RU2776949C2 - Survivin-targeted anti-cancer vaccines and applications thereof - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: presented are a nucleic acid molecule encoding a synthetic consensus Survivin antigen (variants), a synthetic consensus Survivin antigen (variants), an expression vector, an immunogenic composition (variants), a vaccine for treating or preventing Survivin-expressing cancer, a method for treating a subject with a Survivin-expressing cancer cell, and a method for vaccinating a subject against a Survivin-expressing cancer cell.

EFFECT: production of safe and effective vaccines and methods for application thereof for preventing and/or treating cancer and reducing the mortality in subjects suffering from cancer.

22 cl, 14 tbl, 32 dwg, 5 ex

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority over US Patent Application No. 62/598,267, filed December 13, 2017, the contents of which are incorporated herein by reference in their entirety.

SEQUENCE LIST

[0002] The application contains a sequence listing, which is in electronic ASCII format and is incorporated herein by reference in its entirety. The specified ASCII copy, created on December 13, 2018, is named 104409_000448_sequence_listing.txt and is 8,797 bytes in size.

FIELD OF TECHNOLOGY

[0003] The present invention relates to Survivin antigens and nucleic acid molecules encoding them. The present invention also relates to vaccines containing such Survivin antigens and/or nucleic acid molecules. The present invention further relates to methods for using vaccines to induce immune responses and prevent and/or treat subjects having cancer cells and/or tumors that express survivin.

BACKGROUND OF THE INVENTION

[0004] Cancer is one of the leading causes of death worldwide. In the United States, cancer is the second most common cause of death, accounting for approximately 1 in every 4 deaths. Cancer arises from a single cell that has transformed from a normal cell into a cancer cell. This transformation is often a multi-stage process, moving from a precancerous lesion to a malignant neoplasm. Many factors contribute to the progression of this, including aging, genetic contributions, and exposure to environmental factors such as physical carcinogens (eg, ultraviolet and ionizing radiation), chemical carcinogens (eg, asbestos, tobacco smoke components, etc.), and biological carcinogens (eg. , some viruses, bacteria and parasites).

[0005] Prevention, diagnosis, and treatment of cancer can take many forms. Prevention may include screening for predisposing factors (eg, specific genetic variants), behavior modification (eg, smoking, diet, and physical activity levels), and vaccination against viruses (eg, hepatitis B virus, human papillomavirus). Treatment may include chemotherapy, radiation therapy, and surgical removal of the tumor or cancerous tissue. Despite the existence of numerous prevention and treatment methods, such methods often have limited success in effectively preventing and/or treating cancer.

[0006] Survivin, also known as baculovirus apoptosis inhibitor containing repeat 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 plays a significant evolutionarily conserved role in mitosis. (Li, F. et al. Control of apoptosis and mitotic spindle checkpoint by Survivin. Nature 396, 580-584, doi:10.1038/25141 (1998).) Overexpression of Survivin is associated with tumor cell proliferation, progression, angiogenesis, therapeutic resistance and unfavorable prognosis. In healthy cells and tissues, Survivin expression is either absent or present at low levels. However, Survivin is a member of the apoptosis protein (IAP) inhibitor family and IAP genes are highly expressed in a variety of cancer cells and primary tumor biopsies. Among the IAPs, Survivin shows the most pronounced overexpression in tumors and embryonic tissues. In numerous studies of ovarian cancer, the number of patient samples with a positive result for the expression of Survivin ranged from 74 to 92%. ( See 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); ovarian cancer patients.Journal of ovarian research 4, 20, doi:10.1186/1757-2215-4-20 (2011).) The contribution of survivin to oncogenesis, combined with its limited expression and overexpression pattern in various tumors, makes it an attractive target for immunotherapy cancer.

[0007] Survivin is the smallest member of the IAP family. It is a 16.3 kDa protein, consisting of 142 amino acids and characterized by the presence of one BIR repeat. It also lacks the carboxyl terminal finger domain RING in its protein structure. (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 isoforms of Survivin have been identified, with Survivin isoform 1 being the dominant transcript. Survivin is expressed during fetal development but is not expressed in fully differentiated tissues. However, it is expressed at a high level in many cancer cells. Thus, Survivin is a potential target antigen for cancer treatment.

[0008] Vaccines are of interest for the treatment and prevention of cancer, in particular epithelial ovarian cancer (ECC). However, existing vaccines that target tumor cell antigens are limited by poor antigen expression in vivo . Accordingly, there remains a need in the art for safe and effective vaccines and methods of using them to prevent and/or treat cancer and reduce mortality in subjects suffering from cancer.

DISCLOSURE OF THE INVENTION

[0009] This document proposes:

[0010] Nucleic acid molecules comprising one or more nucleic acid sequences selected from the group consisting of: (a) a nucleic acid sequence that encodes amino acids 19-159 of SEQ ID NO:2; (b) a nucleic acid sequence that encodes amino acids 19-210 of SEQ ID NO:4; (c) a nucleic acid sequence that encodes amino acids 19-232 of SEQ ID NO:8; (d) a nucleic acid sequence that encodes 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 that encodes a fragment containing at least 90% of the total length of amino acids 19-210 of SEQ ID NO:4; (e) a nucleic acid sequence that encodes 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 that encodes a protein that is at least 95% identical to amino acids 19-159 of SEQ ID NO:2; (h) a nucleic acid sequence that encodes a protein that is at least 95% identical to amino acids 19-210 of SEQ ID NO:4; (i) a nucleic acid sequence that encodes a protein that is at least 95% identical to amino acids 19-232 of SEQ ID NO:8; (j) a nucleic acid sequence that encodes a fragment containing at least 90% of the entire length of the protein, which is at least 95% identical to amino acids 19-159 of SEQ ID NO:2; (l) a nucleic acid sequence that encodes a fragment containing at least 90% of the entire length of the protein, which is at least 95% identical to amino acids 19-210 of SEQ ID NO:4; and (m) a nucleic acid sequence that encodes a fragment containing at least 90% of the entire length of the protein, which is at least 95% identical to amino acids 19-232 of SEQ ID NO:8.

[0011] Nucleic acid molecules containing 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 total length of nucleotides 55-423 of SEQ ID NO:1; (d) a fragment containing at least 90% of the total 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; (e) 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.

[0012] Nucleic acid molecules containing one or more nucleic acid sequences selected from the group consisting of: (a) a nucleic acid sequence that encodes SEQ ID NO:2; (b) a nucleic acid sequence that encodes SEQ ID NO:4; (c) a nucleic acid sequence that encodes SEQ ID NO:8; (d) a nucleic acid sequence that encodes a fragment containing at least 90% of the entire length of SEQ ID NO:2; (e) a nucleic acid sequence that encodes a fragment containing at least 90% of the entire length of SEQ ID NO:4; (e) a nucleic acid sequence that encodes a fragment containing at least 90% of the entire length of SEQ ID NO:8; (g) a nucleic acid sequence that encodes a protein that is at least 95% identical to SEQ ID NO:2; (h) a nucleic acid sequence that encodes a protein that is at least 95% identical to SEQ ID NO:4; (i) a nucleic acid sequence that encodes a protein that is at least 95% identical to SEQ ID NO:8; (j) a nucleic acid sequence that encodes a fragment containing at least 90% of the entire length of the protein, which is at least 95% identical to SEQ ID NO:2; (l) a nucleic acid sequence that encodes a fragment containing at least 90% of the entire length of the protein, which is at least 95% identical to SEQ ID NO:4; and (m) a nucleic acid sequence that encodes a fragment containing at least 90% of the entire length of the protein, which is at least 95% identical to SEQ ID NO:8.

[0013] Nucleic acid molecules containing 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 entire length of SEQ ID NO:1; (d) a fragment containing at least 90% of the entire length of SEQ ID NO:3; (e) a fragment that is at least 95% identical to SEQ ID NO:1; (e) a fragment that is at least 95% identical to SEQ ID NO:3; (g) a fragment containing at least 90% of the entire length of the nucleic acid sequence, which is at least 95% identical to SEQ ID NO:1; and (h) a fragment containing at least 90% of the entire length of the nucleic acid sequence, which is at least 95% identical to SEQ ID NO:3.

[0014] Nucleic acid molecules containing the nucleic acid sequence shown in SEQ ID NO:1.

[0015] Nucleic acid molecules containing the nucleic acid sequence shown in SEQ ID NO:3.

[0016] Nucleic acid molecules as described herein for use as a drug.

[0017] Nucleic acid molecules as described herein for use as a drug in the treatment of cancer.

[0018] Nucleic acid molecules, according to the present description, for use in the preparation of a drug.

[0019] Nucleic acid molecules as described herein for use in the preparation of a medicament for the treatment of cancer.

[0020] Vectors containing a nucleic acid molecule as described herein.

[0021] Vectors containing a plasmid or viral vector.

[0022] Compositions containing one or more nucleic acid molecules, according to the present description.

[0023] Compositions as described herein, containing a pharmaceutically acceptable carrier.

[0024] Compositions, according to the present description, containing one or more vectors, according to the present description.

[0025] Proteins containing an amino acid sequence selected from the group consisting of: (a) amino acids 19-159 of SEQ ID NO:2; (b) amino acids 19-210 of SEQ ID NO:4; (c) amino acids 19-232 of SEQ ID NO:8; (d) a fragment containing at least 90% of the total length of amino acids 19-159 of SEQ ID NO:2; (e) a fragment containing at least 90% of the total length of amino acids 19-210 of SEQ ID NO:4; (e) a fragment containing at least 90% of the total length of amino acids 19-232 of SEQ ID NO: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 SEQ ID NO:4; (i) an amino acid sequence that is at least 95% identical to amino acids 19-232 of SEQ ID NO:8; (k) a fragment containing at least 90% of the entire length of the amino acid sequence, which is at least 95% identical to amino acids 19-159 of SEQ ID NO:2; (k) a fragment containing at least 90% of the entire length of the amino acid sequence, which is at least 95% identical to amino acids 19-210 of SEQ ID NO:4; and (m) a fragment containing at least 90% of the entire length of the amino acid sequence, which is at least 95% identical to amino acids 19-232 of SEQ ID NO:8.

[0026] Proteins containing an amino acid sequence selected from the group consisting of: (a) SEQ ID NO:2; (b) SEQ ID NO:4; (c) SEQ ID NO:8; (d) a fragment containing at least 90% of the entire length of SEQ ID NO:2; (e) a fragment containing at least 90% of the entire length of SEQ ID NO:4; (e) a fragment containing at least 90% of the entire length of SEQ ID NO:8; (g) an amino acid sequence that is at least 95% identical to SEQ ID NO:2; (h) an amino acid sequence that is at least 95% identical to SEQ ID NO:4; (i) an amino acid sequence that is at least 95% identical to SEQ ID NO:8; (k) a fragment containing at least 90% of the entire length of the amino acid sequence, which is at least 95% identical to SEQ ID NO:2; (k) a fragment containing at least 90% of the entire length of the amino acid sequence, which is at least 95% identical to SEQ ID NO:4; and (m) a fragment containing at least 90% of the entire length of the amino acid sequence, which is at least 95% identical to SEQ ID NO:8.

[0027] Proteins containing the amino acid sequence shown in SEQ ID NO:2.

[0028] Proteins containing the amino acid sequence shown in SEQ ID NO:4.

[0029] Proteins containing the amino acid sequence shown in SEQ ID NO:8.

[0030] Vaccines containing nucleic acid molecules, according to the present description.

[0031] Vaccines containing the vector, according to the present description.

[0032] Vaccines as described herein, further comprising a pharmaceutically acceptable excipient.

[0033] Vaccines, according to the present description, additionally containing an adjuvant.

[0034] Vaccines as described herein, wherein the adjuvant is IL-12, IL-15, IL-28, or RANTES.

[0035] Methods for treating a subject with a survivin-expressing cancer cell, comprising administering a therapeutically effective amount of a vaccine as described herein.

[0036] Methods as described herein wherein administration includes an electroporation step.

[0037] Methods, according to the present description, in which the introduction is carried out at one or more sites of the subject.

[0038] Methods for vaccinating a subject against a survivin-expressing cancer cell, comprising administering an amount of the vaccine as described herein effective to induce a humoral or cellular immune response.

BRIEF DESCRIPTION OF THE DRAWINGS

[0039] The brief description, as well as the following detailed description, are more understandable when read in conjunction with the accompanying drawings. For purposes of illustrating the invention, the drawings show examples of embodiments of the invention; however, the invention is not limited to the specific methods, compositions and devices disclosed. In graphics:

[0040] FIG. 1 shows a schematic representation of isophome 1 of the synthetic Survivin consensus antigen. Asterisks denote mutations required to eliminate anti-apoptotic activity.

[0041] Figure 2 shows the general structure of monomer A (left) and monomer B (right) of isoform 1 of the synthetic consensus antigen Survivin. Changes relative to native Survivin are indicated by spheres.

[0042] FIG. 3 shows a schematic representation of the 1T3 isophome of the synthetic Survivin consensus antigen. Asterisks denote mutations required to eliminate anti-apoptotic activity. The dark gray region on the right (3') represents a truncated region of the Survivin 3 (T3) isoform.

[0043] Figure 4 shows the pGX1428 construct containing isoform 1 of the synthetic Survivin consensus antigen.

[0044] Figure 5 shows the pGX1429 construct containing the 1T3 isoform of the synthetic Survivin consensus antigen.

[0045] Figure 6 shows the expression of Survivin 1 synthetic consensus antigen and 1T3 synthetic consensus antigen Survivin proteins in human rhabdomyosarcoma (RD) cells transfected with pGX1428 and pGX1429, respectively, by immunoblotting. Protein bands of the expected molecular weight were found for pGX1428 (17.5 kDa) and pGX1429 (25.3 kDa). β-Actin was used as a control for application.

[0046] FIG. 7A-7H show the immunogenicity of Survivin 1 synthetic consensus antigen and Survivin 1T3 synthetic consensus antigen. CB6F1 females were immunized 3 times 3 weeks apart with the indicated doses of Survivin 1 (pGX1428) (FIGS. 7A-7D), Survivin 1T3 pGX1429 (E-H) (n=8/group) or pGX0001 (empty vector) (n=4) . In FIG. 7A: Survivin 1-specific IFNγ responses by ELISpot at indicated pGX1428 dose amounts. In FIG. 7B: Survivin 1-specific CD4+ T cell responses. In FIG. 7C: Survivin 1-specific CD8+ T cell responses. In FIG. 7D: Survivin 1T3-specific IFNγ responses by ELISpot at indicated pGX1429 dose amounts. In FIG. 7E: Survivin 1T3-specific CD4+ T cell responses. In FIG. 7F: Survivin 1T3-specific CD8+ T cell responses. In FIG. 7G: Cytokine response profile of Survivin 1-specific CD4+ T cells and CD8+ T cells. In FIG. 7H: Cytokine response profile of Survivin 1T3-specific CD4+ T cells and CD8+ T cells.

[0047] FIG. 8 shows a gating strategy for flow cytometry.

[0048] FIG. 9 shows the relative frequency of CD4+ and CD8+ T cells. Cellular immune responses induced by pGX1428 and pGX1429 were predominantly in the CD4+T cell compartment relative to the CD8+T cell compartment.

[0049] FIG. 10A-10F show the cytolytic potential of Survivin-specific T cells. Cytolytic potential of antigen-specific T cells induced by Survivin 1 and Survivin 1T3. In FIG. 10A: Frequency of Survivin 1-specific CD4+CD107a+T cells. In FIG. 10B: Frequency of Survivin 1T3-specific CD4+CD107a+T cells. In FIG. 10C: Cytokine profile of CD4+CD107a+ T cells. In FIG. 10D: Frequency of Survivin 1-specific CD8+CD107a+T cells. In FIG. 10E: Frequency of Survivin 1T3-specific CD8+CD107a+T cells. In FIG. 10F: Cytokine profile of CD4+CD107a+ T cells.

[0050] FIG. 11A-11D show epitope mapping of IFN-gamma responses to Survivin. Width of IFNγ responses induced by Survivin 1 and Survivin 1T3. In FIG. 11A: Survivin 1 peptide pools after treatment with 50 μg pGX1428, pGX1429 or pGX0001. In FIG. 11B: Survivin T3 peptide pools after treatment with 50 μg pGX1429 or pGX0001. Comparison of pGX1428 and pGX1429 sequences with the position of the template pools indicated by dotted lines. In FIG. 11C: Sequence comparison of pGX1428 and pGX1429. Amino acids in red text indicate epitopes identified by matrix mapping. The underlined text represents parts of the sequence that are added to the synthetic Survivin consensus antigen: The N-terminus indicates the furin cleavage site for IgELS and RGRRKRRS. In FIG. 11D: Matrix design of Survivin 1 and Survivin T3. Peptides that elicited a response are indicated by dotted lines.

[0051] FIG. 12 shows Survivin-specific IFNγ SFU/10 ⁶ PBMCs from selected non-human primates immunized using an embodiment of the invention containing the synthetic consensus Survivin 1T3.

