain 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. Immune checkpoint inhibitors are any nucleic acids or proteins that prevent 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 may be nucleic acid sequences, amino acid sequences, small molecules, or combinations thereof. Nucleic acid sequences may be DNA, RNA, cDNA, their variants, their fragments, or combinations thereof. Nucleic acids may also include additional sequences encoding linker or tag sequences that are linked to immune checkpoint inhibitors by peptide bonds. Small molecules may be low molecular weight, e.g., enzyme substrates, proteins, or ligands (or analogs thereof) bound by nucleic acids, or organic or inorganic compounds that function as regulators of biological processes. Amino acid sequences may be proteins, peptides, their variants, their fragments, or combinations thereof.

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

[0104] Immune checkpoint molecules Immune checkpoint molecules can be nucleic acid sequences, amino acid sequences, small molecules, or combinations thereof. Nucleic acid sequences can be DNA, RNA, cDNA, their variants, their fragments, or combinations thereof. Nucleic acids can also contain additional sequences encoding linker or tag sequences that are linked to immune checkpoint inhibitors by peptide bonds. Small molecules can be low molecular weight, e.g., enzyme substrates, proteins, or ligands (or analogs thereof) bound by nucleic acids, or organic or inorganic compounds that function as regulators of biological processes. Amino acid sequences can be proteins, peptides, their variants, their fragments, or combinations thereof.

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

[0106] PD-1 has two ligands, PD-L1 and PD-L2, and is a member 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, mast cell tumors, and melanoma.

[0107] Anti-immune checkpoint molecules (antibodies) As described above, immune checkpoint inhibitors can be antibodies. Antibodies can bind to or react with antigens (i.e., the immune checkpoint molecules described above). Therefore, antibodies can be considered anti-immune checkpoint molecule antibodies or immune checkpoint molecule antibodies. Antibodies can be encoded by the nucleic acid sequence they contain.

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

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

[0110] A heavy chain polypeptide may contain a set of complementarity-determining regions ("CDRs"). The CDR set may contain three hypervariable regions within the VH region. These CDRs, in order from the N-terminus of the heavy chain polypeptide, are denoted as "CDR1," "CDR2," and "CDR3," respectively. CDR1, CDR2, and CDR3 of the heavy chain polypeptide can contribute to antigen binding or recognition.

[0111] Light chain polypeptides may contain a variable light chain (VL) region and / or a constant light chain (CL) region. Light chain polypeptides may also contain a set of complementarity-determining regions ("CDRs"). A set of CDRs may contain three hypervariable regions from the VL region. These CDRs, in order from the N-terminus of the light chain polypeptide, are denoted "CDR1," "CDR2," and "CDR3," respectively. CDR1, CDR2, and CDR3 of the light chain polypeptide can contribute to antigen binding or recognition.

[0112] The antibody may contain sets of heavy-chain and light-chain complementarity-determining regions ("CDRs"), which are inserted between sets of heavy-chain and light-chain frameworks ("FRs") that provide support to the CDRs and mutually define the spatial relationships of the CDRs. A set of CDRs may contain three hypervariable regions from either the heavy or light-chain V region. Starting from the N-terminus of the heavy or light chain, these regions are denoted as "CDR1," "CDR2," and "CDR3," respectively. Thus, the antigen-binding site may contain six CDRs, including sets of CDRs from each of the heavy and light-chain V regions.

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

[0114] Additionally, the proteolytic enzyme papain preferentially cleaves the IgG molecule to produce 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 the F(ab')2 fragment, which contains both antigen-binding sites. Therefore, the antibody can be either Fab or F(ab')2. Fab can include heavy-chain polypeptides and light-chain polypeptides. The heavy-chain polypeptide of Fab can include a VH region and a CH1 region. The light chain of Fab can include a VL region and a CL region.

[0115] Antibodies may be polyclonal or monoclonal antibodies. Antibodies may be chimeric antibodies, single-chain antibodies, affinity mature antibodies, human antibodies, humanized antibodies, or fully human antibodies. Humanized antibodies may be antibodies from non-human species that bind 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 Antiimmune checkpoint molecule antibodies may be anti-PD-1 antibodies (also referred to herein as "PD-1 antibodies"), their variants, their fragments, or combinations thereof. The PD-1 antibody may be nivolumab. Anti-PD-1 antibodies can inhibit PD-1 activity, thereby inducing, triggering, or increasing the immune response against tumors or cancer, and reducing tumor growth.

[0117] PD-L1 antibody Antiimmune checkpoint molecule antibodies may be anti-PD-L1 antibodies (also referred to herein as "PD-L1 antibodies"), their variants, their fragments, or combinations thereof. Anti-PD-L1 antibodies can inhibit PD-L1 activity, thereby inducing, triggering, or increasing the immune response against tumors or cancer, and reducing tumor growth.

[0118] antigen As described above, vaccines may contain an antigen or nucleic acid encoding an antigen. The antigen may be a survivor, a fragment thereof, a variant thereof, or a combination of a fragment and its variant.

[0119] Therefore, vaccines can be used to treat subjects suffering from cancer or tumors that express Survivin. In some embodiments, the cancer is ovarian cancer. In some embodiments, the ovarian cancer is epithelial ovarian cancer. The ovarian cancer may be serous epithelial ovarian cancer. Vaccines can also be used to treat subjects having cancer or tumors that express Survivin in order to prevent the development of such tumors in the subject. Synthetic consensus Survivin antigens can be obtained differently from the natural Survivin gene and therefore provide therapy or prevention against tumors that express synthetic consensus Survivin antigens. Accordingly, synthetic consensus Survivin antigen sequences (i.e., mutations or synthetic Survivin genes or sequences) different from the natural Survivin gene are provided herein.

[0120] The transcripts of the natural 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, survivin isoforms are selected based on their expression in ovarian cancer cells. The synthetic consensus survivin antigen sequences described herein avoid selective processing, producing a single full-length transcript, resulting in stronger induction of effector T and B cell responses.

[0121] Isolated nucleic acid molecules containing the heterologous sequences described above are provided. Isolated nucleic acid molecules consisting of the heterologous sequences described above are provided. Isolated nucleic acid molecules containing 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 synthetic consensus survivin antigens are provided herein. The coding sequences encoding synthetic consensus survivin antigens have sequences as described above.

[0122] Protein molecules containing the heterogeneous amino acid sequences described above are provided. Protein molecules consisting of the heterogeneous 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. Synthetic consensus survivin antigens can induce an immune response against tumors expressing survivin.

[0123] In one embodiment, it is desirable that a synthetic consensus survivor antigen provides improved transcription and translation, comprising one or more of the following: a low GC-containing leader sequence for increasing transcription, optimization of mRNA stability and codons, and, where possible, elimination of cis-acting sequence motifs (i.e., internal TATA boxes).

[0124] A synthetic consensus survivin antigen may 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 may include a portion of a first isoform combined with a portion of a second isoform, the portion of the second isoform being, for example, non-homologous to any portion of the first isoform. A synthetic consensus survivin antigen may include consensus sequences and / or modifications to improve expression. Modifications include codon optimization, RNA optimization, addition of Kozak sequences (e.g., GCC ACC) to enhance translation initiation, and / or addition of immunoglobulin reader sequences to enhance the immunogenicity of the synthetic consensus survivin antigen. Synthetic consensus survivin antigens may, but are not limited to, include immunoglobulin signal peptides, such as immunoglobulin E (IgE) or immunoglobulin G (IgG) signal peptides. In some embodiments, synthetic consensus survivin antigens may include localization signal sequences, such as mutations or deletions to nuclear localization signals to disrupt nuclear localization at translation. In some embodiments, survivin consensus antigens may include hemagglutinin (HA) tags. Survivin consensus antigens can be designed to induce more potent and broader cellular and / or humoral immune responses than corresponding non-codon-optimized survivin antigens.