[0052] In FIG. 13 shows Survivin-specific IFNγ SFU/10 ⁶ PBMCs from selected non-human primates immunized using an embodiment of the invention containing synthetic consensus Survivin 1T3 with a low dose of IL-12 (0.04 mg).

[0053] FIG. 14 shows Survivin-specific IFNγ SFU/10 ⁶ PBMCs from selected non-human primates immunized using an embodiment of the invention containing synthetic consensus Survivin 1T3 with a high dose of IL-12 (0.20 mg).

[0054] FIG. 15A-15C show the results of separate NHP animal studies, as well as a comparison between immunization with the synthetic consensus construct Survivin 1 and the synthetic consensus construct Survivin 1T3. In FIG. 15A shows the results for group 1 animals; On figv shows the results for animals of group 2; and in FIG. 15C shows the results for group 3 animals.

DETAILED DESCRIPTION OF THE INVENTION

[0055] The present invention relates to vaccines containing the synthetic consensus antigen Survivin. Survivin is expressed in many tumors. Accordingly, the vaccine provides treatment for cancer or cancers expressing Survivin.

[0056] The synthetic Survivin consensus antigen may be a Survivin consensus antigen derived from Survivin sequences from different species or from different isoforms within a species, and thus the synthetic Survivin consensus antigen is not native. The Survivin consensus antigen can be further modified by introducing one or more mutations into the consensus sequence to create a synthetic consensus sequence. Mutations can destroy or modify specific functional domains of the native Survivin sequence, thereby disrupting or enhancing the structure or function of the functional domains. In some embodiments, additional sequences are added to the synthetic Survivin consensus antigen sequence to introduce new structures or functions. For example, a synthetic Survivin consensus antigen sequence may have a furin cleavage site. The synthetic consensus antigen Survivin sequence may include an additional localization signal to enhance the extracellular transport of the final protein product. The additional localization signal may be an IgELS or other cell transport sequence.

[0057] Survivin's synthetic consensus antigen can induce antigen-specific T cell responses and/or high antibody titer, thereby inducing or eliciting an immune response that is directed to or responsive to a cancer or tumor expressing the antigen. In some embodiments, the induced or elicited immune response may be cellular, humoral, or both cellular and humoral immune responses. In some embodiments, the induced or evoked cellular immune response may include the induction or secretion of interferon-gamma (IFN-γ) and/or tumor necrosis factor alpha (TNF-α) and/or interleukin 2 (IL-2). In other embodiments, the induced or evoked immune response may reduce or inhibit one or more immunosuppressive factors that promote the growth of an antigen-expressing tumor or cancer, such as, but not limited to, factors that suppress MHC presentation, factors that stimulate antigen-specific regulatory T cells (Treg), PD-L1, FasL, cytokines such as IL-10 and TFG-β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by immunosuppressive cells, CTLA-4, PD-1 , MDSC, MCP-1 and an immune checkpoint molecule.

[0058] The vaccine of the invention may provide any combination of specific cancer antigens for a specific prevention or treatment of cancer in a subject in need of treatment.

[0059] One way to construct a nucleic acid and its encoded recombinant cancer antigen amino acid sequence is to introduce mutations that change specific amino acids in the overall amino acid sequence of the native cancer antigen. The introduction of mutations does not alter the cancer antigen so much that it cannot be universally applied in a mammalian subject, and preferably a human or canine subject, but alters it so much that the resulting amino acid sequence is intolerant or considered a foreign antigen in the body in order to generate an immune response. Another way would be to create a consensus recombinant cancer antigen that has at least 85% to 99% amino acid sequence identity compared to its corresponding native cancer antigen; preferably at least 90% and up to 98% sequence identity; more preferably at least 93% and up to 98% sequence identity; or even more preferably at least 95% and up to 98% sequence identity. In some instances, the recombinant cancer antigen has 95%, 96%, 97%, 98%, or 99% amino acid sequence identity with its corresponding native cancer antigen. A native cancer antigen is an antigen usually associated with a particular cancer or cancer. Depending on the cancer antigen, the cancer antigen consensus sequence may occur in different mammalian species, or within subtypes of a species, or among viral strains or serotypes. Some cancer antigens do not differ much from the amino acid sequence of the wild-type cancer antigen. Some cancer antigens have nucleic acid/amino acid sequences that vary so much between species that a consensus sequence cannot be obtained. In these cases, a recombinant cancer antigen that will break tolerance and generate an immune response is designed to have at least 85% to 99% amino acid sequence identity with its corresponding native cancer antigen; preferably at least 90% and up to 98% sequence identity; more preferably at least 93% and up to 98% sequence identity; or even more preferably at least 95% and up to 98% sequence identity. In some instances, the recombinant cancer antigen has 95%, 96%, 97%, 98%, or 99% amino acid sequence identity with its corresponding native cancer antigen. The above approaches can be combined such that the resulting recombinant cancer antigen has a percentage similarity to the amino acid sequence of the native cancer antigen, as discussed above.

[0060] The vaccine can be further combined with antibodies to checkpoint inhibitors such as PD-1 and PDL-1 to enhance stimulation of both cellular and humoral immune responses. The use of anti-PD-1 or anti-PDL-1 antibodies prevents PD-1 or PDL-1 from suppressing T cell and/or B cell responses. In general, the development of cancer antigens that are recognized by the immune system helps to overcome other forms of immunosuppression by tumor cells, and these vaccines can be used in combination with suppression or inhibition therapies (eg, anti-PD-1 and anti-PDL-1 antibody therapy) to further enhancing T-cell and/or B-cell responses.

[0061] The vaccine can increase tumor free survival by 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, and 45%. The vaccine can reduce tumor mass by 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59% and 60% after immunization. The vaccine can prevent and block the rise of monocyte chemoattractant protein 1 (MCP-1), a cytokine secreted by myeloid-derived suppressor cells. The vaccine can increase tumor-free 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%.

[0062] The vaccine can enhance the cellular immune response in a subject to which the vaccine is administered from about 50-fold to about 6000-fold, from about 50-fold to about 5500-fold, from about 50-fold to about 5000-fold, from about 50 times to about 4500 times, from about 100 to about 6000 times, from about 150 times to about 6000 times, from about 200 times to about 6000 times, from about 250 times to about 6,000 times, or about 300 times to about 6,000 times the cellular immune response in an unvaccinated subject. In some embodiments, the vaccine may enhance the cellular immune response in the subject to which the vaccine is administered by about 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800 , 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 200.2 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 5100, 5200, 5300, 5400, 5500, 5600, 5600, 5600 , 5700, 5800, 5900 times or 6000 times compared to the cellular immune response in the subject who was not injected with the vaccine.

[0063] The vaccine can increase levels of interferon gamma (IFN-γ) in a subject to whom the vaccine is administered from about 50-fold to about 6000-fold, from about 50-fold to about 5500-fold, from about 50-fold to about 5000 times, from about 50 times to about 4500 times, from about 100 to about 6000 times, from about 150 times to about 6000 times, from about 200 times to about 6000 times, from about 250 times to about 6000 times or from about 300 times to about 6000 times compared to the levels of IFN-γ in a subject who did not receive the vaccine. In some embodiments, the vaccine may increase the levels of IFN-γ in the subject to which the vaccine is administered by about 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800 850 900 950 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 2100 2200 2300 2400 2500 2600 200.0 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 5100, 5200, 5300, 5400, 5500, 5500, 5500, 5500, 5500 5600, 5700, 5800, 5900 times or 6000 times compared to the levels of IFN-γ in the subject who was not administered the vaccine.

[0064] As described in more detail below, the vaccine may further comprise one or more inhibitors of one or more immune checkpoint molecules (ie, an immune checkpoint inhibitor). The immune checkpoint molecules are described in more detail below. An immune checkpoint inhibitor is any nucleic acid or protein that prevents the suppression of any component in the immune system such as MHC class presentation, T cell presentation and/or differentiation, B cell presentation and/or differentiation, any cytokine, chemokine, or transmission signals of proliferation and/or differentiation of immune cells. As also described in more detail below, the vaccine can be further combined with antibodies to checkpoint inhibitors such as PD-1 and PDL-1 to enhance stimulation of both cellular and humoral immune responses. The use of anti-PD-1 or anti-PDL-1 antibodies prevents PD-1 or PDL-1 from suppressing T cell and/or B cell responses.

Definitions

[0065] Unless otherwise noted, all technical and scientific terms used in this document have the same meaning as commonly known to a person of ordinary skill in the art. In the event of a conflict, this document, including definitions, will govern. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention. All publications, patent applications, patents and other references mentioned in this document are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and are not intended to be limiting. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0066] The terms "consist(consists)", "include(includes)", "having", "has", "may", "comprises(contains)" and variations thereof, in the context of this document, are intended to be non-limiting translatable phrases, terms or words that do not exclude the possibility of additional actions or structures. The singular noun forms and "and" include plural forms unless the context clearly specifies otherwise. The present disclosure also contemplates other embodiments "comprising", "consisting of" and "consisting essentially of" the embodiments or elements presented herein, whether explicitly stated or not.

[0067] For the listing of numerical ranges herein, each intermediate value having the same degree of accuracy as the minimum and maximum of the given range is expressly provided. For example, for the range 6-9, the numbers 7 and 8 are provided in addition to 6 and 9, and for the range 6.0-7.0 - the numbers 6.0, 6.1, 6.2, 6.3, 6.4 , 6.5, 6.6, 6.7, 6.8, 6.9 and 7.0 are explicitly provided.

[0068] The term "adjuvant", as used herein, means any molecule added to the vaccines described herein to enhance the immunogenicity of an antigen.

[0069] "Antibody", as used herein, means an IgG, IgM, IgA, IgD or IgE class antibody, or fragment or derivative thereof, including Fab, F(ab') ₂ , Fd and single chain antibodies, diabodies, bispecific antibodies, bifunctional antibodies and their derivatives. The antibody may be an antibody isolated from a mammalian serum sample, a polyclonal antibody, an affinity-purified antibody, or any mixture thereof, that exhibits sufficient binding specificity for the desired epitope or sequence derived from it.

[0070] "Antigen" refers to proteins having the amino acid sequences of the synthetic consensus antigen Survivin, including: (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) fragments containing at least 90% of amino acids 19-159 of SEQ ID NO:2; (e) fragments containing at least 90% of amino acids 19-210 of SEQ ID NO:4; (e) fragments containing at least 90% of amino acids 19-232 of SEQ ID NO:8; (g) proteins that are at least 95% identical to amino acids 19-159 of SEQ ID NO:2; (h) proteins that are at least 95% identical to amino acids 19-210 of SEQ ID NO:4; (i) proteins that are at least 95% identical to amino acids 19-232 of SEQ ID NO:8; (j) fragments containing at least 90% of a protein that is at least 95% identical to amino acids 19-159 of SEQ ID NO:2; (l) fragments containing at least 90% protein, which is at least 95% identical to amino acids 19-210 of SEQ ID NO:4; and (m) fragments 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 proteins having the amino acid sequences of the synthetic consensus antigen Survivin, including: (a) SEQ ID NO:2; (b) SEQ ID NO:4; (c) SEQ ID NO:8; (d) fragments containing at least 90% of the total length of SEQ ID NO:2; (e) fragments containing at least 90% of the total length of SEQ ID NO:4; (e) fragments containing at least 90% of the total length of SEQ ID NO:8; (g) proteins that are at least 95% identical to SEQ ID NO:2; (h) proteins that are at least 95% identical to SEQ ID NO:4; (i) proteins that are at least 95% identical to SEQ ID NO:8; (j) fragments containing at least 90% of the entire length of the protein, which is at least 95% identical to SEQ ID NO:2; (k) fragments containing at least 90% of the entire length of the protein, which is at least 95% identical to SEQ ID NO:4; and (m) fragments containing at least 90% of the entire length of the protein, which is at least 95% identical to SEQ ID NO:8. Antigens may optionally include signal peptides such as peptides from other proteins.

[0071] The terms "coding sequence" or "coding nucleic acid", in the context of this document, means nucleic acids (RNA or DNA molecules) that contain a nucleotide sequence encoding a protein. The coding sequence may further include initiation and termination signals operably linked to regulatory elements, including a promoter and polyadenylations, capable of directing expression in cells of the subject or mammal receiving the nucleic acid.

[0072] The terms "complement" or "complementarity" as used herein in relation to a nucleic acid may refer to Watson-Crick base pairing (e.g., A-T/U and C-G) or Hoogsteen base pairing between nucleotides or nucleotide analogs of nucleic acid molecules.

[0073] The terms "consensus" or "consensus sequence", as used herein, means a polypeptide sequence based on multiple sequence alignment analysis for the same gene from different organisms or from different isoforms in an organism. Can be obtained sequences of nucleic acids that encode the consensus polypeptide sequence.

[0074] The term "direct current", in the context of this document, means the current that is perceived or experienced by the tissue or cells that define the specified tissue, during the action of an electrical impulse delivered to the same tissue. An electrical impulse is supplied from the electroporation devices described herein. The current density remains constant in said tissue throughout the duration of the electrical impulse because the electroporation device provided herein has a feedback element, preferably instantaneous feedback. The feedback element allows you to measure the resistance of the tissue (or cells) throughout the duration of the pulse and cause the electroporation device to change the output electrical power (for example, increase the voltage) so that the current density in the same tissue remains constant during the entire exposure time electrical impulse (on the order of microseconds) and from impulse to impulse. In some embodiments, the feedback element comprises a controller.

[0075] The terms "current feedback" or "feedback", in the context of this document, can be used interchangeably and can mean the active response of the provided electroporation devices, which includes measuring the current in the tissue between the electrodes and the corresponding change in the output energy transmitted by the EP device , to maintain the current density at a constant level. This constant level is set by the user before initiating a pulse train or electrotherapy. Feedback can be provided by an electroporation component, such as an electroporation device controller, because the electrical circuit in it is able to continuously monitor the tissue current between the electrodes and compare this controlled current (or tissue current) with a target current and continuously adjust the output power to maintain a controlled current at the given levels. The feedback loop can be instantaneous because it is analog closed loop.

[0076] The term "decentralized current", in the context of this document, can mean a pattern of electric currents supplied from various sets of needle electrodes of the electroporation devices described herein, the circuits minimizing or preferentially eliminating the occurrence of thermal stress associated with electroporation, in any area of tissue that is electroporated.

[0077] The terms "electroporation", "electropermeabilization" or "electrokinetic enhancement" ("EP"), used interchangeably herein, means the effect of a transmembrane electric field pulse to induce microscopic pathways (pores) in a biomembrane; their presence allows biomolecules such as plasmids, oligonucleotides, miRNAs, drugs, ions, and water to pass from one side of the cell membrane to the other.

[0078] As used herein, the term "fragment", in relation to nucleic acid sequences, means a nucleic acid sequence or portion thereof that encodes a polypeptide capable of eliciting an immune response in a mammal that cross-reacts with an antigen disclosed herein. The fragments may be DNA fragments selected from at least one of the various nucleotide sequences that encode the protein fragments set forth below. Fragments may contain at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% , at least 90% or at least 95% of one or more of the nucleic acid sequences listed below, except for the addition of a heterologous signal peptide. The fragment may contain at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of one or more of the nucleic acid sequences listed below, and optionally include a sequence encoding a heterologous signal peptide that does not need to be included when calculating percent identity. The fragments may further comprise coding sequences for a signal peptide, such as an immunoglobulin signal peptide, such as an IgE or IgG signal peptide. A coding sequence encoding an N-terminal methionine and/or a signal peptide may be linked to a coding sequence fragment.

[0079] In some embodiments, fragments may be at least 20 nucleotides or more, at least 30 nucleotides or more, at least 40 nucleotides or more, at least 50 nucleotides or more, at least 60 nucleotides or more, at least 70 nucleotides or more, at least 80 nucleotides or more, at least 90 nucleotides or more, at least 100 nucleotides or more, at least 150 nucleotides or more, at least 200 nucleotides or more, at least at least 250 nucleotides or more, at least 300 nucleotides or more, at least 350 nucleotides or more, at least 400 nucleotides or more, at least 450 nucleotides or more, at least 500 nucleotides or more, at least 550 nucleotides or more, at least 600 nucleotides or more, at least 650 nucleotides or more of at least one of the nucleic acid sequences listed below.

[0080] The terms "fragment" or "immunogenic fragment", in relation to polypeptide sequences, means a polypeptide capable of inducing an immune response in a mammal that cross-reacts with the antigen disclosed herein. Fragments may be polypeptide fragments selected from at least one of the various amino acid sequences listed below. Fragments of consensus proteins can be 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% consensus protein, excluding the addition of any heterologous signal peptide. The fragment may contain at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of one or more of the amino acid sequences listed below, and optionally include a heterologous signal peptide that does not need to be included when calculating percent identity. The fragments may further comprise a signal peptide, such as an immunoglobulin signal peptide, such as an IgE or IgG signal peptide.