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

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

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

[0128] vector A vaccine may contain one or more vectors containing heterologous nucleic acids encoding a synthetic consensus survivor antigen. One or more vectors may be capable of expressing the antigen in amounts effective enough to induce an immune response in mammals. The vectors may contain heterologous nucleic acids encoding the antigen. The vectors may have nucleic acid sequences containing origins of replication. The vectors may be plasmids, bacteriophages, bacterial artificial chromosomes, or yeast artificial chromosomes. The vectors may be either self-replicating extrachromosomal vectors or vectors integrated into the host genome.

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

[0130] Vectors may have expression signals such as strong promoters, strong stop codons, adjustments to the distance between the promoter and the cloned gene, and insertions of transcription termination sequences and PTIS (portable translation initiation sequences).

[0131] The vector may be a circular plasmid or a linear nucleic acid. Both circular plasmids and linear nucleic acids can direct the expression of a specific nucleotide sequence in appropriate target cells. The vector may have a promoter operably linked to the nucleotide sequence encoding an antigen, and may be operably linked to a termination signal. The vector may 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 the nucleotide sequence of interest may be a chimeric vector, meaning that at least one of its components is heterogeneous with respect to at least one of the other components. The expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive or inductive promoter, and transcription may be initiated only when the host cell is exposed to some specific external stimulus. In the case of multicellular organisms, the promoter may also be specific to a particular tissue or organ or developmental stage. In preferred embodiments, the plasmid vector is pGX1428 as described herein, further comprising the nucleic acid sequence of SEQ ID NO: 1, or pGX1429 as described herein, further comprising the nucleic acid sequence of SEQ ID NO: 3.

[0132] The vector can be a plasmid. Plasmids can be useful for transfecting cells with nucleic acids that encode an antigen. Transformed host cells can be cultured and maintained under conditions in which antigen expression occurs.

[0133] A plasmid may comprise nucleic acid sequences encoding one or more of the various antigens disclosed above, including a consensus antigen, a fragment of such protein, a variant of such protein, or a fragment of a variant or fusion protein, which can induce an immune response to a synthetic antigen, and these consist of combinations of consensus proteins and / or fragments of consensus proteins and / or variants of consensus proteins and / or fragments of variants of consensus proteins.

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

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

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

[0137] Plasmids may also include promoters operably ligated to the coding sequence. Promoters operably ligated to the coding sequence may be from human immunodeficiency virus (HIV) promoters such as monkey virus 40 (SV40), mouse mammary cancer virus (MMTV) promoter, bovine immunodeficiency virus (BIV) long-terminal repeat (LTR) promoter, cytomegalovirus (CMV) promoters such as Moloney virus promoter, avian leukemia virus (ALV) promoter, CMV early promoter, Epstein-Barr virus (EBV) promoter, or Roussarcoma virus (RSV) promoter. Promoters may also be from human genes such as human actin, human myosin, human hemoglobin, human muscle creatine, or human metallothionein. Promoters may also be tissue-specific promoters, such as natural or synthetic muscle or skin-specific promoters. Examples of such promoters are described in U.S. Patent Application Publication US2004 / 0175727, the entirety of which is incorporated herein by reference.

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

[0139] Plasmids may also contain enhancers upstream of the coding sequence. Enhancers may be human actin, human myosin, human hemoglobin, human muscle creatine, or viral enhancers such as those from CMV, FMDV, RSV, or EBV. Polynucleotide functional enhancers are described in U.S. Patents 5,593,972, 5,962,428, and W094 / 016737, the contents of which are fully incorporated by reference.

[0140] Plasmids may also contain mammalian origins of replication to maintain the plasmid outside the chromosome and produce multiple copies of the plasmid in cells. Plasmids may be pVAXI, pCEP4, or pREP4 from Invitrogen (San Diego, CA), containing Epstein-Barr virus replication origins and the nuclear antigen EBNA-1 coding region, and capable of generating high-copy episomal replication without incorporation. The plasmid backbone may be pA V0242. Plasmids may also be replication-deficient adenovirus type 5 (Ad5) plasmids.

[0141] Plasmids may also contain regulatory sequences that may be suitable for gene expression in cells to which the plasmid is administered. The coding sequences may contain codons that may enable more efficient transcription of the coding sequences in host cells.

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

[0143] The plasmid may be pSE420 (Invitrogen, San Diego, Calif.), which can be used for protein production in Escherichia coli (E. coli). Alternatively, the plasmid may be YES2 (Invitrogen, San Diego, Calif.), which can be used for protein production in the yeast strain Saccharomyces cerevisiae. The plasmid may also be from the MAXBAC® complete baculovirus expression system (Invitrogen, San Diego, Calif.), which can be used for protein production in insect cells. The plasmid may 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 may transform target cells by being incorporated into the cell genome, or it may be a circular plasmid that exists outside the chromosome (e.g., a self-replicating plasmid with an origin of replication).

[0145] The vector may be pVAX, pcDNA3.0, or provax, or any other expression vector that expresses antigen-coding DNA and allows cells 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 target by electroporation and express one or more desired antigens. An LEC may be any linear DNA lacking any phosphate backbone. The DNA may encode one or more antigens. An LEC may contain a promoter, introns, stop codons, and / or polyadenylation signals. Antigen expression may be controlled by the promoter. An LEC may not contain any antibiotic resistance genes and / or phosphate backbone. An LEC may not contain other nucleic acid sequences unrelated to the expression of the desired antigen gene.

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

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

[0149] A vector may have a promoter. The promoter can be any promoter capable of driving gene expression and controlling the expression of an isolated nucleic acid. Such a promoter is a cis-acting sequence element required for transcription via DNA-dependent RNA polymerase transcribing the antigen sequences described herein. The choice of promoter used to direct the expression of heterologous nucleic acids depends on the specific application. Promoters may be positioned at approximately the same distance from the transcription start site 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 ligated to the transcript, the ribosome binding site, and the nucleic acid sequence encoding the antigen and signal necessary for efficient polyadenylation at translation termination.

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

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

[0153] How to prepare a vector A method for preparing a vector comprising a nucleic acid molecule encoding a synthetic consensus survivor antigen discussed herein is provided herein. The vector may be used to seed into a cell culture in a large-scale fermentation tank using methods known in the art after the final subcloning step.

[0154] The vectors are used with an EP device and can be formulated or manufactured using a combination of known devices and techniques, as described in more detail below, but are preferably manufactured using the optimized plasmid manufacturing technique described in U.S. Provisional Application U.S. 60 / 939,792, filed May 23, 2007 (see U.S. Patent Publication 2009 / 0004716). In some examples, the DNA vectors used in these studies may be formulated at concentrations of 10 mg / mL or higher. The manufacturing techniques also include, or incorporate, a variety of devices and protocols that are generally known to those skilled in the art, in addition to those described in U.S. 60 / 939792, including those described in U.S. 7,238,522, issued July 3, 2007. The applications and patents referenced above, U.S. 60 / 939,792 and U.S. 7,238,522, are incorporated herein by reference in their entirety, respectively.

[0155] Vaccine excipients and other components The vaccine may further contain pharmaceutically acceptable excipients. pharmaceutically acceptable excipients may be functional molecules such as vehicles, carriers, or diluents. pharmaceutically acceptable excipients may be transfection promoters and may include surfactants such as immunostimulatory complexes (ISCOMS), Freund's incomplete adjuvants, 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] Transfection promoters are polyanions, polycations (including poly-L-glutamate (LGS)), or lipids. The transfection promoter is poly-L-glutamate, which may be present in the vaccine at concentrations less than 6 mg / ml. Transfection promoters may also include immunostimulatory complexes (ISCOMS), Freund's incomplete adjuvants, LPS analog-containing monophosphoryl lipid A, muramil peptides, quinone analogs, and surfactants such as squalene and squalene vesicles, and hyaluronic acid administered with the gene construct may also be used. DNA vector vaccines may also contain transfection promoters such as lipids and liposomes, and the DNA-liposome mixture may include 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 accelerator is a polyanion, polycation (including poly-L-glutamate (LGS)), or lipid. The concentration of the transfection agent in the vaccine is less than 4 mg / ml, less than 2 mg / ml, less than 1 mg / ml, less than 0.750 mg / ml, less than 0.500 mg / ml, less than 0.250 mg / ml, less than 0.100 mg / ml, less than 0.050 mg / ml, or less than 0.010 mg / ml.