[0081] In some embodiments, consensus protein fragments may be at least 20 amino acids or more, at least 30 amino acids or more, at least 40 amino acids or more, at least 50 amino acids or more, at least 60 amino acids, or more than, at least 70 amino acids or more, at least 80 amino acids or more, at least 90 amino acids or more, at least 100 amino acids or more, at least 110 amino acids or more, at least 120 amino acids or more, at least 130 amino acids or more, at least 140 amino acids or more, at least 150 amino acids or more, at least 160 amino acids or more, at least 170 amino acids or more, at least 180 amino acids or more, at least at least 200 amino acids or more, at least 220 amino acids or more of the protein sequence disclosed herein.

[0082] As used herein, the term "genetic construct" refers to DNA or RNA molecules that contain a nucleotide sequence encoding a protein. The coding sequence includes initiation and termination signals operably linked to regulatory elements, including a promoter and a polyadenylation signal, capable of directing expression in the cells of the subject to which the nucleic acid is administered. In the context of this document, the term "expressible form" refers to a gene construct that contains the necessary regulatory elements operably linked to a coding sequence that encodes a protein such that when present in a cell of a subject, the coding sequence will be expressed.

[0083] In the context of this document, the term "homology" refers to the degree of complementarity. There may be partial homology or complete homology (that is, identity). A partially complementary sequence that at least partially inhibits hybridization of a fully complementary sequence to a target nucleic acid is referred to by the functional term "substantially homologous ". When used in relation to a double-stranded nucleic acid sequence, such as a cDNA or a genomic clone, the term "substantially homologous ", as used herein, refers to a probe that can hybridize to a strand of a double-stranded nucleic acid sequence under conditions of low stringency. When used with respect to a single stranded nucleic acid sequence, the term "substantially homologous ", as used herein, refers to a probe that can hybridize (i.e., complement) with a single stranded nucleic acid sequence template under conditions of low stringency.

[0084] The terms "identical" or "identity", in the context of this document, with respect to two or more nucleic acid sequences or polypeptides, means that the sequences have a certain percentage of residues that are the same in the specified area. The percentage can be calculated by optimally aligning two sequences, comparing the two sequences in the specified region, determining the number of positions where the same remainder occurs in both sequences to get the number of matching positions, dividing the number of matching positions by the total number of positions in the specified region, and multiplying the result by 100 to get percent sequence identity. In cases where the two sequences are of different lengths or the alignment results in one or more stepped ends and the specified comparison area includes only one sequence, the remainders of one 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. Identification can be done manually or with a computer sequence algorithm such as BLAST or BLAST 2.0.

[0085] The term "impedance", in the context of this document, can be used when considering the details of the feedback mechanism and can be converted to the current value in accordance with Ohm's law, which allows comparison with a given current.

[0086] The term "immune response", in the context of this document, means the activation of the immune system of the host, for example, a mammal, in response to the introduction of an antigen. The immune response may be in the form of a cellular or humoral response, or both.

[0087] The terms "nucleic acid", or "oligonucleotide", or "polynucleotide", in the context of this document, means at least two nucleotides covalently linked to each other. The description of one circuit also determines the sequence of the additional circuit. Thus, the nucleic acid also spans the complementary strand of the described single strand. Many nucleic acid variants can be used for the same purpose as a given nucleic acid. Thus, a nucleic acid also includes substantially identical nucleic acids and their complements. One strand provides a probe that can hybridize to the target sequence under stringent hybridization conditions. Thus, the nucleic acid also includes a probe that hybridizes under stringent hybridization conditions.

[0088] Nucleic acids may be single-stranded or double-stranded, or may contain parts of both a double-stranded and a single-stranded sequence. The nucleic acid can be DNA, either genomic or cDNA, RNA or hybrid, where the nucleic acid can contain combinations of deoxyribo- and ribonucleotides and combinations of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine and isoguanine. Nucleic acids can be produced by chemical synthesis or by recombinant methods.

[0089] "Operably linked", as used herein, means that the expression of a gene is under the control of the promoter to which it is spatially linked. A promoter can be located 5' (left) or 3' (right) from the gene it controls. The distance between a promoter and a gene may be approximately the same as the distance between that promoter and the gene it controls in the gene from which the promoter is derived. As is known in the art, changing this distance can be done without loss of promoter function.

[0090] In the context of this document, the terms "peptide", "protein" or "polypeptide" can mean an associated amino acid sequence and can be natural, synthetic, or be a modification or combination of natural and synthetic.

[0091] The term "promoter", as used herein, means a synthetic or naturally occurring molecule that is capable of providing, activating or enhancing the expression of a nucleic acid in a cell. The promoter may contain one or more specific transcription control sequences to further enhance expression and/or alter the spatial expression and/or temporal expression of the nucleic acid in the cell. The promoter may also contain distal enhancer or repressor elements, which may be up to several thousand base pairs away from the transcriptional start site. The promoter can be obtained from sources including viral, bacterial, fungal, plant, insect and animal sources. A promoter may regulate the expression of a gene component constitutively or differentially with respect to the cell, tissue, or organ in which expression occurs, or with respect to the developmental stage at which expression occurs, or in response to external stimuli such as physiological stresses, pathogens, metal ions, or excitatory agents. Representative examples of promoters include bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, operator lac promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, SV40 early or SV40 late promoter, and CMV IE promoter .

[0092] The terms "signal peptide" and "leader sequence" are used interchangeably herein and refer to an amino acid sequence that can be linked at the amino terminus of a protein as defined herein. Signal peptides/leader sequences typically direct protein localization. As used herein, the signal peptides/leader sequences preferably facilitate the secretion of the protein from the cell in which it is produced. Signal peptides/leader sequences are often cleaved from a remnant of the protein, often referred to as the mature protein, upon secretion from the cell. The signal peptides/leader sequences are linked at the amino-terminus (ie, N-terminus) of the protein.

[0093] "Stringent hybridization conditions", as used herein, means the conditions under which a first nucleic acid sequence (eg, probe) will hybridize to a second nucleic acid sequence (eg, target), such as in a complex mixture of nucleic acids. Stringent conditions are sequence dependent and will be different in different circumstances. Stringent conditions can be chosen to be about 5-10°C lower than the melting point (Tm) for a particular sequence at a pH of a particular ionic strength. Tm can be the temperature (at a certain ionic strength, pH and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence in equilibrium (because the target sequences are present in excess, at Tm, 50% of the probes are occupied in equilibrium). Stringent conditions may be those in which the salt concentration is less than about 1.0 M sodium ion, for example, the sodium ion concentration is about 0.01-1.0 M (or other salts) at pH 7.0-8.3, and the temperature is at least about 30° C. for short probes (eg, about 10-50 nucleotides) and at least about 60° C. for long probes (eg, greater than about 50 nucleotides). Harsh conditions can also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal can be at least 2-10 times the background hybridization. Exemplary stringent hybridization conditions include the following: 50% formamide, 5x SSC and 1% SDS, incubated at 42°C or 5x SSC, 1% SDS, incubated at 65°C, with a wash in 0.2x SSC and 0.1% SDS at 65°C.

[0094] The term "subject", in the context of this document, can mean a mammal that needs or requires immunization, presented in this document vaccines. The mammal may be a human, chimpanzee, dog, cat, horse, cow, mouse, or rat.

[0095] The term "substantially complementary", as used herein, means that the first sequence is at least 60, 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 , 90, 91, 92, 93, 94, 95, 96, 97, 98% or 99% identical to the complement of the second sequence in the region 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 180, 270, 360, 450, 540 or more nucleotides or amino acids, or that the two sequences hybridize under stringent hybridization conditions.

[0096] The term "substantially identical", as used herein, means that the first and second sequence 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 at 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 , 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 180, 270, 360, 450 , 540 or more nucleotides or amino acids, or that the two sequences hybridize under stringent hybridization conditions.

[0097] The terms "treat", "treating", or "treating", as used herein, may mean protecting an animal from a disease by means of preventing, suppressing, suppressing, or eliminating the disease. Prevention of a disease includes administering the vaccine of the present invention to an animal prior to the onset of the disease. Suppression of a disease includes administering the vaccine of the present invention to an animal after the induction of the disease but before its clinical manifestation. Suppression of a disease includes administering the vaccine of the present invention to an animal after a clinical manifestation of the disease.

[0098] As used herein, the term "variant", in relation to a nucleic acid, means (and) a portion or fragment of a reference nucleotide sequence; (ii) the complement of the reference nucleotide sequence or part thereof; (iii) a nucleic acid that is substantially identical to the reference nucleic acid or its complement; or (iv) a nucleic acid that hybridizes under stringent conditions to a reference nucleic acid, its complement, or a sequence substantially identical to it.

[0099] In the context of this document, the term "variant", in relation to a peptide or polypeptide, means a peptide or polypeptide that differs in amino acid sequence by an insertion, deletion, or conservative amino acid substitution, but retains at least one biological activity. A variant can also mean a protein with an amino acid sequence that is substantially identical to a reference protein with an amino acid sequence that retains at least one biological activity. Conservative amino acid substitution, that is, substitution of an amino acid for another amino acid with similar properties (eg, hydrophilicity, degree and distribution of charged regions), is known in the art to typically involve minor changes. These minor changes can be identified, in part, in terms of the amino acid hydrophobicity index as understood in the art. Kyte et al., J. Mol. Biol. 157:105-132 (1982). The hydrophobicity index of an amino acid is based on an assessment of its hydrophobicity and charge. It is known in the art that amino acids with similar hydrophobicity indices can be substituted and still retain protein function. In one aspect, amino acids with hydrophobicity indices ± 2 are substituted. The hydrophilicity of amino acids can also be used to identify substitutions that cause proteins to retain biological function. Assessment of amino acid hydrophilicity in the context of a peptide allows the calculation of the greatest local average hydrophilicity of that peptide, which is a useful measurement that has been reported to correlate well with antigenicity and immunogenicity. US Patent No. 4,554,101 is incorporated herein by reference in its entirety. Substitution of amino acids having similar hydrophilicity values can result in peptides retaining biological activity, eg immunogenicity, as understood in the art. Substitutions can be made with amino acids having each other's hydrophilicity values within ±2. Both the hydrophobicity index and hydrophilicity value of amino acids are affected by the specific side chain of that amino acid. In accordance with this observation, it is believed that amino acid substitutions that are compatible with biological function depend on the relative similarity of amino acids and, in particular, on the side chains of these amino acids, which manifests itself in hydrophobicity, hydrophilicity, charge, size and other properties.

[00100] A variant may be a nucleic acid sequence that is substantially identical over the entire length of the complete gene sequence or fragment thereof. The nucleic acid sequence can be 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or 100% identical along the entire length of the gene sequence or its fragment. A variant may be an amino acid sequence that is substantially identical over the entire length of the amino acid sequence or fragment thereof. The amino acid sequence can be 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, or 100% identical in the entire length of the amino acid sequence or its fragment.

[00101] The term "vector", in the context of this document, means a nucleic acid sequence containing a source of replication. The vector may be a viral vector, a bacteriophage, a bacterial artificial chromosome, or a yeast artificial chromosome. The vector may be a DNA or RNA vector. The vector may be a self-replicating extrachromosomal vector and is preferably a DNA plasmid. The vector may contain or include one or more heterologous nucleic acid sequences.

Vaccines

[00102] Provided herein are vaccines comprising the synthetic Survivin consensus antigen as described herein, a nucleic acid molecule encoding an antigen, a nucleic acid molecule encoding an antigen fragment, a nucleic acid molecule encoding a variant antigen, or combinations thereof. Vaccines may be capable of eliciting an immune response against an antigen in a subject. The immune response may be a therapeutic or prophylactic immune response. Vaccines may contain a vector or a plurality of vectors, as described in more detail below.

[00103] In some embodiments, the vaccine comprises a nucleic acid molecule. In some embodiments, the nucleic acid molecule encodes a synthetic Survivin consensus antigen. In some embodiments, the nucleic acid molecule contains a nucleic acid sequence that encodes SEQ ID NO: 3; a nucleic acid sequence that encodes a fragment containing at least 90% of the length of SEQ ID NO 3; a nucleic acid sequence that encodes a protein that is at least 95% identical to SEQ ID NO: 3; or a nucleic acid sequence that encodes a fragment containing at least 90% of the entire length of the protein, which is at least 95% identical to SEQ ID NO: 3. In some embodiments, the nucleic acid molecule contains SEQ ID NO: 1; a fragment containing at least 90% of the entire 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 entire length of a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 1.

[00104] In some embodiments, the nucleic acid molecule contains a nucleic acid sequence that encodes SEQ ID NO: 4; a nucleic acid sequence that encodes a fragment containing at least 90% of the length of SEQ ID NO 4; a nucleic acid sequence that encodes a protein that is at least 95% identical to SEQ ID NO: 4; or a nucleic acid sequence that encodes a fragment containing at least 90% of the entire length of the protein, which is at least 95% identical to SEQ ID NO: 4. In some embodiments, the nucleic acid molecule contains SEQ ID NO: 2; a fragment containing at least 90% of the entire 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 entire length of a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 2.

[00105] In some embodiments, the vaccine contains a synthetic consensus antigen Survivin, where the antigen contains SEQ ID NO: 3; a fragment containing 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 containing at least 90% of the entire length of the protein, which is at least 95% identical to SEQ ID NO: 3.

[00106] In some embodiments, the vaccine contains a synthetic consensus antigen Survivin, where the antigen contains SEQ ID NO: 4; a fragment containing 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 containing at least 90% of the entire length of the protein, which is at least 95% identical to SEQ ID NO: 4.

[00107] Vaccines can be used to protect against cancer, such as cancer or tumor expressing Survivin. The vaccines can be used to prevent and/or treat a tumor expressing Survivin in a subject in need thereof. The vaccines can induce cellular and/or antibody responses against Survivin and against tumors expressing Survivin.

[00108] In one embodiment, the vaccines can be used to protect, prevent and/or treat, or to induce a cellular and/or antibody response against Survivin-expressing ovarian cancer cells, in particular Survivin-expressing ovarian epithelial cells, more specifically cells serous ovarian cancer expressing Survivin.

[00109] The development of a cancer vaccine as described herein involves the identification of a cancer antigen, such as Survivin, which is not recognized by the immune system and is a tumor associated antigen ("cancer/testis", "C/T"). The identified cancer antigen is changed from a self antigen to a foreign antigen for recognition by the immune system. Rearrangement of the nucleic acid and amino acid sequence of the recombinant cancer antigen from auto- to foreign antigen disrupts the antigen's tolerance by the immune system. To break tolerance, more redesign measures can be applied to the cancer antigen, as described below.

[00110] The recombinant vaccine cancer antigen is not recognized as a self antigen, thereby breaking tolerance. Impaired tolerance can induce antigen-specific T cell responses and/or high antibody titer, thereby inducing or inducing an immune response that is directed to or responsive to the antigen-expressing cancer or tumor. In some embodiments, the induced or elicited immune response may be cellular, humoral, or both cellular and humoral immune responses. In some embodiments, the induced or evoked cellular immune response may include the induction or secretion of interferon-gamma (IFN-γ) and/or tumor necrosis factor alpha (TNF-α) and/or interleukin 2 (IL-2). In other embodiments, the induced or evoked immune response may reduce or inhibit one or more immunosuppressive factors that promote the growth of an antigen-expressing tumor or cancer, such as, but not limited to, factors that suppress MHC presentation, factors that stimulate antigen-specific regulatory T cells (Treg), PD-L1, FasL, cytokines such as IL-10 and TFG-β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by immunosuppressive cells, CTLA-4, PD-1 , MDSC, MCP-1 and an immune checkpoint molecule.

[00111] In a specific embodiment, the vaccine can provide clearance or prevent the growth of tumor cells by (1) increasing cytotoxic T-lymphocytes, such as CD8 ⁺ and/or CD107a ⁺ (CTL), to attack and kill tumor cells; (2) increase in T-helper responses; and/or (3) enhancing inflammatory responses by IFN-γ, IL-2 and TFN-α, or preferably all of the above.

[00112] The vaccine may be a DNA vaccine. DNA vaccines are described in US Pat. Nos. 5,593,972; 5,739,118; 5,817,637; 5,830,876; The DNA vaccine may additionally contain elements or reagents that prevent its integration into the chromosome.

[00113] The vaccine may contain RNA encoding a cancer antigen. The RNA vaccine can be introduced into the cell.

[00114] The vaccine may be an attenuated live vaccine, a vaccine using recombinant antigen delivery vectors, subunit vaccines, and glycoprotein vaccines, such as, but not limited to, the vaccines described in US Pat. Nos.: 4,510,245; 4797368; 4722848; 4790987; 4920209; 5017487; 5077044; 5110587; 5112749; 5174993; 5223424; 5225336; 5240703; 5242829; 5294441; 5294548; 5310668; 5387744; 5389368; 5424065; 5451499; 5,453,364; 5462734; 5470734; 5474935; 5482713; 5591439; 5643579; 5650309; 5698202; 5955088; 6034298; 6042836; 6156319 and 6589529, each of which is incorporated herein by reference.

[00115] In some embodiments, the nucleic acid vaccine may further comprise a coding sequence for a molecular adjuvant, in some cases the molecular adjuvant may be IL-12, IL-15, IL-28, IL-31, IL-33 and/or RANTES , and in some cases, the molecular adjuvant is a checkpoint inhibitor, including against cytotoxic T-lymphocyte antigen 4 (CTLA-4), against programmed death receptor-1 (PD-1), and against the lymphocyte activation gene (LAG-3). The coding sequence for IL-12, IL-15, IL-28, IL-31, IL-33 and/or RANTES may be included in one or more nucleic acid molecules that contain the coding sequence for one or more antigens. The coding sequence for IL-12, IL-15, IL-28, IL-31, IL-33 and/or RANTES may be included in one or more single nucleic acid molecules, such as one or more single plasmids or vectors, and administered in combination with a nucleic acid vaccine.

[00116] The vaccines of the present invention may have the properties required for effective vaccines, such as safety, such that the vaccine itself does not cause disease or death; protection from diseases; induction of a neutralizing antibody; induction of protective T-cell responses; and providing ease of administration, few side effects, biological stability, and low cost per dose. A vaccine may achieve some or all of these properties by containing nucleic acid molecule(s) encoding a cancer antigen, as discussed below.

Vaccine in combination with immune checkpoint inhibitor

[00117] The vaccine may further comprise one or more inhibitors of one or more immune checkpoint molecules (ie, an immune checkpoint inhibitor). The immune checkpoint molecules are described in more detail below. An immune checkpoint inhibitor is any nucleic acid or protein that prevents the suppression of any component in the immune system such as MHC class presentation, T cell presentation and/or differentiation, B cell presentation and/or differentiation, any cytokine, chemokine, or transmission signals of proliferation and/or differentiation of immune cells.

[00118] Such an inhibitor may be a nucleic acid sequence, an amino acid sequence, a small molecule, or a combination thereof. The nucleic acid sequence may be DNA, RNA, cDNA, a variant thereof, a fragment thereof, or a combination thereof. The nucleic acid may also include additional sequences that encode linker or tag sequences that are linked to the immune checkpoint inhibitor by a peptide bond. The small molecule can be a low molecular weight, for example, less than 800 Daltons, organic or inorganic compound that can serve as an enzyme substrate, a ligand (or equivalent) associated with a protein or nucleic acid, or a biological process regulator. The amino acid sequence may be a protein, a peptide, a variant thereof, a fragment thereof, or a combination thereof.

[00119] In some embodiments, an immune checkpoint inhibitor may be one or more nucleic acid sequences encoding an antibody, a variant thereof, a fragment thereof, or a combination thereof. In other embodiments, the immune checkpoint inhibitor may be an antibody, a variant thereof, a fragment thereof, or a combination thereof.

Immune checkpoint molecule

[00120] An immune checkpoint molecule can be a nucleic acid sequence, an amino acid sequence, a small molecule, or a combination thereof. The nucleic acid sequence may be DNA, RNA, cDNA, a variant thereof, a fragment thereof, or a combination thereof. The nucleic acid may also include additional sequences that encode linker or tag sequences that are linked to the immune checkpoint inhibitor by a peptide bond. The small molecule can be a low molecular weight, for example, less than 800 Daltons, organic or inorganic compound that can serve as an enzyme substrate, a ligand (or equivalent) associated with a protein or nucleic acid, or a biological process regulator. The amino acid sequence may be a protein, a peptide, a variant thereof, a fragment thereof, or a combination thereof.

PD-1 and PD-L1

[00121] The immune checkpoint molecule can be programmed cell death protein 1 (PD-1), programmed cell death ligand 1 (PD-L1), a fragment thereof, a variant thereof, or a combination thereof. PD-1 is a cell surface protein encoded by the PDCD1 gene. PD-1 is a member of the immunoglobulin superfamily and is expressed on T cells and pro-B cells and thus promotes recruitment and/or differentiation of these cells. In particular, PD-1 is a type 1 membrane protein of the CD28/CTLA-4 T cell regulator family and negatively regulates T cell receptor (TCR) signals, thereby negatively regulating immune responses. PD-1 can negatively regulate CD8 ⁺ T cell responses and thus inhibit CD8-mediated cytotoxicity and enhance tumor growth.

[00122] PD-1 has two ligands, PD-L1 and PD-L2, which are members of the B7 family. PD-L1 is upregulated on macrophages and dendritic cells (DCs) in response to LPS and GM-CSF treatment and on T cells and B cells upon TCR and B cell receptor signaling. PD-L1 is expressed by many tumor cell lines, including myelomas, mastocytomas, and melanomas.

Antibody against immune checkpoint molecule

[00123] As described above, the immune checkpoint inhibitor may be an antibody. An antibody may bind or react with an antigen (ie, the immune checkpoint molecule described above). Accordingly, an antibody may be considered an anti-immune checkpoint antibody or an immune checkpoint antibody. An antibody may be encoded by a nucleic acid sequence contained herein.

[00124] The antibody may include a heavy chain polypeptide and a light chain polypeptide. The heavy chain polypeptide may include a heavy chain variable region (VH) and/or at least one heavy chain constant region (CH). The at least one heavy chain constant region may include a heavy chain constant region 1 (CH1), a heavy chain constant region 2 (CH2), and a heavy chain constant region 3 (CH3) and/or a hinge region.

[00125] 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.

[00126] The heavy chain polypeptide may include a set of complementarity determining regions ("CDRs"). A set of CDRs may contain three hypervariable regions of the VH region. Starting at the N-terminus of the heavy chain polypeptide, these CDRs are designated "CDR1", "CDR2", and "CDR3", respectively. The CDR1, CDR2, and CDR3 of a heavy chain polypeptide may contribute to antigen binding or recognition.

[00127] The light chain polypeptide may include a variable light chain (VL) region and/or a constant light chain (CL) region. The light chain polypeptide may include a set of complementarity determining regions ("CDRs"). The CDR set may contain three hypervariable regions of the VL region. Starting at the N-terminus of the light chain polypeptide, these CDRs are designated "CDR1", "CDR2", and "CDR3", respectively. The CDR1, CDR2, and CDR3 of a light chain polypeptide can contribute to antigen binding or recognition.

[00128] An antibody may contain a set of heavy chain and light chain complementarity determining ("CDR") regions, and accordingly be placed between a set of framework heavy chain and light chain ("FR"), which provide support for the CDR and determine the spatial relationship of the CDR relative to each other. A set of CDRs may contain three heavy or light chain V region hypervariable regions. Starting at the N-terminus of the heavy or light chain, these regions are designated "CDR1", "CDR2", and "CDR3", respectively. Therefore, an antigen binding site may include six CDRs containing a set of CDRs from each heavy and light chain V region.

[00129] The antibody may be an immunoglobulin (Ig). The Ig may be, for example, IgA, IgM, IgD, IgE and IgG. The immunoglobulin may include a heavy chain polypeptide and a light chain polypeptide. An immunoglobulin heavy chain polypeptide may include a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region. An immunoglobulin light chain polypeptide may include a VL region and a CL region.

[00130] In addition, the proteolytic enzyme papain preferentially cleaves IgG molecules to form multiple fragments, two of which (F(ab) fragments) contain a covalent heterodimer that includes an intact antigen-binding site. The pepsin enzyme is able to cleave IgG molecules into several fragments, including the F(ab') ₂ fragment, which contains both antigen-binding sites. Accordingly, the antibody may be Fab or F(ab') ₂ . The Fab may include a heavy chain polypeptide and a light chain polypeptide. The Fab heavy chain polypeptide may include a VH region and a CH1 region. The light chain Fab may include a VL region and a CL region.

[00131] The antibody may be a polyclonal or monoclonal antibody. The antibody can be a chimeric antibody, a single chain antibody, an affinity matured antibody, a human antibody, a humanized antibody, or a fully human antibody. The humanized antibody may be a non-human antibody that binds the desired antigen, having one or more complementarity determining regions (CDRs) from a non-human species and framework regions from a human immunoglobulin molecule.

Antibody to PD-1

[00132] An anti-immune checkpoint antibody can be an anti-PD-1 antibody (also referred to herein as "anti-PD-1 antibody"), a variant thereof, a fragment thereof, or a combination thereof. The anti-PD-1 antibody may be nivolumab. An anti-PD-1 antibody can inhibit the activity of PD-1, thereby inducing, eliciting or enhancing an immune response against a tumor or cancer and reducing tumor growth.

Antibody to PD-L1

[00133] An anti-immune checkpoint antibody can be an anti-PD-L1 antibody (also referred to herein as "anti-PD-L1 antibody"), a variant thereof, a fragment thereof, or a combination thereof. An anti-PD-L1 antibody can inhibit PD-L1 activity, thereby inducing, eliciting or enhancing an immune response against a tumor or cancer and reducing tumor growth.

Antigens

[00134] As described above, the vaccine may contain an antigen or a nucleic acid encoding an antigen. The antigen may be Survivin, a fragment thereof, a variant thereof, or a combination of a fragment and a variant thereof.

[00135] Accordingly, the vaccine can be used to treat subjects suffering from cancer or tumors that express Survivin. In some embodiments, the cancer is ovarian cancer. In some embodiments, the ovarian cancer is epithelial ovarian cancer. Ovarian cancer may be serous epithelial ovarian cancer. The vaccine may also be used to treat subjects with cancers or tumors that express Survivin to prevent the subjects from developing such tumors. The synthetic Survivin consensus antigen may differ from the native Survivin gene and thus provide therapy or prophylaxis against a tumor expressing the synthetic Survivin consensus antigen. Accordingly, provided herein are synthetic Survivin antigen consensus sequences that differ from the native Survivin gene (i.e., mutated or synthetic Survivin genes or sequences).

[00136] Transcripts of the native Survivin gene are processed into a variety of mRNAs. Specific Survivin mRNA isoforms can be selected based on, for example, their expression in cancer cells. In specific embodiments, the Survivin isoform is selected based on its expression in ovarian cancer cells. The synthetic Survivin antigen consensus sequences described herein avoid alternative processing, producing a single full-length transcript, resulting in stronger induction of effector T and B cell responses.

[00137] Proposed isolated nucleic acid molecules containing the heterologous sequences described above. Proposed isolated nucleic acid molecules consisting of the heterologous sequences described above. Isolated nucleic acid molecules containing the heterologous sequences described above can be inserted into vectors such as plasmids, viral vectors, and other forms of nucleic acid molecules, as described below. This document provides nucleic acid sequences that encode synthetic Survivin consensus antigens. The coding sequences encoding the synthetic Survivin consensus antigens have the sequences as described above.

[00138] Proposed protein molecules containing the heterologous amino acid sequences described above. Proposed protein molecules consisting of the heterologous amino acid sequences described above. Provided herein are proteins and polypeptides having the sequences described above. The proteins and polypeptide may be referred to as synthetic Survivin consensus antigens and Survivin immunogens. Survivin consensus synthetic antigens are capable of eliciting an immune response against tumors expressing Survivin.

[00139] In one aspect, it is desirable that the synthetic Survivin consensus antigen provide improved transcription and translation, including having one or more of the following: a low GC leader sequence to increase transcription; mRNA stability and codon optimization; and, as far as possible, eliminating cis-acting sequence motifs (i.e. internal TATA boxes).

[00140] A synthetic Survivin consensus 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 different species of Survivin isoforms. The consensus sequence is derived from Survivin sequences collected from GenBank or other similar DNA or protein sequence database. In some embodiments, the consensus antigen may comprise a portion of the first isoform combined with a portion of the second isoform, with the portion of the second isoform, for example, being non-homologous to any portion of the first isoform. The synthetic Survivin consensus antigen may contain a consensus sequence and/or modification(s) to improve expression. The modification may include codon optimization, RNA optimization, addition of a Kozak sequence (eg, GCC ACC) to enhance translation initiation, and/or addition of an immunoglobulin leader sequence to increase the immunogenicity of the synthetic Survivin consensus antigen. The synthetic Survivin consensus antigen may comprise a signal peptide, such as an immunoglobulin signal peptide, for example, but not limited to, an immunoglobulin E (IgE) or immunoglobulin G (IgG) signal peptide. In some embodiments, the synthetic Survivin consensus antigen may include mutations or deletions in localization signal sequences, eg, nuclear localization signal, for example, to disrupt nuclear localization upon translation. In some embodiments, the Survivin consensus antigen may contain a hemagglutinin (HA) tag. The Survivin consensus antigen can be engineered to elicit stronger and broader cellular and/or humoral immune responses than the corresponding non-codon optimized Survivin antigen.

[00141] The Survivin consensus sequence can be mutated to destroy and/or enhance certain structures and/or functions of native Survivin to produce a synthetic Survivin antigen consensus sequence. In one embodiment, the mutations are introduced to eliminate the anti-apoptotic activity of Survivin. In a specific embodiment, the T34A, T48A and C84A mutations are introduced into the Survivin consensus isoform 1 sequence to abolish anti-apoptotic function. ( See Muchmore, SW et al. Crystal structure and mutagenic analysis of the inhibitor-of-apoptosis protein Survivin. Molecular cell 6, 173-182 (2000); O'Connor, DS et al. Regulation of apoptosis at cell division by p34cdc2 phosphorylation of Survivin Proceedings of the National Academy of Sciences of the United States of America 97, 13103-13107, doi:10.1073/pnas.240390697 (2000) Barrett, RM, Colnaghi, R. & Wheatley, SP Threonine 48 in the BIR domain of Survivin is critical to its mitotic and anti-apoptotic activities and can be phosphorylated by CK2 in vitro Cell cycle (Georgetown, Tex.) 10, 538-548 (2011).)

[00142] In some embodiments, the synthetic Survivin consensus antigen sequence may be generated from a single isoform, e.g., the dominant Survivin isoform, or the consensus sequence may comprise a combination of a portion of the first isoform and a portion of the second isoform, or a truncated portion of the second isoform. In one embodiment, the synthetic consensus sequence is derived from Suvivin isoform 1 (Survivin 1). In another embodiment, the synthetic consensus sequence is derived from Suvivin isoform 3 (Survivin 3). In one embodiment, the Survivin 3 synthetic consensus antigen sequence is a truncated portion of Survivin 3 (Survivin 3T). In one embodiment, the synthetic consensus sequence is a combination of Survivin 1 and Survivin T3 or Survivin 1T3.

[00143] In a preferred embodiment, the synthetic consensus antigen Survivin sequence has 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 antigen Survivin sequence has 95.0% or more identity, 95.2% or more identity, 95.4% or more identity, 95.6% or more identity, 95.8% or more identity, 96.0% or more identical, 96.2% or more identical, 96.4% or more identical, 96.6% or more identical, 96.8% or more identical, 97.0% or more identical, 97, 2% or more identical, 97.4% or more identical, 97.6% or more identical, 97.8% or more identical, 98.0% or more identical, 98.2% or more identical, 98.4% or more identical, or 98.6% or more identical, 98.8% or more identical, 99.0% or more identical, 99.2% or more identical, 99.4% or more identical, 99.6% or more than identical, 99.8% or more identical, or 100% identical to SEQ ID NO:1 or SEQ ID NO:3.

Vectors

[00144] The vaccine may contain one or more vectors that include a heterologous nucleic acid encoding a synthetic Survivin consensus antigen. One or more vectors may be capable of expressing an antigen in an amount effective to elicit an immune response in a mammal. The vector may contain a heterologous nucleic acid encoding an antigen. The vector may have a nucleic acid sequence containing an origin of replication. The vector may be a plasmid, a bacteriophage, a bacterial artificial chromosome, or a yeast artificial chromosome. The vector can either be a self-replicating extrachromosomal vector or a vector that integrates into the host's genome.

[00145] One or more vectors may be an expression construct, which is a plasmid that is typically used to introduce a specific gene into a target cell. Once the expression vector is inside the cell, the protein encoded by the gene is produced by the ribosomal complexes of cellular transcription and translation machinery. The plasmid is often designed to contain regulatory sequences that act as enhancer and promoter regions and result in efficient transcription of the gene carried on the expression vector. The vectors of the present invention express large amounts of stable messenger RNA and hence proteins.

[00146] Vectors may have expression signals such as a strong promoter, a strong termination codon, regulation of the distance between the promoter and the cloned gene, and the insertion of a transcription termination sequence and PTIS (portable translation initiation sequence).

[00147] The vector can be a circular plasmid or a linear nucleic acid. The circular plasmid and the linear nucleic acid are capable of directing the expression of a particular nucleotide sequence in the corresponding cell of the subject. The vector may have a promoter operably linked to an antigen-coding nucleotide sequence which may be operably linked to termination signals. The vector may also contain sequences necessary for the correct translation of the nucleotide sequence, as well as sequences for cloning and subcloning of the vector and its fragments. The vector containing the nucleotide sequence of interest may be chimeric, which means that at least one of its components is heterologous with respect to at least one of its other components. Expression of a nucleotide sequence in an expression cassette may be under the control of a constitutive promoter or an inducible promoter that initiates transcription only when a particular external stimulus is applied to the host cell. In the case of a multicellular organism, the promoter may also be specific to a particular tissue or organ or developmental stage. In a preferred embodiment, the plasmid vector is pGX1428 described herein, further comprising the nucleic acid sequence of SEQ ID NO: 1; or pGX1429 described herein, further comprising the nucleic acid sequence of SEQ ID NO: 3.