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

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

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

[0160] The vaccine may further contain a gene vaccine enhancer as described in U.S. Patent No. 021,579 (fully incorporated by reference), filed on April 1, 1994.

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

[0162] Vaccines can be formulated according to the mode of administration used. Injectable vaccine pharmaceutical compositions may be sterile, pyrogenic, and particulate-free. Isotonic formulations or solutions may be used. Additives for isotonicity may include sodium chloride, dextrose, mannitol, sorbitol, and lactose. Vaccines may contain vasoconstrictors. Isotonic solutions may include phosphate-buffered saline. Vaccines may further contain stabilizers, including gelatin and albumin. Stabilizers, including LGS or polycations or polyanions, can stabilize formulations for extended periods at room temperature or ambient temperature.

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

[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 vaccine nucleic acid molecules. In some embodiments, the pharmaceutical composition contains 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, and 23 0, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 605, 610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695, 700, 705, 710, 715, 720, 725, 730, 735, 740, 745, 750, 755, 760, 765, 770, 775, 780, 785, 790, 795, 800, 8 May contain 05, 810, 815, 820, 825, 830, 835, 840, 845, 850, 855, 860, 865, 870, 875, 880, 885, 890, 895, 900, 905, 910, 915, 920, 925, 930, 935, 940, 945, 950, 955, 960, 965, 970, 975, 980, 985, 990, 995, or 1000 micrograms of vaccine nucleic acid molecules(s). In some embodiments, the pharmaceutical composition may contain at least 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 mg or more of vaccine nucleic acid molecules.

[0165] In other embodiments, the pharmaceutical composition may include 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) of nucleic acid molecules(s) of the vaccine. In some embodiments, the pharmaceutical composition is up to 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235 ,240,245,250,255,260,265,270,275,280,285,290,295,300,305,310,315,320,325,330,335,340,345,350,355,360,365,370,375,380,385,390,395,400,405,410,415,420,425,430,435,440,445,450,455,460,465,470,47 5, 480, 485, 490, 495, 500, 605, 610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695, 700, 705, 710, 715, 720, 725, 730, 735, 740, 745, 750, 755, 760, 765, 770, 775, 780, 785, 790, 795, 800, 805, 810, 8 It may contain 15, 820, 825, 830, 835, 840, 845, 850, 855, 860, 865, 870, 875, 880, 885, 890, 895, 900, 905, 910, 915, 920, 925, 930, 935, 940, 945, 950, 955, 960, 965, 970, 975, 980, 985, 990, 995, or 1000 micrograms (including these values) of vaccine nucleic acid molecules(s). In some embodiments, the pharmaceutical composition may contain up to 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 mg (including these values) of the nucleic acid molecule(s) of the vaccine.

[0166] Pharmaceutical compositions may further contain other agents for the formulation purpose depending on the mode of administration used. If the pharmaceutical composition is an injectable drug composition, it is sterile, pyrogenic, 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. Gelatin and albumin are included as stabilizers. In some embodiments, vasoconstrictors are added to the formulation.

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

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

[0169] Vaccination methods Methods for treating and / or preventing cancer using the pharmaceutical formulations described herein are provided herein. Methods for using the pharmaceutical formulations described herein to treat and / or prevent cancer in subjects are also described herein. Methods for vaccinating subjects are also described herein. Methods for administering the pharmaceutical formulations described herein to subjects in need are also described herein. Methods described herein, collectively referred to as methods for treatment using the pharmaceutical formulations described herein, may include administering one or more vaccines described herein to subjects in need to induce a therapeutic and / or prophylactic immune response. Vaccines may be administered to subjects to modulate the activity of the subject's immune system and enhance the immune response. Vaccine administration may be transfection of cancer antigens disclosed herein as nucleic acid molecules that are expressed in cells and, upon delivery to the cell surface, cause the immune system to recognize and induce cellular, humoral, or cellular and humoral responses. Vaccine administration may be used to induce or trigger an immune response in a subject to one or more of the cancer antigens disclosed herein by administering the vaccines discussed herein to the subject.

[0170] Vaccines are 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 mammal is administered a vaccine, thereby introducing the vector into mammalian cells, the transfected cells express and secrete one or more of the cancer antigens disclosed herein. These secreted proteins or synthetic antigens are recognized as foreign by the immune system, which initiates an immune response that may include antibodies produced against one or more cancer antigens and a T-cell response specific to one or more cancer antigens. In some examples, a mammal vaccinated with the vaccines discussed herein has an antigen-stimulated immune system, and when inoculated with one or more of the cancer antigens disclosed herein, the antigen-stimulated immune system can rapidly clear the subsequent cancer antigens disclosed herein, whether by humoral, cellular, or both cellular and humoral immune responses.

[0171] Methods for administering DNA vaccines are described in U.S. Patents No. 4,945,050 and No. 5,036,006, both of which are incorporated herein by reference in their entirety.

[0172] Vaccines can be administered to mammals to induce an immune response in them. Mammals may include humans, non-human primates, cattle, pigs, sheep, goats, antelopes, bison, buffalo, deer, hedgehogs, elephants, llamas, alpacas, mice, rats, preferably humans, cattle, or pigs. Vaccines can similarly be administered to non-human subjects, such as chickens, to induce an immune response.

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

[0174] Methods for generating an immune response through vaccines Vaccines can be used to generate immune responses in mammalian or non-mammalian subjects, including therapeutic or prophylactic immune responses. These immune responses can generate antibodies and / or killer T cells directed to one or more cancer antigens, as disclosed herein. Such antibodies and T cells can be isolated.

[0175] Some embodiments provide methods for generating an immune response to one or more cancer antigens disclosed herein, and such embodiments include administering a vaccine to a subject. Some embodiments provide methods for prophylactically vaccinating a subject against cancer or tumors expressing one or more of the aforementioned cancer antigens, and such embodiments include administering a vaccine. Some embodiments provide methods for therapeutically vaccinating a subject suffering from cancer or tumors expressing one or more of the cancer antigens, and such embodiments include administering a vaccine. Diagnosis of cancer or tumors expressing one or more cancer antigens disclosed herein can be performed routinely before vaccine administration.

[0176] Methods of cancer treatment using vaccines Vaccines can be used to generate or induce an immune response in mammals that are reactive to or targeted to cancer or tumors (e.g., ovarian cancer) that express survivin in the mammal or in a target that requires it. 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. Therefore, vaccines can be used in ways to treat and / or prevent cancer or tumors in vaccinated mammals or subjects.

[0178] Route of administration Vaccines or pharmaceutical compositions may be administered orally, parenterally, sublingually, percutaneously, rectally, transmucosally, topically, by inhalation, buccally, intrapleurally, intravenously, intraarterially, intraperitoneally, subcutaneously, intramuscularly, intranasally, subarachnoidally, and / or intraarticularly, or in combination thereof. For veterinary use, compositions may be administered as formulations appropriately acceptable according to the usual veterinary practice. Veterinarians can readily determine the most appropriate administration regimen and route of administration for a particular animal. Vaccines may be administered by conventional syringes, needleless injectors, "microprojectile bombardment gene guns," or by other physical methods such as electroporation ("EP"), "hydrodynamics," or ultrasound.

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

[0180] Vaccines or pharmaceutical compositions containing vaccines may be administered by electroporation. Electroporation of vaccines can be achieved using an electroporation device configured to deliver pulses of energy effective in causing reversible pores to form in the cell membrane to desired tissues of a mammal, preferably a constant current equivalent to a preset current input by the user. The electroporation device may comprise electroporation components and electrode assemblies or handle assemblies. Electroporation components may include, and can incorporate, one or more of the various elements of the electroporation device, including controllers, current waveform generators, impedance testers, waveform loggers, input elements, status reporting elements, communication ports, memory components, power supplies, and power switches. Electroporation may be achieved using in vivo electroporation devices, such as the CELLECTRA® EP system (Inovio Pharmaceuticals, Inc., Blue Bell, PA) or the Elgen electroporator (Inovio Pharmaceuticals, Inc.), to facilitate the transfection of cells with vectors.