[00148] The vector may be a plasmid. The plasmid can be used to transfect cells with a nucleic acid encoding an antigen. Transformed host cells can be cultured and maintained under conditions where antigen expression occurs.

[00149] The plasmid may contain a nucleic acid sequence that encodes one or more of the various antigens disclosed above, including coding sequences that encode a synthetic consensus antigen capable of eliciting an immune response against the antigen, fragments of such proteins, variants of such proteins, fragments of variants, or fusions. proteins that consist of combinations of consensus proteins and/or consensus protein fragments and/or consensus protein variants and/or consensus protein variant fragments.

[00150] One plasmid may contain a coding sequence for one antigen, a coding sequence for two antigens, a coding sequence for three antigens, or a coding sequence for four antigens.

[00151] In some embodiments, the plasmid may further comprise a coding sequence that encodes CCR20 alone or as part of one of these plasmids. Similarly, plasmids may further contain coding sequences for IL-12, IL-15 and/or IL-28.

[00152] The plasmid may further contain an initiation codon, which may be to the left of the coding sequence, and a stop codon, which may be to the right of the coding sequence. The initiation and termination codon may be within the frame of the coding sequence.

[00153] The plasmid may also contain a promoter that is operably linked to a coding sequence. A promoter operably linked to a coding sequence can be a simian virus 40 (SV40) promoter, a mouse mammary tumor virus (MMTV) promoter, a human immunodeficiency virus (HIV) promoter such as the bovine immunodeficiency virus long terminal repeat (LTR) promoter ( BIV), a Moloney virus promoter, an avian leukemia virus (ALV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter, an Epstein-Barr virus (EBV) promoter, or a Rous sarcoma virus (RSV) promoter. The promoter may also be a human gene promoter such as human actin, human myosin, human hemoglobin, human muscle creatine, or human metallothionein. The promoter may also be a tissue-specific promoter, such as a muscle or skin specific promoter, natural or synthetic. Examples of such promoters are described in US Patent Application Publication No. 20040175727, the contents of which are incorporated herein in their entirety.

[00154] The plasmid may also contain a polyadenylation signal, which may be to the right of the coding sequence. The polyadenylation signal can be an SV40 polyadenylation signal, an LTR polyadenylation signal, a bovine growth hormone (bGH) polyadenylation signal, a human growth hormone (hGH) polyadenylation signal, or a human β-globin polyadenylation signal. The SV40 polyadenylation signal may be the polyadenylation signal from the plasmid pCEP4 (Invitrogen, San Diego, CA).

[00155] The plasmid may also contain an enhancer to the left of the coding sequence. The enhancer can be a human actin enhancer, human myosin enhancer, human hemoglobin enhancer, human muscle creatine enhancer, or a viral enhancer such as a CMV, FMDV, RSV or EBV enhancer. Polynucleotide function enhancers are described in US Pat.

[00156] The plasmid may also contain a mammalian origin of replication for maintaining the extrachromosomal plasmid and making multiple copies of the plasmid in the cell. The plasmid may be pVAXI, pCEP4, or pREP4 from Invitrogen (San Diego, CA), which may include an Epstein-Barr virus origin of replication and an EBNA-1 nuclear antigen coding region, which may result in high-copy episomal replication without integration. The basis of the plasmid can be pA V0242. The plasmid may be a replication defective adenovirus type 5 (Ad5) plasmid.

[00157] The plasmid may also contain a regulatory sequence that may be well suited for gene expression in the cell into which the plasmid is introduced. The coding sequence may contain a codon that can provide more efficient transcription of the coding sequence in the host cell.

[00158] The coding sequence may also contain an Ig leader sequence. The leader sequence may be a 5' coding sequence. Consensus antigens encoded by this sequence may contain an N-terminal Ig leader followed by an antigen consensus protein. The leader of the N-terminal Ig may be IgE or IgG.

[00159] The plasmid can be pSE420 (Invitrogen, San Diego, Calif.), which can be used to produce protein in Escherichia coli (E. coli). The plasmid can also be p YES2 (Invitrogen, San Diego, CA) which can be used to produce protein in strains of the yeast Saccharomyces cerevisiae. The plasmid can also be a complete MAXBAC™ baculovirus expression system (Invitrogen, San Diego, CA) that can be used for protein production in insect cells. The plasmid can also be pcDNA I or pcDNA3 (Invitrogen, San Diego, Calif.), which can be used for protein production in mammalian cells such as Chinese hamster ovary (CHO) cells.

[00160] The vector can be a circular plasmid that can transform the target cell by integrating into the cellular genome or exist extrachromosomally (eg, an autonomous replicating plasmid with an origin of replication).

[00161] The vector can be pVAX, pcDNA3.0 or provax, or any other expression vector capable of expressing the DNA encoding the antigen and allowing the cell to translate the sequence into an antigen that is recognized by the immune system.

[00162] Also provided herein is a linear nucleic acid vaccine or linear expression cassette ("LEC") that is capable of being efficiently delivered to a subject by electroporation and expression of one or more desired antigens. The LEC can be any linear DNA lacking any phosphate backbone. DNA may encode one or more antigens. The LEC may contain a promoter, an intron, a stop codon, and/or a polyadenylation signal. Expression of an antigen can be controlled by a promoter. The LEC may not contain antibiotic resistance and/or phosphate backbone genes. The LEC may not contain other nucleic acid sequences not associated with the desired expression of the antigen gene.

[00163] LEC can be obtained from any plasmid capable of linearization. The plasmid may be capable of expressing the antigen. The plasmid can be pNP (Puerto Rico/34) or pM2 (New Caledonia/99). The plasmid can be WLV009, pVAX, pcDNA3.0 or provax, or any other expression vector capable of expressing the DNA encoding the antigen and allowing the cell to translate the sequence into an antigen that is recognized by the immune system.

[00164] The LEC may be pcrM2. The LEC may be pcrNP. pcrNP and pcrMR can be obtained from pNP (Puerto Rico/34) and pM2 (New Caledonia/99), respectively.

[00165] The vector may have a promoter. The promoter can be any promoter that is capable of directing gene expression and regulating the expression of the isolated nucleic acid. Such a promoter is a cis-acting sequence element required for transcription through a DNA-dependent RNA polymerase that transcribes the antigen sequence described herein. The choice of promoter used for direct expression of a heterologous nucleic acid depends on the particular application. The promoter can be located approximately the same distance from the transcription start in the vector as it is from the transcription start site in its natural environment. However, changing this distance can be achieved without loss of promoter function.

[00166] A promoter may be operably linked to a nucleic acid sequence encoding an antigen and signals necessary for efficient polyadenylation of the transcript, ribosome binding sites, and translation termination.

[00167] The promoter can be a CMV promoter, an SV40 early promoter, an SV40 late promoter, a metallothionein promoter, a mouse mammary tumor virus promoter, a Rous sarcoma virus promoter, a polyhedrin promoter, or another promoter shown to be effective for expression in eukaryotic cells.

[00168] The vector may include an enhancer and an intron with functional splicing donor and acceptor sites. The vector may contain a transcription termination region to the right of the structural gene to ensure efficient termination. The termination region may be derived from the same gene as the promoter sequence, or may be derived from different genes.

Methods for obtaining a vector

[00169] Provided herein are methods for producing a vector that contains nucleic acid molecules encoding the synthetic Survivin consensus antigen discussed herein. The vector after the final subcloning step can be used to inoculate the cell culture in a large scale fermentation tank using methods known in the art.

[00170] A vector for use with EP devices, which are described in more detail below, can be prepared or made using a combination of known devices and techniques, but are preferably made using an optimized plasmid manufacturing technique as described in U.S. Provisional Application Serial No. 60 /939792, which was filed May 23, 2007 (see US Pat. Publication No. 20090004716). In some examples, the DNA vectors used in these studies can be prepared at concentrations greater than or equal to 10 mg/ml. The manufacturing techniques also include or combine various devices and protocols that are generally known to those skilled in the art, in addition to those described in US application serial number 60/939792, including those described in US patent No. 7238522, which is issued July 3, 2007 The above application and US Pat. No. 60/939,792 and US Pat. No. 7,238,522, respectively, are incorporated herein in their entirety.

Excipients and other components of the vaccine

[00171] The vaccine may further comprise a pharmaceutically acceptable excipient. A pharmaceutically acceptable excipient may be a functional molecule such as a filler, carrier or diluent. The pharmaceutically acceptable excipient may be a transfection facilitating agent which may include surfactants such as immunostimulatory complexes (ISCOMS), incomplete Freund's adjuvant, LPS analog including monophosphoryl lipid A, muramyl peptides, quinone analogs, vesicles such as squalene and squalene , hyaluronic acid, lipids, liposomes, calcium ions, viral proteins, polyanions, polycations or nanoparticles, or other known transfection aids.

[00172] The transfection facilitator is a polyanion, a polycation, including poly-L-glutamate (LGS), or a lipid. The transfection facilitator is poly-L-glutamate, and poly-L-glutamate may be present in the vaccine at a concentration of less than 6 mg/ml. The transfection facilitator may also include surfactants such as immunostimulatory complexes (ISCOMS), incomplete Freund's adjuvant, an LPS analog including monophosphoryl lipid A, muramyl peptides, quinone analogs and vesicles such as squalene and squalene, and hyaluronic acid may also be used in conjunction with a genetic construct. DNA vector vaccines may also include a transfection facilitator such as lipids, liposomes, including lecithin liposomes or other liposomes known in the art, as a mixture of DNA liposomes (see e.g. W09324640), calcium ions, viral proteins, polyanions, polycations or nanoparticles, or other known means to facilitate transfection. The transfection facilitator is a polyanion, a polycation including poly-L-glutamate (LGS) or a lipid. The concentration of the transfection agent in the vaccine is less than 4 mg/ml, less than 2 mg/ml, less than 1 mg/ml, 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.

[00173] The pharmaceutically acceptable excipient may be one or more adjuvants. The adjuvant may be other genes that are expressed in an alternative vector or delivered as proteins in combination with the above vector in a vaccine. One or more adjuvants may be selected from the group consisting of: CCL20, α-interferon (IFN-α), β-interferon (IFN-β), γ-interferon, platelet growth factor (PDGF), TNFα, TNFβ, GM- CSF, epidermal growth factor (EGF), skin T-cell-attracting chemokine (CTACK), epithelial thymus-expressed chemokine (TECK), epithelial mucosal-associated chemokine (MEC), IL-12, IL-15, IL- 28, MHC, CD80, CD86, IL-l, IL-2, IL-4, IL-5, IL-6, IL-10, IL-18, IL-33, MCP-1, MIP-la, MIP- 1~, IL-8, 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, mutants of IL-18, CD40, CD40L, vascular growth factor, fibroblast growth factor, IL-7, nerve growth factor, vascular endothelial growth, Fas, TNF receptor, Flt, Apo-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 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 a coding sequence that encodes a deleted signal sequence and optionally includes another signal peptide such as from IgE, or a coding sequence that encodes another signal peptide, such as from IgE, and functional fragments thereof, or a combination thereof. The adjuvant can be IL-12, IL-15, IL-28, CTACK, TECK, platelet-derived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermal growth factor (EGF), IL-1, IL-2, IL -4, IL-5, IL-6, IL-10, IL-12, IL-18, or a combination thereof.

[00174] In some embodiments, the adjuvant may be one or more nucleic acid molecules that encode proteins selected from the group consisting of: CCL-20, IL-12, IL-15, IL-28, CTACK, TECK, MEC or RANTES. Exemplary IL-12 constructs and sequences are disclosed in PCT Application No. PCT/US1997/019502 and corresponding US Serial No. 08/956,865 and US Provisional Application No. 61/569,600, filed December 12, 2011, each of which is incorporated herein by links. Exemplary IL-15 constructs and sequences are disclosed in PCT Application No. PCT/US04/18962 and corresponding U.S. Application Serial No. 10/560650 and PCT Application No. PCT/US07/00886 and corresponding U.S. Application Serial No. 12/160766, and in PCT Application No. PCT/USI0/048827, each of which is incorporated herein by reference. Examples of IL-28 constructs and sequences are disclosed in PCT Application No. PCT/US09/039648 and corresponding US Application Serial No. 12/936192, each of which is incorporated herein by reference. Examples of RANTES and other constructs and sequences are disclosed in PCT Application No. PCT/US1999/004332 and corresponding US Application Serial No. 09/622452, each of which is incorporated herein by reference. Other examples of RANTES constructs and sequences are disclosed in PCT Application No. PCT/US11/024098, which is incorporated herein by reference. Examples of RANTES and other constructs and sequences are disclosed in PCT Application No. PCT/US1999/004332 and corresponding US Application Serial No. 09/622452, each of which is incorporated herein by reference. Other examples of RANTES constructs and sequences are disclosed in PCT Application No. PCT/US11/024098, which is incorporated herein by reference. Exemplary chemokine CTACK, TECK and MEC constructs and sequences are disclosed in PCT Application No. PCT/US2005/042231 and corresponding US Application Serial No. 11/719646, each of which is incorporated herein by reference. Examples of OX40 and other immunomodulators are disclosed in US application No. 10/560653, which is incorporated herein by reference. Examples of DR5 and other immunomodulators are disclosed in US application No. 09/622452, which is incorporated herein by reference.

[00175] Other genes that can be used as adjuvants include those that encode: 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, IL-18 mutants, CD40, CD40L, vascular growth factor, fibroblast growth factor, IL-7, IL-22, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Flt, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, ICE caspase, Fos, c-jun, Sp-1 , Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, inactive NIK, SAP K, SAP-1, JNK, interferon response genes, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3 , TRAIL-R4, RANK, RANK LIGAND, Ox40, Ox40 LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1, TAP2 and their functional fragments.

[00176] The vaccine may further comprise a genetic vaccine promoter as described in U.S. Serial No. 021579, filed April 1, 1994, which is incorporated herein by reference in its entirety.

[00177] The vaccine may contain plasmids in an amount of from about 1 nanogram to 100 milligrams; about 1 microgram to about 10 milligrams; or preferably from about 0.1 micrograms to about 10 milligrams; or more preferably from about 1 to about 2 mg. In some preferred embodiments, the vaccine of the present invention contains from about 5 nanograms to about 1000 micrograms of DNA. In some preferred embodiments, the vaccine may contain from about 10 nanograms to about 800 micrograms of DNA. In some preferred embodiments, the vaccine may contain from about 0.1 to about 500 micrograms of DNA. In some preferred embodiments, the vaccine may contain from about 1 to about 350 micrograms of DNA. In some preferred embodiments, the vaccine may contain from about 25 to about 250 micrograms, from about 100 to about 200 micrograms, from about 1 nanogram to 100 milligrams; about 1 microgram to about 10 milligrams; from about 0.1 micrograms to about 10 milligrams; from about 1 to about 2 mg, from about 5 to about 1000 micrograms, from about 10 to about 800 micrograms, from about 0.1 to about 500 micrograms, from about 1 to about 350 micrograms, from about 25 to about 250 micrograms from about 100 to about 200 micrograms of antigen or plasmid encoding it.

[00178] The vaccine may be formulated according to the route of administration employed. The pharmaceutical composition for injectable vaccines may be sterile, pyrogen-free and free of particulate matter. You can use an isotonic composition or solution. Isotonicity supplements may include sodium chloride, dextrose, mannitol, sorbitol, and lactose. The vaccine may contain a vasoconstrictor. Isotonic solutions may include phosphate buffered saline. The vaccine may additionally contain stabilizers, including gelatin and albumin. Stabilizers can provide formulation stability at room or ambient temperature for extended periods of time, including LGS or polycations or polyanions.

Vaccine Pharmaceutical Compositions

[00179] The vaccine may be in the form of a pharmaceutical composition. The pharmaceutical composition may contain a vaccine. Pharmaceutical compositions may contain from about 5 nanograms (ng) to about 10 milligrams (mg) of vaccine nucleic acid molecule(s). In some embodiments, the pharmaceutical compositions of the present invention contain from about 25 ng to about 5 mg of vaccine nucleic acid molecule(s). In some embodiments, the pharmaceutical compositions contain from about 50 ng to about 1 mg of vaccine nucleic acid molecule(s). In some embodiments, the pharmaceutical compositions contain from about 0.1 to about 500 micrograms of vaccine nucleic acid molecule(s). In some embodiments, the pharmaceutical compositions contain from about 1 to about 350 micrograms of vaccine nucleic acid molecule(s). In some embodiments, the pharmaceutical compositions contain from about 5 to about 250 micrograms of vaccine nucleic acid molecule(s). In some embodiments, the pharmaceutical compositions contain from about 10 to about 200 micrograms of vaccine nucleic acid molecule(s). In some embodiments, the pharmaceutical compositions contain from about 15 to about 150 micrograms of vaccine nucleic acid molecule(s). In some embodiments, the pharmaceutical compositions contain from about 20 to about 100 micrograms of vaccine nucleic acid molecule(s). In some embodiments, the pharmaceutical compositions contain from about 25 to about 75 micrograms of vaccine nucleic acid molecule(s). In some embodiments, the pharmaceutical compositions contain from about 30 to about 50 micrograms of vaccine nucleic acid molecule(s). In some embodiments, the pharmaceutical compositions contain from about 35 to about 40 micrograms of vaccine nucleic acid molecule(s). In some embodiments, the pharmaceutical compositions contain from about 100 to about 200 micrograms of vaccine nucleic acid molecule(s). In some embodiments, the pharmaceutical compositions contain from about 10 to about 100 micrograms of vaccine nucleic acid molecule(s). In some embodiments, the pharmaceutical compositions contain from about 20 to about 80 micrograms of vaccine nucleic acid molecule(s). In some embodiments, the pharmaceutical compositions contain from about 25 to about 60 micrograms of vaccine nucleic acid molecule(s). In some embodiments, the pharmaceutical compositions contain from about 30 ng to about 50 micrograms of vaccine nucleic acid molecule(s). In some embodiments, the pharmaceutical compositions contain from about 35 ng to about 45 micrograms of vaccine nucleic acid molecule(s). In some preferred embodiments, the pharmaceutical compositions contain from about 0.1 to about 500 micrograms of vaccine nucleic acid molecule(s). In some preferred embodiments, the pharmaceutical compositions contain from about 1 to about 350 micrograms of vaccine nucleic acid molecule(s). In some preferred embodiments, the pharmaceutical compositions contain from about 25 to about 250 micrograms of vaccine nucleic acid molecule(s). In some preferred embodiments, the pharmaceutical compositions contain from about 100 to about 200 micrograms of vaccine nucleic acid molecule(s).

[00180] In some embodiments, the pharmaceutical compositions of the present invention comprise at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 ng vaccine nucleic acid molecule. In some embodiments, pharmaceutical compositions may contain at least 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,100 , 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225 , 230,235,240,245,250,255,260,265,270,275,280,285,290,295,300,305,310,315,320,325,330,335,340,345,350 , 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475 , 480, 485, 490, 495, 500, 605, 610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695, 700 ,705,710,715,720,725,730,735,740,745,750,755,760,765,770,775,780,785,790,795,800,805,810,815,820,825 830 835 840 845 850 855 860 865 870 875 880 885 890 895 900 905 910 915 920 925 930 935 940 945 950 , 955, 960, 965, 970, 975, 980, 985, 990, 995 or 1000 micrograms of a molecule (mo molecules) of the vaccine nucleic acid. In some embodiments, pharmaceutical compositions may contain at least 1.5; 2; 2.5; 3; 3.5; four; 4.5; 5; 5.5; 6; 6.5; 7; 7.5; eight; 8.5; 9; 9.5 or 10 mg or more vaccine nucleic acid molecule(s).

[00181] In other embodiments, the pharmaceutical compositions may contain up to and including 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 ng vaccine nucleic acid molecule(s). In some embodiments, the pharmaceutical compositions may contain up to and including 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,100 105 110 115 120 125 130 135 140 145 150 155 160 165 170 175 180 185 190 195 200 205 210 215 220 225 ,230,235,240,245,250,255,260,265,270,275,280,285,290,295,300,305,310,315,320,325,330,335,340,345,350 , 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475 , 480, 485, 490, 495, 500, 605, 610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695, 700 ,705,710,715,720,725,730,735,740,745,750,755,760,765,770,775,780,785,790,795,800,805,810,815,820,825 830 835 840 845 850 855 860 865 870 875 880 885 890 895 900 905 910 915 920 925 930 935 940 945 950 , 955, 960, 965, 970, 975, 980, 985, 990, 995 or 1000 microgram molecule(s) st) nucleic acid vaccine. In some embodiments, pharmaceutical compositions may contain up to and including 1.5; 2; 2.5; 3; 3.5; four; 4.5; 5; 5.5; 6; 6.5; 7; 7.5; eight; 8.5; 9; 9.5 or 10 mg of vaccine nucleic acid molecule(s).

[00182] The pharmaceutical composition may additionally contain other agents for formulation purposes in accordance with the route of administration used. Where the pharmaceutical compositions are injectable pharmaceutical compositions, they are sterile, non-pyrogenic and free of particulate matter. It is preferable to use an isotonic composition. Typically, isotonicity additives may include sodium chloride, dextrose, mannitol, sorbitol, and lactose. In some cases, isotonic solutions such as phosphate buffered saline are preferred. Stabilizers include gelatin and albumin. In some embodiments, a vasoconstrictor agent is added to the formulation.

[00183] The pharmaceutical composition may further comprise a pharmaceutically acceptable excipient as set forth in Section 2 above. For example, a pharmaceutically acceptable excipient may include functional molecules, carriers, adjuvants, carriers, diluents, or transfection aids, as proposed in Section 2.

Indications for use

[00184] The vaccines and pharmaceutical compositions containing the vaccines provided herein can be used to treat or prevent cancer cells and tumors expressing Survivin. In particular, the vaccines and pharmaceutical compositions containing the vaccines provided herein can be used to treat or prevent ovarian cancer, more specifically epithelial ovarian cancer, most specifically serous ovarian cancer.

Methods of vaccination

[00185] This document provides methods for the treatment and/or prevention of cancer using the pharmaceutical compositions described above. Also described herein are methods of using the pharmaceutical compositions described above for the treatment and/or prevention of cancer in a subject. This document also describes methods for vaccinating a subject. Also described herein are methods for administering the pharmaceutical compositions described herein to a subject in need thereof. The methods described herein, collectively referred to as methods of treatment using the pharmaceutical compositions described herein, may include administering one or more vaccines as described herein to a subject in need thereof to induce a therapeutic and/or prophylactic immune response. The vaccine may be administered to a subject to modulate the activity of the subject's immune system and enhance the immune response. The administration of the vaccine may be transfection of cancer antigens, as described herein, in the form of a nucleic acid molecule that is expressed in a cell and delivered to the cell surface, whereupon the immune system recognizes and induces a cellular, humoral or cellular and humoral response. Vaccine administration can be used to induce or elicit an immune response in subjects against one or more cancer antigens as described herein by administering the vaccine to the subject as described herein.

[00186] A vaccine may be administered to a subject to modulate the activity of the subject's immune system and thus enhance the immune response. In some embodiments, the subject is a mammal. When a vaccine is administered to a mammal, and thereby the vector is introduced into mammalian cells, the transfected cells will express and secrete one or more cancer antigens as described herein. These secreted proteins or synthetic antigens will be recognized as foreign by the immune system, leading to an immune response that may include: antibodies raised against one or more cancer antigens and a T cell response, specifically against one or more cancer antigens. In some examples, a mammal vaccinated with the vaccines discussed herein will have a primed immune system, and when challenged with one or more cancer antigens as described herein, the primed immune system will rapidly clear subsequent cancer antigens as described herein, whether or not , through humoral, cellular, or both cellular and humoral immune responses.

[00187] Methods for administering a DNA vaccine are described in US Pat. Nos. 4,945,050 and 5,036,006, both of which are incorporated herein by reference in their entirety.

[00188] The vaccine may be administered to a mammal to elicit an immune response in the mammal. The mammal may be human, non-human primate, cow, pig, sheep, goat, antelope, bison, water buffalo, bull, deer, hedgehogs, elephants, llama, alpaca, mice, rats, and preferably human, cow or pig. Vaccine also may be administered to a non-mammalian subject, such as a chicken, to elicit an immune response.

[00189] The dose of the vaccine may be from 1 microgram to 10 mg of active ingredient per kilogram (kg) of body weight over time (component/kg of body weight/time) and can be from 20 micrograms to 10 mg of ingredient/kg of body weight/time. The vaccine can be given every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31 days. The number of doses of the vaccine for effective treatment may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more doses.

Ways to generate an immune response using a vaccine

[00190] The vaccine can be used to generate an immune response in a mammal or non-mammalian subject, including a therapeutic or prophylactic immune response. The immune response may generate antibodies and/or killer T cells directed to one or more cancer antigens as described herein. Such antibodies and T cells can be isolated.

[00191] Some embodiments provide methods for generating immune responses against one or more cancer antigens as described herein, which embodiments include administering a vaccine to a subject. Some embodiments provide methods for prophylactically vaccinating a subject against a cancer or tumor expressing one or more cancer antigens as described above, which embodiments include administering the vaccine. Some embodiments provide methods for therapeutically vaccinating a subject who is suffering from a cancer or tumor expressing one or more cancer antigens, which embodiments include administering the vaccine. Diagnosis of a cancer or tumor expressing one or more cancer antigens as described herein prior to administration of a vaccine can be routinely performed.

Ways to treat cancer with a vaccine

[00192] The vaccine can be used to generate or induce an immune response in a mammal that is reactive or directed against a Survivin-expressing cancer or tumor (eg, ovarian cancer) of the mammal or a subject in need thereof. The triggered immune response can prevent cancer or tumor growth.

[00193] The elicited immune response can prevent and/or reduce the metastasis of cancer or tumor cells. Accordingly, the vaccine may be used in a method that treats and/or prevents cancer or tumors in a mammal or subject to which the vaccine is administered.

Routes of administration

[00194] The vaccine or pharmaceutical composition may be administered by a variety of routes, including oral, parenteral, sublingual, transdermal, rectal, transmucosal, topical, inhaled, buccal, intrapleural, intravenous, intraarterial, intraperitoneal, subcutaneous, intramuscular, intranasal, intrathecal, and /or intra-articular, or combinations thereof. For veterinary use, the composition may be administered as a suitable acceptable composition in accordance with normal veterinary practice. The veterinarian can easily determine the dosing regimen and route of administration that is most appropriate for a particular animal. The vaccine can be administered using conventional syringes, needleless injection devices, "microparticle bombardment gene guns" or other physical methods such as electroporation ("EP"), "hydrodynamic method" or ultrasound.

[00195] The vaccine vector can be administered to a mammal using several well-known technologies, including DNA injection (also referred to as DNA vaccination) with and without in vivo electroporation, liposome-mediated transfection, nanoparticle-assisted transfection, and the use of recombinant vectors such as as recombinant adenovirus, recombinant adenovirus-associated virus and recombinant vaccination. One or more vaccine cancer antigens can be administered by DNA injection together with in vivo electroporation.

[00196] The vaccine or pharmaceutical composition containing the vaccine can be administered by electroporation. Vaccine administration by electroporation can be accomplished using electroporation devices that can be configured to deliver to the desired mammalian tissue an energy pulse effective to form reversible pores in cell membranes, and preferably the energy pulse is a direct current. corresponding to the incoming current set by the user. The electroporation device may comprise an electroporation component and an electrode setup or manual setup. The electroporation component may include and combine one or more of the various elements of the electroporation devices, including: a controller, a current signal generator, an impedance meter, a signal recorder, an input element, a status message element, a communication port, a memory component, a power supply, and a switch. Electroporation can be performed using an in vivo electroporation device such as the CELLECTRA® EP system (Inovio Pharmaceuticals, Inc., Blue Bell, PA) or the Elgen electroporator (Inovio Pharmaceuticals, Inc.) to facilitate transfection of cells with the vector.

[00197] Examples of electroporation devices and electroporation methods that can facilitate the administration of the DNA vaccines of the present invention include those described in US Pat. No. 7,245,963 to Draghia-Akli et al., US Patent Publication. 2005/0052630 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 disclosed in concurrently pending and co-owned U.S. Patent Application Serial No. 11/874072, filed Oct. 17, 2007, which claims benefit under 35 USC 119(e) to Provisional Applications US Ser. No. 60/852149, filed October 17, 2006, and 60/978982, filed October 10, 2007, all of which are incorporated herein in their entirety.

[00198] US Patent No. 7245963, Draghia-Akli, et al. describes modular electrode systems and their use to ensure the introduction of a biomolecule into the cells of a selected tissue of an organism or plant. Modular electrode systems may include a plurality of needle electrodes; hypodermic needle; an electrical connector that provides a conductive connection from the DC programmable switching controller to the plurality of needle electrodes; and power supply. The operator can wrap around a plurality of needle electrodes that are mounted on a support structure and firmly insert them into the selected tissue in the body or plant. The biomolecules are then injected through a hypodermic needle into the selected tissue. The programmable DC pulse controller is activated and a DC electrical pulse is applied to the plurality of needle electrodes. The direct current electrical pulse applied facilitates the introduction of the biomolecule into the cell between the electrode array. The entire contents of US Pat. No. 7,245,963 are incorporated herein by reference in their entirety.

[00199] US Patent Publication 2005/0052630 to Smith et al. describes an electroporation device that can be used to efficiently provide the introduction of a biomolecule into the cells of a selected tissue of an organism or plant. The electroporation device contains an electrokinetic device ("EKD device"), the operation of which is set by external software or firmware. The EKD generates a series of programmable DC pulse patterns between the electrodes in the array based on user control and input of pulse parameters, and allows the storage and retrieval of current waveform data. The electroporation device also includes a replaceable electrode disc having a row of needle electrodes, a central channel for an injection needle, and a removable guide disc. All content of US Patent Publication. 2005/0052630 is incorporated herein by reference in its entirety.

[00200] The electrode arrays and methods described in US Pat. No. 7,245,963 and US Patent Publication 2005/0052630 can be adapted for deep penetration not only into tissues such as muscle, but also into other tissues or organs. Due to the configuration of the electrode array, the injection needle is also fully inserted into the target organ, and the injection is administered perpendicular to the target problem area at the places that are initially set by the electrodes. The electrodes described in US Pat. No. 7,245,963 and US Patent Publication 2005/005263 are preferably 20 mm long and have 21 needle gauges.

[00201] In addition, in some embodiments that include electroporation devices and their use, there are electroporation devices that are described in the following patents: US patent 5,273,525 issued December 28, 1993; August 2000, 6,261,281, issued July 17, 2001, and 6,958,060, issued October 25, 2005, and U.S. Patent 6,939,862, issued September 6, 2005. covering the subject matter of US Pat. . The above patents are incorporated by reference in their entirety.

[00202] Provided herein are methods for producing vectors that contain nucleic acid molecule(s) encoding the synthetic Survivin consensus antigen discussed herein. After the final step of subcloning into a mammalian expression plasmid, the vectors can be used to inoculate the cell culture in a large scale fermentation tank using methods known in the art.

[00203] DNA vectors for use with the EP devices of the present invention can be formulated or manufactured using a combination of known devices and technologies, but are preferably manufactured using an optimized manufacturing technique as described in US Published Application No. 20090004716, filed May 23, 2007 of the year. In some examples, the DNA vectors used in these studies can be prepared at concentrations greater than or equal to 10 mg/ml. The manufacturing techniques also include or combine various devices and protocols that are widely known to those skilled in the art, in addition to those described in US application serial number 60/939792, including those described in US patent No. 7238522, which is issued to July 3, 2007 The above application and US Pat. No. 60/939,792 and US Pat. No. 7,238,522, respectively, are incorporated herein in their entirety.

Examples

[00204] The present invention is further illustrated in the following examples. It should be understood that these examples, while depicting preferred embodiments of the invention, are provided by way of illustration only. From the above discussion and these examples, a person skilled in the art can determine the main characteristics of this invention and, without departing from its essence and scope, can make various changes and modifications to the invention in order to adapt it to various applications and conditions. 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 should also fall within the scope of the appended claims.

Example 1 Preparation of Isoform 1 Survivin Consensus

[00205] Twenty-nine Survivin isoform 1 sequences were obtained from GenBank (www.ncbi.nlm.nih.gov/genbank/). The GenBank access numbers for the selected Survivin isoform 1 sequences are: NP_001125727.1, 3UIG, 1F3H, BAC22748.2, NP_001159.2, CAG46540.1, BAD97148.1, XP_002748841.1, XP_003818322.1, NP_00128.XP_003818322.1, NP_00128.18 1, XP_011832690.1, XP_001083183.1, XP_003786134.1, XP_012516801.1, XP_007957800.1, XP_001915435.1, XP_008523102.1, AAT37504.1, XP_004432883.1, XP_003417277.1, XP_004374167.1, NP_999306.1, XP_002918421.1, NP_001003348.1, NP_001009280.1, XP_004748859.1, NP_001001855.2 and AAU89275.1.

[00206] The consensus sequence was generated using the DNASTAR® Lasergene software package (version 13.0.0.357). The twenty-nine sequences listed above were imported into MegAlign and aligned using the ClustalW multiple sequence alignment program. The resulting sequence of Survivin consensus isoform 1 has a homology of 97.2-97.9% with native human Survivin isoform 1. To eliminate the potential biological function of the resulting Survivin consensus isoform 1 protein, three mutations were introduced to eliminate the anti-apoptotic activity of Survivin. Three mutations are T34A, T48A and C84A. Additionally, in order to have a higher level of expression, a left Kozak sequence and an IgE leader sequence were added to the N-terminus. In addition, the codon usage of this gene has been 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: regions with very high (>80%) or very low (<30%) GC content and cis-acting sequence motifs such as TATA inner boxes, chi sites, and ribosome entry sites were avoided. As a result, the isoform 1 protein of the synthetic Survivin consensus antigen has 95.1-95.8% identity with the native human Survivin isoform 1 protein. The nucleotide sequence of isoform 1 of the synthetic Survivin consensus antigen is shown in SEQ ID NO: 1. The amino acid sequence of isoform 1T3 of the synthetic Survivin consensus antigen is shown in SEQ ID NO: 2.