[0181] Examples of electroporation devices and electroporation methods that can facilitate the administration of DNA vaccines of the present invention include those described in U.S. Patent No. 7,245,963 by Draghia-Akli et al. and U.S. Patent Publication No. 2005 / 0052630 filed by Smith et al., the contents of which are incorporated herein by reference in their entirety. Other electroporation devices and electroporation methods that can be used to facilitate the administration of DNA vaccines include those provided in concurrently pending and jointly owned U.S. Patent Application No. 11 / 874072 filed on 17 October 2007, which claims benefit under U.S. Provisional Application No. 60 / 852,149 filed on 17 October 2006 and U.S. Provisional Application No. 60 / 978,982 filed on 10 October 2007, under Section 119(e) of the U.S. Patent Act, all of which are incorporated herein 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 plant. The modular electrode system may comprise multiple needle electrodes, a subcutaneous needle, an electrical connector providing conductive connections from a programmable constant-current pulse controller to the multiple needle electrodes, and a power supply. An operator can grasp the multiple needle electrodes mounted on a support structure and firmly insert them into selected tissue of the body or plant. The biomolecule is then administered into the selected tissue via the subcutaneous needle. The programmable constant-current pulse controller is activated to apply constant-current electrical pulses to the multiple needle electrodes. The applied constant-current electrical pulses facilitate the introduction of biomolecules into cells between the multiple electrodes. The entirety of U.S. Patent No. 7,245,963 is incorporated herein by reference.

[0183] U.S. Patent Publication 2005 / 0052630, filed by Smith et al., describes an electroporation device that can be used to effectively facilitate the introduction of biomolecules into cells of selected tissues of the body or plants. The electroporation device comprises an electrodynamic device ("EKD device") whose operation is specified by software or firmware. The EKD device generates a series of programmable constant-current pulse patterns between a row of electrodes and pulse parameter inputs based on user control, enabling the storage and retrieval 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 entirety of U.S. Patent Publication 2005 / 0052630 is fully incorporated herein by reference.

[0184] The electrode array and method described in U.S. Patent No. 7,245,963 and U.S. Patent Publication No. 2005 / 0052630 may be suitable for deep penetration into other tissues or organs as well as muscle tissue. The configuration of the electrode array allows the injection needle to be fully inserted into the target organ, and the injection is delivered perpendicularly to the target tissue in a pre-defined area 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, contemplated in several embodiments incorporating electroporation devices and their use: U.S. Patent No. 5,273,525 issued December 28, 1993; U.S. Patent No. 6,110,161 issued August 29, 2000; U.S. Patent No. 6,261,281 issued July 17, 2001; and U.S. Patent No. 6,958,060 issued October 25, 2005; and the electroporation device described in U.S. Patent No. 6,939,862 issued September 6, 2005. Furthermore, patents covering 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 the various devices, and U.S. Patent No. 7,328,064 issued February 5, 2008, which focuses on a method of injecting DNA, are contemplated herein. The above patents are incorporated in their entirety by reference.

[0186] Methods for preparing vectors comprising nucleic acid molecules(or more) encoding synthetic consensus survivor antigens discussed herein are provided herein. The vectors may be used to seed cell cultures in large-scale fermentation tanks using methods known in the art, after the final subcloning step to mammalian expression plasmids.

[0187] 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 technique described in U.S. Publication Application No. 2009 / 0004716, filed on 23 May 2007. In some examples, the DNA vectors used in these studies may be formulated at concentrations of 10 mg / mL or higher. The manufacturing technique also includes, or incorporates, various devices and protocols that are generally known to those skilled in the art, in addition to those described in U.S. Patent No. 60 / 939792, including those described in U.S. Patent No. 7,238,522, issued on 3 July 2007. The applications and patents referenced above, U.S. Patent No. 60 / 939,792 and U.S. Patent No. 7,238,522, are incorporated herein by reference in their entirety, respectively. [Examples]

[0188] The present invention is further illustrated in the following embodiments. These embodiments illustrate preferred embodiments of the present invention, but should be understood to be provided merely as examples. From the above considerations and these embodiments, those skilled in the art will be able to identify the essential features of the present invention and adapt various changes and modifications to the invention to various uses and conditions without departing from its spirit and scope. Thus, various modifications of the invention, in addition to those shown and described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

[0189] Example 1 - Generation of Consensus Survivine Isoform 1 Twenty-nine survivor isoform 1 sequences were collected from GenBank (www.ncbi.nlm.nih.gov / genbank / ). The GenBank acceptance numbers for the selected survivor 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_012 These are 516801.1, XP_007957800.1, XP_001915435.1, XP_008523102.1, AAT37504.1, XP_004432883.1, XP_003417277.1, XP_004374167.1, NP_999306.1, XP_002918421.1, NP_001003348.1, NP_001009280.1, XP_004748859.1, NP_001001855.2, and AAU89275.1.

[0190] Consensus sequences were 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 multiplex sequence alignment program. The resulting consensus survivin isoform 1 sequence shares 97.2–97.9% homology with native human survivin isoform 1. Three mutations were introduced to eliminate the potential biological function of the resulting consensus survivin isoform 1 protein, thereby abolishing survivin's anti-apoptotic activity. The three mutations are T34A, T48A, and C84A. Furthermore, upstream Kozak sequences and IgE reader sequences were added to the N-terminus to obtain higher levels of expression. In addition, 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, avoiding regions with very high (>80%) or very low (<30%) GC content, as well as cis-operating 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 synthetic consensus survivin antigen isoform 1 is shown in Sequence ID No. 1. Synthetic consensus survivin antigen isoform. 1 The amino acid sequence is shown in Sequence ID No. 2.

[0191] Synthetic consensus survivorbin antigen isoform 1 was digested with BamHI and XhoI and cloned into a proprietary expression vector pGX0001 with an expression cassette under transcriptional control of the cytomegalovirus pre-initial 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 survivorbin antigen isoform 1 construct is shown in Figure 1. The overall structure of synthetic consensus survivorbin antigen isoform 1 is shown in Figure 2. [Table 1] [Table 2]

[0192] Example 2 - Generation of Consensus Survivin Isoform 1T3 To generate a human consensus survivorbin isoform 3, eight survivorbin isoform 3 sequences were collected from GenBank (www.ncbi.nlm.nih.gov / genbank / ). The GenBank acceptance numbers for the selected survivorbin 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] Consensus sequences were 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 multiplex sequence alignment program. Six of these sequences from lower animals contained 11 extra amino acid residues at their C-terminus that are not present in the human sequence. These extra residues were removed when generating consensus human survivin isoform 3 to prevent the induction of off-target immune responses in humans. After generating human consensus survivin isoform 3, identical amino acid sequences between survivin isoforms 1 and 3 were removed from consensus survivin isoform 3. The cleaved 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 1 T3 immunogen was generated by adding a furin cleavage site between survivin antigen 1 (described above) and survivin antigen T3.

[0194] Once the consensus-synthesized consensus-survivin antigen isoform 1T3 DNA sequence was obtained, the upstream Kozak sequence and IgE reader sequence were added to the N-terminus to obtain higher levels of expression. (Yang. genes(Andre,S.et al.Increased immune response elicited by DNA vaccination with a synthetic gp120 sequence with optimized codon usage.Journal of virology 72,1497-1503(1998), Deml,L.et al.Multiple effects of codon usage optimization on expression and immunogenicity of DNA candidate vaccines encoding the human immunodeficiency virus type 1 Gag protein.Journal of virology 75,10991-11001,doi:10.1128 / JVI.75.22.10991-11001.2001(2001)). In addition, RNA optimization was performed to avoid regions with very high (>80%) or very low (<30%) GC content, as well as cis-operating sequence motifs such as internal TATA boxes, chi-sites, and ribosome entry sites in 11 and 12.Synthetic consensus survivorbin antigen isoform 1T3 was digested with BamHI and XhoI and cloned into expression vector pGX0001, which contained an expression cassette under transcriptional control of the cytomegalovirus pre-initial 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 survivorbin antigen isoform 1T3 is shown as SEQ ID NO: 3. As shown in Table 1, the amino acid sequence of synthetic consensus survivorbin antigen isoform 1T3 is shown as SEQ ID NO: 8. A schematic diagram of the synthetic consensus survivorbin antigen isoform 1T3 construct is shown in Figure 3. The characteristics of the synthetic consensus survivorbin antigen isoform 1T3 construct are shown in Table 3. [Table 3]

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

[0196] Modifications were introduced into pVAX1 to construct 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 respect to plasmid amplification, as well as antigen transcription and translation. To date, no changes have been observed in the pGX0001 sequence in any data of plasmid products in platforms using pGX0001 as a backbone. [Table 4-1] Base pairs 2, 3, and 4 are changed from ACT to CTG in the upstream skeleton of the CMV promoter.