[00207] Survivin's synthetic consensus antigen isoform 1 was digested with BamHI and XhoI and cloned into the proprietary expression vector pGX0001 with an expression cassette placed under transcriptional control of the cytomegalovirus immediate-early promoter. The resulting plasmid was designated pGX1428. Full length sequencing was performed and then analyzed and confirmed to be correct. A schematic representation of the isoform 1 construct of the synthetic Survivin consensus antigen is shown in FIG. The general structure of isoform 1 of the synthetic Survivin consensus antigen is shown in FIG. 2.

Table 1.

Features SEQ ID NO:3 Peculiarity Amino acid position IgE leader sequence 1-18 Survivin coding sequence 19-159 Mutations to eliminate anti-apoptotic activity T51A
T65A
C101A Features SEQ ID NO:8 Peculiarity Amino acid position IgE leader sequence 1-18 Survivin isoform 1 coding sequence 19-161 Mutations to eliminate anti-apoptotic activity T51A
T65A
C101A Furin cleavage site 160-166 Shortened coding sequence of Survivin isoform 3 167-232

Table 2. Characteristics of isoform 1 of the synthetic consensus antigen Survivin

Characteristics Isoforms 1 of the synthetic consensus antigen Survivin Identity to native human Survivin 95.1% - 95.8% Identity to native Survivin rhesus monkey 95.8% Identity to native mouse Survivin 70.0% - 83.6% Number of amino acid mutations (compared to native human) 6 Number of built-in mutations (non-consensus origin) 3 Molecular mass 161 aa (18 kDa) Coding sequence length (bp) 483

Example 2 - Preparation of Survivin consensus isoform 1T3

[00208] To create Survivin human consensus isoform 3, 8 Survivin isoform 3 sequences were obtained from GenBank (www.ncbi.nlm.nih.gov/genbank/). The GenBank access numbers for the selected Survivin isoform 3 sequences are: NP_001012270.1, XP_008969790.1, XP_008011275.1, XP_009189614.1, XP_011897653.1, XP_011810642.1, XP_011844315.1, and XP35.01171

[00209] The consensus sequence was generated using the DNASTAR® Lasergene software package (version 13.0.0.357). The eight sequences listed above were imported into MegAlign and aligned using the ClustalW multiple sequence alignment program. Six of these sequences from lower animals contained an additional 11 amino acid residues at their C-terminal end, which are not found in human sequences. These additional residues were not taken into account when creating consensus human Survivin isoform 3 in order to avoid the occurrence of a non-targeted immune response in humans. After the creation of isoform 3 of the human consensus Survivin, the identical amino acid sequence between isoform 1 and 3 of Survivin was removed from isoform 3 of the Survivin consensus. The truncated sequence of Survivin consensus isoform 3 (Survivin T3 antigen) has 96.8% sequence homology with the native sequence of human Survivin T3 isoform. Survivin antigen isoform 1T3 immunogen was prepared by adding a furin cleavage site between Survivin antigen 1 (described above) and Survivin T3 antigen.

[00210] Once the DNA sequence of the 1T3 isoform of the synthetic Survivin consensus antigen was obtained, in order to have a higher level of expression, a left Kozak sequence and an IgE leader sequence were added to the N-terminus. (Yang, J. S. et al. Induction of potent Th1-type immune responses from a novel DNA vaccine for West Nile virus New York isolate (WNV-NY1999). The Journal of infectious diseases 184, 809-816, doi:10.1086/323395 ( 2001)). In addition, the codon usage of this gene has been adapted to the codon shift 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: regions with very high (>80%) or very low (<30%) GC content and cis-acting sequence motifs such as internal TATA boxes, chi sites, and ribosome entry sites were avoided. 11.12. The 1T3 isoform of the synthetic Survivin consensus antigen was digested with BamHI and XhoI and cloned into the pGX0001 expression vector with an expression cassette placed under transcriptional control of the cytomegalovirus immediate-early promoter. The resulting plasmid was designated pGX1429. Full length sequencing was performed and then analyzed and confirmed to be correct. The nucleotide sequence of isoform 1T3 of the synthetic Survivin consensus antigen is shown in SEQ ID NO:3 as shown in Table 1. The amino acid sequence of the isoform 1T3 of the synthetic Survivin consensus antigen is shown in SEQ ID NO:8 as shown in Table 1. consensus antigen Survivin shown in Fig.3. The design characteristics of the 1T3 isoform of the synthetic consensus antigen Survivin are shown in Table 3.

Table 3. Characteristics of the 1T3 isoform of the synthetic consensus antigen Survivin

Characteristics Isoforms 1T3 of the synthetic consensus antigen Survivin Identity to native human Survivin 95.1%-95.8% (iso 1 area); 96.8% (T3 area) Identity to native Survivin rhesus monkey 95.9% (ISO 1 area); 9.4% (T3 area) Identity to native mouse Survivin 70.0%-84.9% (iso 1 area); 10.9%-13.0% (T3 area) Number of amino acid mutations (compared to native human) 6 (iso area 1); 2 (area T3) Number of built-in mutations (non-consensus origin) 3 (iso area 1); 0 (T3 area) Molecular mass 232 aa (26 kDa) Coding sequence length (bp) 696

Example 3 - Construction of pGX Survivin Expression Vectors

[00211] pGX0001 (modified pVAX1 expression vector) under control of human cytomegalovirus immediate-early promoter (hCMV promoter) was used as a framework vector. The original pVAX1 was obtained from Thermo Fisher Scientific.

[00212] Modifications were introduced into pVAX1 to create pGX0001 and identified based on the described pVAX1 sequence available from Thermo Fisher Scientific. These modifications are listed below and no problems were found regarding plasmid amplification and transcription and translation of the antigen. No additional changes in the sequence of pGX0001 have been observed to date in any of the plasmid products on the platform using pGX0001 as backbone.

Modification Base pair Description

C>G 241 in CMV promoter

C>T 1158 backbone, to the right of the bovine growth hormone polyadenylation signal (bGH polyA)

A> - 2092 main chain, to the right of the kanamycin resistance gene

gene

C>T 2493 at pUC replication origin (pUC ori)

G>C 2969 at the very end of the Ori pUC to the left of the RNASeH site

Base pairs 2, 3 and 4 change from ACT to CTG in the main chain to the left of the CMV promoter.

[00213] pGX1428 is a DNA plasmid encoding the isoform 1 protein of the synthetic Survivin consensus antigen (Survivin 1 antigen). The 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 includes a kanamycin resistance gene (KanR) and a plasmid origin of replication (pUC ori) for manufacturing purposes. These elements do not function in eukaryotic cells. pGX1428 was generated by cloning the synthetic Survivin consensus antigen isoform 1 DNA sequence (Survivin 1 antigen) into pGX0001 at the BamHI and XhoI sites as shown in FIG. four.

[00214] pGX1429 is a DNA plasmid encoding the 1T3 protein of the synthetic Survivin consensus antigen (Survivin 1T3 antigen). The 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 includes a kanamycin resistance gene (KanR) and a plasmid origin of replication (pUC ori) for manufacturing purposes. These elements do not function in eukaryotic cells. pGX1429 was obtained by cloning the 1T3 isoform DNA sequence of the synthetic Survivin consensus antigen (Survivin 1T3 antigen) into pGX0001 at the BamHI and XhoI sites as shown in FIG.

Example 4 Immunogenicity of Survivin Synthetic Consensus Antigen Constructs

[00215] The immunogenicity of vaccine constructs designed to target human Survivin, Survivin 1 synthetic consensus antigen (pGX1428), and Survivin 1T3 synthetic consensus antigen (pGX1429) was evaluated in mice. Expression of proteins of the antigen by construct was also assessed in vitro by Western blotting.

Materials and methods

Plasmids

[00216] Survivin synthetic consensus antigen 1 (pGX1428) and Survivin synthetic consensus antigen 1T3 (pGX1429) were constructed as described herein. For in vitro and in vivo studies, plasmids (10 mg) were ordered from GenScript for both pGX1428 (Lot # 786114S-1/G52238) and pGX1429 (Lot # 786114S-2/G52239). Antigen sequences from 10 mg plasmid stocks were confirmed by Sanger sequencing.

Expression of the antigen in vitro

[00217] Expression of antigen proteins by pGX1428 and pGX1429 was confirmed by Western blotting. As shown in Figure 6, human rhabdomyosarcoma (RD) cells (ATCC, CCL-136) maintained in DMEM with 10% FBS (ThermoFisher) were transfected with pGX1428, pGX1429 or pGX0001 (6 μg/10 cm2 plate) using Turbofectin 8 (Origin). Forty-eight hours after transfection, the cells were lysed using RIPA cell lysis buffer (ThermoFisher) and the cell lysate was collected. After BCA analysis (ThermoFisher) to determine the concentration of total protein, 15 μg of cell lysate was subjected to electrophoresis on a 4-12% SDS-PAGE gel (ThermoFisher). Detection was performed with a monoclonal antibody against amino acids 1-142 Human Survivin (Santa Cruz Biotech, clone D8, sc-17779), then visualized with horseradish peroxidase (HRP) conjugated goat anti-mouse IgG (Santa Cruz Biotech, sc-2005) with using the Western blot analysis system ECL (GE Amersham). As a loading control, blots were re-examined for actin expression using an anti-β-actin monoclonal antibody (Santa Cruz Biotech, clone C4, sc-47778 HRP).

Animals and immunizations

[00218] Female 8-week-old CB6F1 mice were purchased from the Jackson laboratory. All animals were kept in a temperature controlled, light cycling facility at BTS Research (San Diego, CA). Animal care was provided in accordance with National Institutes of Health guidelines and Animal Care and Use Proposal (ACUP) (BTS ACUP #15-091). The mice were divided into nine groups as shown in Table 4.

Table 4. Groups of studies

Group n Design Construct Dose (mcg) Injection volume (µl) one four pGX0001 thirty thirty 2 eight pGX1428 ten thirty 3 eight pGX1428 twenty thirty four eight pGX1428 thirty thirty 5 eight pGX1428 fifty thirty 6 eight pGX1429 ten thirty 7 eight pGX1429 twenty thirty eight eight pGX1429 thirty thirty 9 eight pGX1429 fifty thirty

[00219] Mice in the immunized groups were vaccinated with the indicated doses of pGX0001, pGX1428 or pGX1429 according to SOP R20-003147 CELLECTRA® 3P by treating mice. Briefly, plasmids were prepared in sterile water for injection (VetOne) such that the indicated dose was delivered by intramuscular injection into the tibialis anterior muscle in an injection volume of 30 μl. Each intramuscular injection was immediately followed by electroporation (EP) using the adaptive DC electroporation device CELLECTRA® 2000 with 3P array (Inovio Pharmaceuticals). The device was set to deliver two 0.1 A pulses with a pulse width of 52 ms, separated by 1 second. Mice received 3 immunizations 3 weeks apart. Mice were sacrificed one week after the last immunization and spleens were collected for immune cell counts. No other tissue was collected.

Isolation of spleen lymphocytes

[00220] Splenocytes were aseptically isolated and placed in 5 ml R10 medium (Rosewell Park Memorial Institute 1640 medium supplemented with 10% fetal bovine serum and 1% antimycotic antibiotic). Splenocytes were isolated by mechanical disruption of the spleen using a Stomacher apparatus (Seward Laboratory Systems Inc.) and the resulting product was filtered using a 40 μm cell sieve (BD Falcon). The resulting product was centrifuged and the pellet was treated for 5 minutes with ACK lysis buffer (Lonza) to lyse the erythrocytes. The splenocytes were then centrifuged, washed in PBS and then resuspended in R10 medium and immediately used for further analysis.

IFNγ ELISpot

[00221] Mouse IFNγ ELISpot analysis was performed using a kit from MabTech (MabTech, No. 3321-4APW-10) to assess antigen-specific cellular responses. Briefly, 96-well plates precoated with anti-mouse IFNγ antibody (mAb AN18) were washed in PBS and blocked for 2 hours at room temperature with complete culture medium (RPMI 1640 supplemented with 10% FBS and antibiotics). Spleen lymphocytes were resuspended in R10 medium (and then added in triplicate at 2 × 10 ⁵ cells per well injected. A set of peptides (GenScript) was synthesized, each containing 15 amino acid residues overlapping with 11 amino acids representing the complete protein sequences of the synthetic consensus of Survivin antigen 1 and Survivin synthetic consensus antigen 1T3. These sets of peptides were resuspended in DMSO (Sigma) and pooled at a peptide concentration of ~2 μg/ml into two peptide pools. One peptide pool contained peptides corresponding to the Survivin synthetic consensus antigen 1 protein, and the second the peptide pool contained peptides corresponding to the protein of the synthetic consensus antigen 1T3 Survivin.Concavalin A (Sigma) at a concentration of 5 μg/ml was used as a positive control, and the complete culture medium was used as a negative control.The plates were incubated for 18 hours at 37°C in and incubator with 5% CO ₂ . Biotinylated anti-mouse IFNγ antibody (MabTech mAb R4-6A2) was then added and the plates were incubated for 2 hours at room temperature. The plates were washed, streptavidin-ALP (MabTech) was added and the plates were incubated for 1 hour at room temperature. Spot detection was performed using BCIP/NBT substrate according to the kit manufacturer's (MabTech) instructions. The spots on the plates were counted using an ELISPOT automatic reader (Cellular Technology). The mean spot formation units (SFU) were adjusted to 1 x 10 ⁶ splenocytes for display purposes.

[00222] FIG. 7A-7H, antigen-specific IFNγ ELISpot responses are presented as the number of IFNγ spotting units (SFUs) per 1 x 10 ⁶ splenocytes, greater than the SFU in the medium-only control.

flow cytometry

[00223] Cellular immune responses induced by Survivin synthetic consensus antigen 1 and Survivin synthetic consensus antigen 1T3 were further characterized by flow cytometry. Briefly, 2 x 10 ⁶ splenocytes from vaccinated and naive mice were immediately stimulated after isolation of Survivin synthetic consensus antigen 1 and Survivin 1T3 peptides, respectively, for 6 hours in the presence of Brefeldin A (BD Biosciences), Monensin (BD Biosciences) and FITC against mouse anti-CD107a antibody (BD Biosciences, clone 1D4B). After stimulation with peptides, splenocytes were centrifuged and resuspended in 20 μl per well of mouse BD Fc Block (BD Biosciences) solution. The Fc block is used at an initial dilution of 1:40 in PBS and incubated at 4° C. for 5 minutes. After incubation, the remaining extracellular antibodies (in PBS) are added at 30 μl per well and allowed to incubate at 4° C. for 30 minutes. After addition of extracellular dye, the final volume in each well is 50 µl, consisting of the Fc block at a final dilution of 1:100 and extracellular antibodies at their respective working dilutions. Cells were then stained with viability dye (Vivid V450, Thermo-Fisher) and the following extracellular antibodies: PerCP-Cy5.5 anti-mouse CD4 (BD Biosciences, clone RM4-5) and APC anti-mouse CD8a (BD Biosciences, clone 63-6.7 ). Cells were fixed and permeabilized (BD Biosciences, No. 554714) for 20 minutes at 4°C. Intracellular staining was subsequently completed with the following antibodies: APC-Cy7 anti-mouse and 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 with anti-mouse TNF-α (BD Biosciences, clone MP6-XT22). ICS data were collected on 10-color FACS CANTO (BD Biosciences) and analysis completed using FlowJo software. The flow cytometry gating strategy is shown in FIG. eight.

[00224] In order for a cell to be called antigen-specific by flow cytometry, the frequency of the described parameter must exceed the frequency of the medium-only control. In order for a cell to be identified as producing antigen-specific CD107a, the cell must also be identified as positive for antigen-specific production of IFNγ and/or IL-2 and/or TNFα, as identified by Boolean gating.

Statistical analysis

[00225] Statistical analysis was performed using IBM SPSS Statistics 22 (IBM Corporation). Intergroup analyzes were performed using Tukey's Honest Significant Difference (HSD) ANOVA for ad hoc evaluation to adjust for multiple comparisons. Homogeneity of variance was confirmed using the F-statistic before multiple comparisons. For all statistical analysis, a p value of 0.050 was considered significant.

results

Expression of proteins of the synthetic consensus antigen Survivin

[00226] Two constructs were designed to target human Survivin, Survivin synthetic consensus antigen 1 (pGX1428) and Survivin synthetic consensus antigen 1T3 (pGX1429). Expression of antigenic proteins of Survivin synthetic consensus antigen 1 and Survivin synthetic consensus antigen 1T3 by pGX1428 and pGX1429, respectively, was confirmed by Western blotting. Briefly, human rhabdomyosarcoma (RD) cells were transfected with plasmids pGX1428, pGX1429 or pGX0001 (empty vector, negative control). Cell lysates were tested for expression of synthetic anti-human Survivin consensus antigen proteins (BIRC5). Protein bands with the expected molecular weight for Survivin synthetic consensus antigen 1 (17.5 kDa) and Survivin synthetic consensus antigen 1T3 (25.3 kDa) were detected (FIG. 6). A weak band was found in the negative control (pGX0001), which is most likely due to the low level of expression of the endogenous Survivin protein in the RD cell line. The anti-β-actin bands were found with equal intensity, indicating that equal amounts of protein were loaded in each band. Thus, pGX1428 and pGX1429 were found to express their respective antigenic proteins.