[0197] pGX1428 is a DNA plasmid encoding the synthetic consensus survivin antigen isoform 1 (survivin antigen 1) protein. Associated mRNA production is driven by the human CMV promoter (hCMV promoter) and terminated by the bovine growth hormone 3'-end polyadenylation signal (bGH polyA). The pGX0001 backbone contains a kanamycin resistance gene (KanR) and a plasmid origin of replication (pUC ori) for production purposes. These elements are non-functional in eukaryotic cells. pGX1428 was constructed by cloning the synthetic consensus survivin antigen isoform 1 (survivin antigen 1) DNA sequences at the BamHI and XhoI sites into pGX0001, as shown in Figure 4.

[0198] pGX1429 is a DNA plasmid encoding the synthetic consensus survivin antigen isoform 1T3 (survivin antigen 1T3) protein. Associated mRNA production is driven by the human CMV promoter (hCMV promoter) and terminated by the bovine growth hormone 3'-end polyadenylation signal (bGH polyA). The pGX0001 backbone contains a kanamycin resistance gene (KanR) and a plasmid origin of replication (pUC ori) for production purposes. These elements are non-functional in eukaryotic cells. pGX1429 was constructed by cloning the synthetic consensus survivin antigen isoform 1T3 (survivin antigen 1T3) DNA sequences at the BamHI and XhoI sites into pGX0001, as shown in Figure 5.

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

[0200] material and method plasmid Synthetic Consensus Survivin Antigen 1 (pGX1428) and Synthetic Consensus Survivin Antigen 1T3 (pGX1429) were designed as described in this document. 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 The expression of antigen proteins mediated 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 treated with Turbofectin 8 (Origene) to express pGX1428, pGX1429, or pGX0001 (6 μg / 10 cm³). 2 The cells were transfected with (Dish). 48 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 total protein concentration, 15 μg of the cell lysates were 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), followed by horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Santa Cruz Biotech, sc-2005), and visualized using an ECL Western blot analysis system (GE Amersham). Actin expression in the blots was reprobed using an anti-β-actin monoclonal antibody (Santa Cruz Biotech, clone C4, sc-47778 HRP) as a loading control.

[0202] Animals and Immunization Eight-week-old female 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 carried out according to the National Institutes of Health and the Suggestions for Animal Care and Use (ACUP) guidelines (BTS ACUP #15-091). The mice were divided into nine groups as shown in Table 4. [Table 4-2]

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

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

[0205] IFNγ ELISpot The mouse IFNγ ELISpot assay was performed using the MabTech kit (MabTech, #3321-4APW-10) to evaluate the antigen-specific cellular response. Briefly, 96-well plates pre-coated with anti-mouse IFNγ antibody (mAb AN18) were washed in PBS and blocked in complete culture medium (RPMI 1640 supplemented with 10% FBS and antibiotics) at room temperature for 2 hours. Splenic lymphocytes were resuspended in R10 medium (then added in triplicates with an input cell count of 2 × 10⁵ cells per well). A set of peptides was synthesized (GenScript), each containing 15 amino acid residues with 11 amino acids duplicated, representing the entire synthetic consensus survivorbin antigen 1 and the synthetic consensus survivorbin antigen 1 T3 protein sequence. 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 survivorbin antigen 1 antigen protein, and the second peptide pool contained peptides corresponding to the synthetic consensus survivorbin antigen 1 T3 antigen protein. 5 μg / ml concavalin A (Sigma) was used as a positive control, and complete culture medium was used as a negative control. Plates were incubated in a 5% CO₂ atmosphere incubator at 37°C for 18 hours. Next, biotinylated anti-mouse IFNγ detection antibody (MabTech mAb R4-6A2) was added, and the plate was incubated at room temperature for 2 hours. The plate was washed, streptavidin-ALP (MabTech) was added, and the plate was incubated at room temperature for 1 hour. Spot detection was completed using BCIP / NBT substrate according to the kit manufacturer's instructions (MabTech). Spots on the plate were counted using an automated ELISPOT reader (Cellular Technology). The average number of spot-forming units (SFUs) was 1 × 10⁻⁶ for data display. 6 It was adjusted to spleen cells.

[0206] Figures 7A to 7H show that the antigen-specific response induced by IFNγ Erispot is 1 × 10⁶, which is larger than the SFU in the control with culture medium alone. 6The number of IFNγ spot-forming units (SFUs) per spleen cell was reported.

[0207] Flow cytometry Cellular immune responses induced by synthetic consensus survivin antigen 1 and synthetic consensus survivin antigen 1T3 were further characterized by flow cytometry. In short, 2 × 10⁶ mice were vaccinated and untreated. 6Splenocytes 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) for each group immediately after isolation with synthetic consensus survivor antigen 1 and survivor 1T3 peptide, depending on the group. After peptide stimulation, the splenocytes were centrifuged and resuspended in 20 μL per well of mouse BD Fc Block (BD Biosciences) solution. The 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. After adding the extracellular stain, the final volume of each well was 50 μL, consisting of the Fc Block at a final dilution of 1:100 and the extracellular antibody at the appropriate dilution. Next, the cells were stained with 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 was collected using a 10-color FACS CANTO (BD Biosciences), and analysis was completed using FlowJo software. The flow cytometry gating strategy is shown in Figure 8.

[0208] For cells to be considered antigen-specific by flow cytometry, the reported frequency of the parameter must exceed the frequency of the control in the culture medium alone. For cells to be identified as producing antigen-specific CD107a, they must be identified as positive for antigen-specific production of IFNγ and / or IL-2 and / or TNFα, as identified by Boulevard gating.

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

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

[0211] Immunogenicity of synthetic consensus survivorbin antigen vaccine constructs IFNγ ELISpot The immunogenicity of two synthetic consensus survivorbin 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). A blank vector skeleton (pGX0001) was used as a negative control (n=4 / group) and mice were immunized. Vaccination with the synthetic consensus survivorbin antigen resulted in a significant IFNγ response compared to mice vaccinated with the negative control. However, synthetic consensus survivorbin antigen 1 (Figure) 7Evidence for dose-dependent increase in IFNγ production induced by A) is minimal, suggesting that the maximum response was achieved at the lowest dose. In detail, the synthetic consensus survivorbin antigen 1 IFNγ SFUs were 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 survivorbin antigen 1 IFNγ response was significantly greater in pGX1428 at 10 μg (p=0.031), 20 μg (p=0.015), and 30 μg (p=0.021) doses compared to untreated (2±3), but not at the 50 μg (p=0.134) dose. Vaccination with synthetic consensus survivor antigen 1T3 resulted in a significant IFNγ response, and there was some evidence of a dose-dependent increase with increasing dose levels (Figure). 7 D). Survivin 1T3 IFNγ SFUs were 516±156, 812±534, 1,016±654, and 818±339 at doses of 10 μg, 20 μg, 30 μg, and 50 μg, respectively. The synthetic consensus Survivin antigen 1T3 IFNγ response was significantly greater than the untreated (5±6) pGX1429 at doses of 20 μg (p=0.039), 30 μg (p=0.006), and 50 μg (p=0.037), but not at the 10 μg (p=0.337) dose. The IFNγ response is summarized in Table 5. [Table 5]

[0212] Flow cytometry Both synthetic consensus survivorbin antigen 1 and synthetic consensus survivorbin antigen 1 T3 induced a more robust response in the CD4+ T cell compartment compared to the response in the CD8+ T cell compartment (Figure 9). Synthetic consensus survivorbin antigen 1 induced a significantly stronger antigen-specific CD4+ T cell response frequency in the 20 μg (1.08% ± 0.65%) (p=0.024) and 50 μg (1.21% ± 0.73%) (p=0.009) dose groups compared to the untreated group (0.03% ± 0.05%), 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 survivorin antigen 1T3 induced a significantly stronger antigen-specific CD4+ T cell response at doses of 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) compared to the untreated group (0.05% ± 0.05%), but not at the 10 μg (0.62% ± 0.35%) dose group (p=0.248) (Figure 7E). The cytokine profiles of synthetic consensus survivorin antigen-specific CD4+ T cells were similar across 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 frequencies of antigen-specific CD4+ T cells are 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 1 T3 induced a significantly higher frequency of antigen-specific CD8+ T cells between dose groups (p=0.043). The frequency of antigen-specific CD8+ T responses in groups immunized with 10 μg (0.15% ± 0.09%) (p=0.919), 20 μg (0.27% ± 0.21%) (p=0.274), or 30 μg (0.25% ± 0.14%) (p=0.377) of pGX1429 did not approach statistical significance compared to the untreated group (0.07% ± 0.01%). The frequency of Survivin 1 T3-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 1 T3 induced similar CD8+ T cell responses in terms of size and phenotype. Antigen-specific CD8+ T cells were mainly IFNγ+IL-2-TNFα- and IFNγ+IL-2-TNFα+ (Figures 7G, ​​7H). The frequency of antigen-specific CD8+ T cells is 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 (FIG. 10A) and synthetic consensus survivin antigen 1T3 (FIG. 10B) are also positive for CD107a, indicating the possibility of a cytolytic function mediated by CD4+ T cells. All doses of the synthetic consensus survivin antigen 1 induced significantly higher frequencies 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 (FIG. 10A). Synthetic consensus survivin antigen 1T3 induced significantly higher frequencies of CD4+CD107a+ T cells than untreated (0.01±0.01) in all groups except the 10 μg dose group (0.18%±0.09%) (p=0.147). Antigen-specific CD4 + CD107a + + T cell frequencies 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 (FIG. 10B). The frequency of antigen-specific CD4 + + T cells with cytolytic capacity is described in further detail in Table 8.

Table 8

[0215] Similar to the size of antigen-specific CD8+ T cells, synthetic consensus survivorin antigen 1 did not induce a significant change in the frequency of CD8+CD107a+ T cells across all groups (p=0.101) (Figure 10C). Synthetic consensus survivorin antigen 1T3 did induce a significant change in the frequency of CD8+CD107a+ T cells across 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 the untreated group (0.03 ± 0.01) (p = 0.026), but this was not the case in the groups immunized with synthetic consensus survivorin antigen 1T3 at 10 μg (0.11% ± 0.08%) (p = 0.813), 20 μg (0.16% ± 0.11%) (p = 0.450), and 30 μg (0.16% ± 0.07%) (p = 0.424). The cytokine profiles of synthetic consensus survivorin antigen-specific CD8+CD107a+ T cells were similar across dose groups in both constructs and consisted mainly of IFNγ+IL-2-TNFα+ and IFNγ+IL-2-TNFα- cells (Figure 10E, Figure 10F). Antigen-specific CD8 with cytolytic ability + The frequency of T cells is described in more detail in Table 9. [Table 9] Width of the IFNγ response induced by synthetic consensus survivor antigen constructs

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

[0217] The following synthetic consensus survivorbin antigen 1 epitopes were present in both pGX1428 and pGX1429: LPPAWQLFLKDHRISTFKN (Sequence ID 5) (Matrix Pools 1, 2, 3, 4, and 7) LKLDRERAKNKIAKETNNK (Sequence ID 6) (Matrix Pools 1, 2, 3, 4, and 11) Synthetic consensus survivorin antigen T3 epitope present in pGX1429: EWLHHFQGLFP(Sequence ID 7) (Matrix Pools 3, 4, and 7)

[0218] The combined magnitude of the cellular immune responses induced by synthetic consensus survivorbin antigen 1 and synthetic consensus survivorbin antigen 1T3 was similar, but the response induced by the synthetic consensus survivorbin antigen 1T3 (pGX1429) region against the synthetic consensus survivorbin antigen 1 (pGX1428) region of synthetic consensus survivorbin antigen 1T3 was approximately half the magnitude of the response induced by the synthetic consensus survivorbin antigen 1 construct (pGX1428). Epitope mapping using IFNγ ELISpot with a matrix approach revealed that both synthetic consensus survivorbin antigen constructs produced responses to the same epitopes of the synthetic consensus survivorbin antigen 1 antigen, but the response driven by synthetic consensus survivorbin antigen 1T3 to these epitopes was smaller in magnitude. The existence of epitopes specific to the T3 region of the synthetic consensus survivorbin antigen 1T3 antigen was also confirmed.

[0219] The total magnitude of the cellular immune responses induced by synthetic consensus survivorbin antigen 1 (pGX1428) and synthetic consensus survivorbin antigen 1T3 (pGX1429) was similar, but the response induced by the synthetic consensus survivorbin antigen 1T3 (pGX1429) region against the synthetic consensus survivorbin antigen 1 region of the synthetic consensus survivorbin antigen 1T3 antigen was about half the magnitude of the response induced by the synthetic consensus survivorbin antigen 1 construct (pGX1428). Epitope mapping using IFNγ ELISpot with a matrix approach revealed that both synthetic consensus survivorbin antigen constructs produced responses to the same epitopes as the synthetic consensus survivorbin antigen 1 antigen, but the response driven by synthetic consensus survivorbin antigen 1T3 to these epitopes was smaller in magnitude. Using this approach, we also confirmed the existence of an epitope specific to the T3 region of the synthetic consensus survivin antigen 1T3 antigen. Synthetic consensus survivin antigen 1T3 also significantly increased the frequency of antigen-specific CD8+ and CD8+CD107a+ T cells compared to untreated cells, while synthetic consensus survivin antigen 1 did not significantly increase antigen-specific CD8+ T cells in mice. Based on its potential to further broaden the range of cellular immune responses to survivin compared to mouse pGX1428, we selected synthetic consensus survivin antigen 1T3 (pGX1429) to move to monovalent non-human primate studies.

[0220] Example 5 - Non-human primate study using synthetic consensus survivor antigen 1T3 (pGX1429) To investigate the potential of synthetic consensus survivor 1T3 alone and in combination with low and high doses of IL-12, 18 adult rhesus monkeys were identified by their unique NHP ID numbers and divided into three groups of six, and immunized with pGX1429 as follows: 6 animals were immunized with 3.0 mg of pGX1429, 6 animals with 3.0 mg of pGX1429 plus 0.04 mg of pGX6006 (opt rh IL-12) as an adjuvant, and 6 animals with 3.0 mg of pGX1429 plus 0.20 mg of pGX6006 (opt rh IL-12) as an adjuvant. The immunizations were formulated in 1.0 mL infusions at the Survival Surgical Center (SSC). Immunotherapy infusions were administered at weeks 0, 4, 8, and 12. All immunizations were performed intramuscularly in alternating contralateral limbs using a CELLECTRA® 2000 5P-IM EP device with a 1 ml injection volume formulated in sterile WFI. EP conditions were as follows: OpBlock 0070-IM, 0.5 Amp, 3 pulses, 52 ms, 0.2 sec intervals between pulses. Survivin immunogenicity was evaluated at weeks 2, 6, 10, and 14. Survivin-specific IFN-gamma responses are shown in Figures 12 and 13. Homology between natural rhesus monkey survivin and pGX1429 is shown in Table 10. As shown in Figures 13 and 14, an increased 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 Whole blood from non-human primates was collected in sodium citrate cell preparation tubes (CPTs from BD Biosciences) containing an anticoagulant and gel barrier. The whole blood was centrifuged immediately after collection (within 2 hours) to isolate and concentrate PMMCs before overnight transport. Red blood cells and neutrophils settled at the bottom of the tube and were retained in place by the gel barrier. Plasma and lymphocytes remained on top of the gel barrier. Each CPT could hold approximately 8 mL of blood and was transported at room temperature. The rotated CPT tubes were processed for PBMC isolation. After lysing red blood cells with ammonium chloride-potassium (ACK) buffer, viable cells were counted using Invitrogen's Countess® Automated Cell Counter and resuspended in complete culture medium (RPMI 1640 supplemented with 10% FBS, antibiotics, and β-mercaptoethanol). After the assays described herein were completed, the remaining PBMCs were frozen in cryovials in a freezing medium (10% DMSO from Sigma in 90% FBS from Seradigm) and stored in liquid nitrogen for an extended period.

[0222] IFNγ ELISpot To evaluate the antigen-specific cellular response induced by the vaccine, the monkey IFNγ ELISpot assay was performed using a kit (MabTech IFNγ ELISpotPro, #3421M-2APW-10) with PMBCs isolated at each time point. 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 in complete culture medium (RPMI 1640 supplemented with 10% FBS, antibiotics, and β-mercaptoethanol). NHP PBMCs were then resuspended in R10 medium (2 × 10⁶ per well). 5The cells were added in triplicates based on the input cell count. A set of peptides (GenScript) was synthesized, each containing 15 amino acid residues with 11 duplicate amino acids representing the entire synthetic consensus protein sequence. These peptide sets were resuspended in DMSO (Sigma) and pooled in pools at a concentration of approximately 2 μg / mL for each 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 variations in the size of each antigen protein, two peptide pools were obtained for the survivor.

[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. Plates were incubated in a 5% CO2 atmosphere incubator at 37°C for approximately 18 hours. After removing the cells and adding ALP-complex anti-monkey IFNγ detection antibody (MabTech Ab 7-B6-1-ALP), the plates were incubated at room temperature for 2 hours. Next, a sandwich immunoenzyme assay was performed using BCIP / NBT substrate solution according to the kit manufacturer's instructions (MabTech). A bluish-black precipitate formed as spots, revealing individual IFNγ-producing cells. The spots were then scanned and counted by a trained operator using CTLimmunoSpot® analyzer and software (Cellular Technology) for quality control. An IFNγ response greater than 1x10⁶ SFU in the medium-only control was used. 6 It was reported as a spot-forming unit (SFU) for 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). Formulated in IM at SSC with an injection volume of 1.0 mL. Group 2-3.0 mg of pGX1429 (synthetic consensus survivin 1T3) + 0.04 pGX6006 (opt. rIL-12). Formulated in IM at SSC with an injection volume of 1.0 mL. Group 3-3.0 mg of pGX1429 (synthetic consensus survivin 1 T3) + 0.20 pGX6006 (opt. rIL-12). Formulated in IM at SSC with an injection volume of 1.0 mL.

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

[0226] result Survivin-specific IFNγ responses for each of the three groups (1-3) are shown in Figures 12-14. Results show the response at each time point 2 weeks after dose. Overall, all groups and individual animals showed an increase in response by the end of the study 2 weeks after dose 4, compared to pre-collected baseline. Adding a 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 early PD2 response was also observed with Survivin + 0.2 mg of IL-12.

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

Table 12

Table 13

Table 14

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

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

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

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

[10] A nucleic acid molecule as described in any one of items 1-6 and 9, for use in the preparation of drugs for the treatment of cancer.

[11] A vector containing a nucleic acid molecule as described in any one of items 1 to 10.

[12] A vector as described in item 11, including a plasmid or viral vector.

[13] A composition comprising one or more nucleic acid molecules as described in 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 as shown in item 11 or 12.

[16] (a) Amino acids 19-159 of sequence number 2, (b) Amino acids 19-210 of sequence number 4, (c) Amino acids 19-232 of sequence number 8, (d) A fragment containing at least 90% of the full length of amino acids 19-159 of SEQ ID NO: 2, (e) A fragment containing at least 90% of the full length of amino acids 19-210 of SEQ ID NO: 4, (f) A fragment containing at least 90% of the full length of amino acids 19-232 of sequence number 8, (g) An amino acid sequence that is at least 95% identical to amino acids 19-159 of SEQ ID NO: 2, (h) An amino acid sequence that is at least 95% identical to amino acids 19-210 of sequence number 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 set forth in SEQ ID NO: 2.

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

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

[21] A vaccine comprising the nucleic molecule according to any one of items 1 to 10.

[22] A vaccine containing the vector described in item 11 or 12.

[23] A vaccine as described in any one of items 18-20, further comprising pharmaceutically acceptable excipients.

[24] A vaccine as described in any one of items 21-23, further containing an adjuvant.

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

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

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

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

[29] A method of vaccinating cancer cells expressing survivor, comprising administering an amount of the vaccine described in any one of items 20 to 25 that is effective in inducing a humoral or cellular immune response.

Claims

1. The promoter and the following: (a) The nucleic acid sequence encoding amino acids 19-159 of SEQ ID NO: 2; (b) Nucleic acid sequence encoding amino acids 19-210 of SEQ ID NO: 4; (c) Nucleic acid sequence encoding amino acids 19-232 of Sequence ID No. 8; (d) A nucleic acid sequence that comprises at least 90% of the full length of amino acids 19-159 of SEQ ID NO: 2 and encodes a fragment containing SEQ ID NO: 5; (e) A nucleic acid sequence comprising at least 90% of the full length of amino acids 19-210 of SEQ ID NO: 4, and encoding a fragment containing SEQ ID NO: 5; (f) A nucleic acid sequence that comprises at least 90% of the full length of amino acids 19-232 of SEQ ID NO: 8 and encodes a fragment containing SEQ ID NO: 5; (g) A nucleic acid sequence encoding a protein that is at least 99% identical to amino acids 19-159 of SEQ ID NO: 2, wherein the protein comprises SEQ ID NO: 5 and, relative to SEQ ID NO: 2, alanine at positions 51, 65, and 101, and cysteine ​​at position 150; (h) A nucleic acid sequence encoding a protein that is at least 99% identical to amino acids 19-210 of SEQ ID NO: 4, wherein the protein comprises SEQ ID NO: 5, and with respect to SEQ ID NO: 4, alanine at positions 51, 65, and 101, and cysteine ​​at position 150; and (i) A nucleic acid sequence encoding a protein that is at least 99% identical to amino acids 19-232 of SEQ ID NO: 8, wherein the protein comprises SEQ ID NO: 5 and, relative to SEQ ID NO: 8, comprises alanine at positions 51, 65 and 101, and cysteine ​​at position 150. A host cell comprising a vector, which comprises a nucleic acid molecule containing one or more nucleic acid sequences selected from the group consisting of the following.

2. The promoter and the following: (a) Nucleotides 55–423 of Sequence ID No. 1; (b) Nucleotides 55–636 of Sequence ID No. 3; (c) A fragment comprising at least 90% of the full length of nucleotides 55–423 of SEQ ID NO: 1, and encoding SEQ ID NO: 5; (d) A fragment comprising at least 90% of the full length of nucleotides 55–636 of SEQ ID NO: 3, and encoding SEQ ID NO: 5; (e) A fragment that is at least 99% identical to nucleotides 55-423 of SEQ ID NO: 1, wherein the fragment codes for SEQ ID NO: 5, and codes for alanine at amino acid positions 51, 65, and 101 and cysteine ​​at position 150 relative to SEQ ID NO: 2; and (f) A fragment that is at least 99% identical to nucleotides 55-636 of SEQ ID NO: 3, wherein the fragment encodes SEQ ID NO: 5, and encodes alanine at amino acid positions 51, 65, and 101, and cysteine ​​at position 150, relative to SEQ ID NO:

8. A host cell comprising a vector, which comprises a nucleic acid molecule containing one or more nucleic acid sequences selected from the group consisting of the following.

3. The promoter and the following: (a) The nucleic acid sequence encoding Sequence ID No. 2; (b) The nucleic acid sequence encoding Sequence ID No. 4; (c) Nucleic acid sequence encoding Sequence ID No. 8; (d) A nucleic acid sequence encoding a fragment, comprising at least 90% of the full length of SEQ ID NO: 2 and including SEQ ID NO: 5; (e) A nucleic acid sequence encoding a fragment, comprising at least 90% of the full length 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 full length of SEQ ID NO: 8 and including SEQ ID NO: 5; (g) A nucleic acid sequence encoding a protein that is at least 99% identical to SEQ ID NO: 2, wherein the protein comprises SEQ ID NO: 5, and with respect to SEQ ID NO: 2, alanine at positions 51, 65, and 101, and cysteine ​​at position 150; (h) A nucleic acid sequence encoding a protein that is at least 99% identical to SEQ ID NO: 4, wherein the protein comprises SEQ ID NO: 5, and with respect to SEQ ID NO: 4, alanine at positions 51, 65, and 101, and cysteine ​​at position 150; and (i) A nucleic acid sequence encoding a protein that is at least 99% identical to SEQ ID NO: 8, wherein the protein comprises SEQ ID NO: 5 and, relative to SEQ ID NO: 8, comprises alanine at positions 51, 65, and 101, and cysteine ​​at position 150. A host cell comprising a vector, which comprises a nucleic acid molecule containing one or more nucleic acid sequences selected from the group consisting of the following.

4. The promoter and the following: (a) Sequence ID 1; (b) Sequence ID 3; (c) A fragment comprising at least 90% of the full length of Sequence ID No. 1 and encoding Sequence ID No. 5; (d) A fragment comprising at least 90% of the full length of Sequence ID No. 3 and encoding Sequence ID No. 5; (e) A fragment that is at least 99% identical to SEQ ID NO: 1, wherein the fragment codes for SEQ ID NO: 5, and for SEQ ID NO: 2, alanine at amino acid positions 51, 65, and 101, and cysteine ​​at position 150; and (f) A fragment that is at least 99% identical to SEQ ID NO: 3, wherein the fragment codes for SEQ ID NO: 5, and for SEQ ID NO: 8, alanine is at amino acid positions 51, 65, and 101, and cysteine ​​is at position 150. A host cell comprising a vector, which comprises a nucleic acid molecule containing one or more nucleic acid sequences selected from the group consisting of the following.

5. The promoter and the following amino acid sequence: WPFLEGCACAPERMAEAGFIHCPAENEPDLAQCFFCFKELEGWEPDDDPIEEHKKHSSGAAFLSVKKQFEELTLSEF A host cell comprising a vector containing a nucleic acid molecule encoding a synthetic consensus survivin antigen, which includes 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 containing the amino acid sequences of SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, wherein Sequence IDs 5 and 6 have the following amino acid sequences: WPFLEGCACAPERMAEAGFIHCPAENEPDLAQCFFCFKELEGWEPDDDPIEEHKKHSSGAAFLSVKKQFEELTLSEF It is separated by a polypeptide consisting of, and Sequence IDs 6 and 7 have the following amino acid sequences: EFEETAKKVRCAIEQLAAMDRGRKRRSMQRKPTIRRKNLRKLRRKCAVPSSSWLPWTEASGWSCLVP It is separated by a polypeptide consisting of A host cell containing the aforementioned vector.

7. A host cell comprising a vector containing a promoter and a nucleic acid molecule containing the nucleic acid sequence described in Sequence ID No.

1.

8. A host cell comprising a vector containing a promoter and a nucleic acid molecule containing the nucleic acid sequence described in Sequence ID No.

3.

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

10. The host cell according to claim 9, wherein the nucleic acid molecule is operably linked to a regulatory element containing a polyadenylation signal.

11. The host cell according to claim 9, wherein the promoter is selected from the group consisting of the human cytomegalovirus (hCMV) promoter, monkey virus 40 (SV40) promoter, mouse mammary cancer virus (MMTV) promoter, human immunodeficiency virus (HIV) promoter, bovine immunodeficiency virus (BIV) long-terminal repeat (LTR) promoter, Moloney virus promoter, avian leukemia virus (ALV) promoter, Epstein-Barr virus (EBV) promoter, Roussarcoma 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. The host cell according to claim 9, wherein the promoter is a modified human cytomegalovirus (hCMV) promoter.

13. The host cell according to claim 10, wherein the polyadenylation signal is the bovine growth hormone polyadenylation signal (bGH polyA).

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

15. A cell culture comprising one or more host cells as described in any one of claims 1 to 14.

16. The vector is as follows: (a) Amino acids 19-159 of Sequence ID No. 2; (b) Amino acids 19-210 of Sequence ID No. 4; (c) Amino acids 19-232 of Sequence ID No. 8; (d) A fragment containing at least 90% of the full length of amino acids 19-159 of SEQ ID NO: 2, and containing SEQ ID NO: 5; (e) A fragment containing at least 90% of the full length of amino acids 19-210 of SEQ ID NO: 4, and containing SEQ ID NO: 5; (f) A fragment containing at least 90% of the full length of amino acids 19-232 of SEQ ID NO: 8, and containing SEQ ID NO: 5; (g) An amino acid sequence that is at least 99% identical to amino acids 19-159 of SEQ ID NO: 2, wherein the amino acid sequence includes alanine at positions 51, 65, and 101, and cysteine ​​at position 150, relative to SEQ ID NO: 5 and SEQ ID NO: 2; (h) an amino acid sequence that is at least 99% identical to amino acids 19-210 of SEQ ID NO: 4, wherein the amino acid sequence includes alanine at positions 51, 65, and 101, and cysteine ​​at position 150, relative to SEQ ID NO: 5 and SEQ ID NO: 4; and (i) An amino acid sequence that is at least 99% identical to amino acids 19-232 of SEQ ID NO: 8, wherein the amino acid sequence contains alanine at positions 51, 65 and 101 and cysteine ​​at position 150 relative to SEQ ID NO: 5 and SEQ ID NO:

8. A host cell according to any one of claims 1 to 14, or a cell culture according to claim 15, encoding a protein comprising an amino acid sequence selected from the group consisting of the following.

17. The vector is as follows: (a) Sequence ID 2; (b) Sequence ID 4; (c) Sequence ID 8; (d) A fragment comprising at least 90% of the full length of Sequence ID No. 2 and comprising Sequence ID No. 5; (e) A fragment comprising at least 90% of the full length of Sequence ID No. 4 and comprising Sequence ID No. 5; (f) A fragment comprising at least 90% of the full length of Sequence ID No. 8 and comprising Sequence ID No. 5; (g) an amino acid sequence that is at least 99% identical to SEQ ID NO: 2, wherein the amino acid sequence contains alanine at positions 51, 65, and 101, and cysteine ​​at position 150, relative to SEQ ID NO: 5 and SEQ ID NO: 2; (h) an amino acid sequence that is at least 99% identical to SEQ ID NO: 4, wherein the amino acid sequence contains alanine at positions 51, 65 and 101 and cysteine ​​at position 150 relative to SEQ ID NO: 5 and SEQ ID NO: 4; and (i) An amino acid sequence that is at least 99% identical to SEQ ID NO: 8, wherein the amino acid sequence contains alanine at positions 51, 65 and 101, and cysteine ​​at position 150, compared to SEQ ID NO: 5 and SEQ ID NO:

8. A host cell according to any one of claims 1 to 14, or a cell culture according to claim 15, encoding a protein comprising an amino acid sequence selected from the group consisting of the following.

18. A host cell according to any one of claims 1 to 14, or a cell culture according to claim 15, for use in the manufacture of a pharmaceutical product.

19. A host cell according to any one of claims 1 to 14, or a cell culture according to claim 15, for use in the manufacture of a pharmaceutical product for cancer treatment.

20. A host cell according to any one of claims 1 to 14, or a cell culture according to claim 15, for use in the manufacture of a pharmaceutical for the treatment of survivin-expressing cancer.

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