Immunogenicity of Survivin Synthetic Consensus Antigen Vaccine Constructs

IFNγ ELISpot

[00227] The immunogenicity of the two Survivin synthetic consensus antigen constructs was assessed at four doses (10, 20, 30, and 50 μg) by IFNγ ELISpot and flow cytometry (n=8/group). Mice were immunized with an empty base vector (pGX0001) as a negative control (n=4/group). Vaccination with Survivin's synthetic consensus antigen 1 resulted in significant IFNγ responses compared to negative control vaccinated mice. However, there was minimal evidence of a dose-dependent increase in IFNγ production induced by Survivin's synthetic consensus antigen 1 (FIG. 10A), indicating that the maximum response was achieved at the lowest dose. In particular, the IFNγ SFU value for the synthetic consensus antigen Survivin was 1082 ± 574, 1186 ± 747, 1135 ± 647 and 848 ± 350 at 10, 20, 30 and 50 μg, respectively. IFNγ responses to synthetic consensus antigen 1 Survivin were significantly greater than naive (2±3) at 10 µg (p=0.031), 20 µg (p=0.015) and 30 µg (p=0.021) pGX1428, but not at dose of 50 mcg (p=0.134). Vaccination with Survivin's synthetic 1T3 consensus antigen resulted in significant IFNγ responses, with some evidence of a dose-dependent increase with increasing dose levels (FIG. 10D). The IFNγ SFU value for Survivin 1T3 was 516 ± 156, 812 ± 534, 1016 ± 654 and 818 ± 339 at 10 μg, 20 μg, 30 μg and 50 μg, respectively. IFNγ responses to the synthetic 1T3 consensus antigen Survivin were significantly greater than naive (5±6) at 20 µg (p=0.039), 30 µg (p=0.006), and 50 µg (p=0.037) pGX1429, but not at dose of 10 µg (p=0.337). IFNγ responses are summarized in Table 5.

Table 5. IFN-gamma responses induced by Survivin synthetic consensus antigen 1 and Survivin synthetic consensus antigen 1T3.

Synthetic consensus antigen 1 Survivin (pGX1428) Synthetic consensus antigen 1T3 Survivin (pGX1429) Design Dose Mean SFU ± St. Off p-value Design Dose Mean SFU ± St. Off p-value pGX0001 30 mcg 2 ± 3 n/a pGX0001 30 mcg 5 ± 6 n/a pGX1428 10 mcg 1082 ± 574 p=0.031 pGX1429 10 mcg 516 ± 156 p=0.337 20 mcg 1186 ± 747 p=0.015 20 mcg 812 ± 534 p=0.039 30 mcg 1 135 ± 647 p=0.021 30 mcg 1016 ± 654 p=0.006 50 mcg 848 ± 350 p=0.134 50 mcg 818 ± 339 p=0.037 The reported p-values are close to naive (pGX0001 immunized mice).

flow cytometry

[00228] Survivin synthetic consensus antigen 1 and Survivin synthetic consensus antigen 1T3 both elicited more robust responses in the CD4 ⁺ T cell compartment compared to those in the CD8 ⁺ T cell compartment (FIG. 9). Survivin's synthetic consensus antigen-1 induced frequencies of antigen-specific CD4+ T cell responses that were significantly more robust than naive (0.03% ± 0.05%) in the 20 μg dose groups (1.08% ± 0, 65%) (p = 0.024) and 50 mcg (1.21% ± 0.73%) (p = 0.009), but not in the 10 mcg dose groups (0.86% ± 0.31%) (p = 0.105 ) or 30 μg (0.82% ± 0.46%) (p=0.134) (Fig. 7B). The synthetic 1T3 consensus antigen Survivin induced antigen-specific CD4+ T cell responses that were significantly more robust than naive (0.05% ± 0.05%) in the 20 µg dose groups (1.02% ± 0.59 %) (p=0.010), 30 mcg (1.08% ± 0.47%) (p=0.006) and 50 mcg (1.13% ± 0.44%) (p=0.004), but not in the group doses of 10 μg (0.62%±0.35%) (p=0.248) (Fig. 7E). The cytokine profile of CD4+ T cells specific to the synthetic consensus antigen Survivin was the same for both constructs in different dose groups and included mainly IFNγ+IL-2+TNFα+, IFNγ+IL-2-TNFα+ or IFNγ+IL- 2-TNFα cells (Fig. 7G, Fig. 7H). The frequency of antigen-specific CD4 ⁺ T cells is described in more detail in Table 6.

Table 6 CD4+ cell responses induced by Survivin synthetic consensus antigen 1 and Survivin synthetic consensus antigen 1T3.

Synthetic consensus antigen 1 Survivin Synthetic consensus antigen 1T3 Survivin Constr-
tion Dose %CD4 ⁺ ± Art. Off p-value Design-
tion Dose %CD4 ⁺ ± Art. Off p-value pGX0001 30 mcg 0.03±0.05 n/a pGX0001 30 mcg 0.05±0.05 n/a pGX1428 10 mcg 0.86±0.31 p=0.105 pGX1429 10 mcg 0.62±0.35 p=0.248 20 mcg 1.08±0.65 p=0.024 20 mcg 1.02±0.59 p=0.010 30 mcg 0.82±0.46 p=0.134 30 mcg 1.08 ± 0.47 p=0.006 50 mcg 1.21±0.73 p=0.009 50 mcg 1.13 ± 0.44 p=0.004 The reported p-values are close to naive (pGX0001 immunized mice).

[00229] There was no significant difference in the frequency of antigen-specific CD8+ T cells induced by Survivin's synthetic consensus antigen 1 (p=0.117) (Fig. 7C). Survivin 1T3 did induce a significantly higher frequency of antigen-specific CD8+ T cells between dose groups (p=0.043). 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) pGX1429 did not reach statistical significance compared to naive (0.07% ± 0.01%). The frequency of Survivin 1T3-specific CD8+ T cells reached statistical significance (0.36%±0.21%) (p=0.051) in the 50 μg pGX1429 immunized group (Fig. 7F). Both Survivin synthetic consensus antigen 1 and Survivin synthetic consensus antigen 1T3 induced CD8+ T cell responses similar in magnitude and phenotype. Antigen-specific CD8+ T cells were mainly IFNγ+IL-2-TNFα-, IFNγ+IL-2-TNFα+ (FIG. 7G, FIG. 7H). The frequency of antigen-specific CD8 ⁺ T cells is described in more detail in Table 7.

Table 7. CD8+ Cell Responses Induced by Survivin Synthetic Consensus Antigen 1 and Survivin Synthetic Consensus Antigen 1T3

Synthetic consensus antigen 1 Survivin Synthetic consensus antigen 1T3 Survivin Design Dose %CD8 ⁺ ± Art. Off p-value Design Dose %CD8 ⁺ ± Art. Off p-value pGX0001 30 mcg 0.03±0.01 n/a
between groups p=0.117 pGX0001 30 mcg 0.07 ± 0.01 n/a pGX1428 10 mcg 0.21 ± 0.11 pGX1429 10 mcg 0.15 ± 0.09 p=0.919 20 mcg 0.35 ± 0.31 20 mcg 0.27 ± 0.21 p=0.274 30 mcg 0.25 ± 0.12 30 mcg 0.25 ± 0.14 p=0.377 50 mcg 0.24 ± 0.15 50 mcg 0.36 ± 0.21 p=0.051 The reported p-values are close to naive (pGX0001 immunized mice).

[00230] Approximately 25% of cytokine-positive CD4+ T cells induced by Survivin synthetic consensus antigen 1 (FIG. 10A) and Survivin synthetic consensus antigen 1T3 (FIG. 10B) were also positive for CD107a, indicating some potential for CD4+T-cell mediated cytolytic function. All doses of the synthetic consensus antigen 1 Survivin induced a frequency of CD4+CD107a+T cells significantly higher than naive (0.01% ± 0.01%). In particular, the frequency of antigen-specific CD4+CD107a+T cells was 0.25% ± 0.08%, 0.36% ± 0.11%, 0.21% ± 0.15%, and 0.38% ± 0.13% in dose groups of 10 μg (p=0.013), 20 μg (p<0.001), 30 μg (p=0.050), and 50 μg (p<0.001), respectively (FIG. 10A). Synthetic consensus antigen 1T3 Survivin induced a frequency of CD4+CD107a+T cells significantly higher than naive (0.01 ± 0.01) in all groups except the 10 µg dose group (0.18% ± 0.09%) (p =0.147). The frequency of antigen-specific CD4+CD107a+ T cells was 0.24% ± 0.12%, 0.27% ± 0.12%, and 0.29% ± 0.16% in the 20 μg dose groups (p = 0.030), 30 μg (p=0.010) and 50 μg (p=0.004), respectively (Fig. 10B). The frequency of antigen-specific CD4 ⁺ T cells with cytolytic potential is described in more detail in Table 8.

Table 8 Cytolytic potential of antigen-specific CD4+ T cells induced with synthetic consensus antigen Survivin 1 and synthetic consensus antigen Survivin 1T3

Synthetic consensus antigen 1 Survivin Synthetic consensus antigen 1T3 Survivin Design Dose %CD4 ⁺ CD107a ⁺ ± Art. Off p-value Design Dose %CD4 ⁺ CD107a ⁺ ± Art. Off p-value pGX0001 30 mcg 0.01 ± 0.01 n/a pGX0001 30 mcg 0.01 ± 0.01 n/a pGX1428 10 mcg 0.25±0.08 p=0.013 pGX1429 10 mcg 0.18 ± 0.09 p=0.147 20 mcg 0.36 ± 0.11 p<0.001 20 mcg 0.24 ± 0.12 p=0.030 30 mcg 0.21 ± 0.15 p=0.050 30 mcg 0.27 ± 0.12 p=0.010 50 mcg 0.38 ± 0.13 p<0.001 50 mcg 0.29 ± 0.16 p=0.004 The reported p-values are close to naive (pGX0001 immunized mice).

[00231] Similar to the magnitude of antigen-specific CD8+T cells, Survivin's synthetic consensus antigen 1 did not cause a significant change in the frequency of CD8+CD107a+T cells among all groups (p=0.101) (Fig. 10C). Survivin's synthetic 1T3 consensus antigen did cause a significant change in CD8+CD107a+T cell frequency between all dose groups (p=0.034) (FIG. 10D). The frequency of antigen-specific CD8+CD107a+ T cells increased significantly above naive (0.03 ± 0.01) in the group immunized with 50 μg pGX1429 (0.28% ± 0.22%) (p=0.026), but not in groups , immunized 10 µg (0.11% ± 0.08%) (p=0.813), 20 µg (0.16% ± 0.11%) (p=0.450), 30 µg (0.16% ± 0, 07%) (p=0.424) of the synthetic consensus antigen 1T3 Survivin. The cytokine profile of CD8+CD107a+ T cells specific for the synthetic consensus antigen of Survivin was similar for both constructs in different dose groups and included mainly IFNγ+IL-2-TNFα+, IFNγ+IL-2-TNFα- (Fig. 10E , Fig. 10F). The frequency of antigen-specific CD8 ⁺ T cells with cytolytic potential is described in more detail in Table 9.

Table 9 Cytolytic potential of antigen-specific CD8+ T cells induced with synthetic consensus antigen Survivin 1 and synthetic consensus antigen Survivin 1T3

Synthetic consensus antigen 1 Survivin Synthetic consensus antigen 1T3 Survivin Design Dose %CD8 ⁺ CD107a ⁺ ± Art. Off p-value Design Dose %CD8 ⁺ CD107a ⁺ ± Art. Off p-value pGX0001 30 mcg 0.01 ± 0.01 n/a
between groups p=0.101 pGX0001 30 mcg 0.03±0.01 n/a pGX1428 10 mcg 0.18±0.10 pGX1429 10 mcg 0.11 ± 0.08 p=0.813 20 mcg 0.31 ± 0.28 20 mcg 0.16 ± 0.11 p=0.450 30 mcg 0.21 ± 0.10 30 mcg 0.16 ± 0.07 p=0.424 50 mcg 0.21 ± 0.16 50 mcg 0.28 ± 0.22 p=0.026 The reported p-values are close to naive (pGX0001 immunized mice).

Width of IFNγ responses induced by Survivin synthetic consensus antigen constructs

[00232] The width of IFNγ responses to Survivin induced by pGX1428 and pGX1429 was examined by epitope mapping using a matrix peptide pool approach (FIGS. 11A-11D). Pooled spleen lymphocytes were examined from mice immunized with the highest dose of pGX1428 (n=8) (FIG. 11A) or pGX1429 (n=8) (FIG. 11B).

[00233] The following epitopes of Survivin synthetic consensus antigen 1 were present in both pGX1428 and pGX1429:

LPPAWQLFLKDHRISTFKN (SEQ ID NO:5) (matrix pools 1, 2, 3, 4 and 7)

LKLDRERAKNKIAKETNNK (SEQ ID NO:6) (matrix pools 1, 2, 3, 4 and 11)

The synthetic T3 consensus antigen of Survivin is presented in pGX1429:

EWLHHFQGLFP (SEQ ID NO:7) (matrix pools 3, 4 and 7)

[00234] Although the overall magnitude of cellular immune responses induced by Survivin synthetic consensus antigen 1 and Survivin synthetic consensus antigen 1T3 was similar, responses induced by Survivin synthetic consensus antigen 1T3 (pGX1429) against the synthetic Survivin consensus antigen 1 (pGX1428) region of the synthetic consensus antigen Survivin's 1T3 was approximately half that induced by the synthetic Survivin consensus antigen 1 (pGX1428) construct. Epitope mapping with the IFNγ ELISpot using a matrix approach showed that both Survivin synthetic consensus antigen constructs generated responses to the same antigenic epitopes in Survivin synthetic consensus antigen 1, but responses driven by Survivin synthetic consensus antigen 1T3 against these epitopes were more low value. It was also found that in the T3 region there is a unique antigenic epitope of the synthetic consensus antigen 1T3 Survivin.

[00235] Although the overall magnitude of cellular immune responses induced by Survivin synthetic consensus antigen 1 (pGX1428) and Survivin synthetic consensus antigen 1T3 (pGX1429) was similar, the responses induced by Survivin synthetic consensus antigen 1T3 antigen (pGX1429) vs. Survivin synthetic consensus antigen 1 region Survivin's synthetic 1T3 antigen consensus was about half that induced by the Survivin synthetic consensus antigen 1 construct (pGX1428). Epitope mapping by IFNγ ELISpot using a matrix approach showed that both Survivin synthetic consensus antigen constructs generate responses to the same antigenic epitopes as in Survivin synthetic consensus antigen 1, but responses induced by Survivin synthetic consensus antigen 1T3 against these epitopes have lower value. Also, using this approach, it was found that in the T3 region there is a unique antigenic epitope of the synthetic consensus antigen 1T3 Survivin. Survivin's synthetic consensus antigen 1T3 also significantly increased the frequency of antigen-specific CD8+ and CD8+CD107a+ T cells compared to naive, while Survivin's synthetic consensus antigen 1 did not significantly increase antigen-specific CD8+ T cells in mice. Survivin's synthetic 1T3 consensus antigen (pGX1429) was chosen to move into a monovalent study in non-human primates based on its potential to further broaden the spectrum of cellular immune responses to Survivin compared to pGX1428 in mice.

Example 5 Non-Human Primate Studies Using Survivin's Synthetic 1T3 Consensus Antigen (pGX1429).

[00236] To explore the potential of synthetic consensus Survivin 1T3 alone and in combination with low and high dose IL-12, eighteen adult rhesus monkeys, each identified by a unique NHP ID number, were divided into 3 groups of 6 and immunized with pGX1429 as follows way. Six animals were immunized with 3.0 mg pGX1429, six with 3.0 mg pGX1429 plus 0.04 mg pGX6006 (opt rh IL-12) adjuvant, and six with 3.0 mg pGX1429 plus 0.20 mg pGX6006 (opt rh IL-12) as an adjuvant that was formulated in SSC in a 1.0 ml injection volume. Immunization injections were administered at 0, 4, 8 and 12 weeks. All immunizations were administered intramuscularly using the CELLECTRA® 2000 5P-IM EP device in a 1 ml injection volume formulated in sterile WFI, alternately into the contralateral limbs, respectively. The EP conditions were as follows: OpBlock 0070 - IM, 0.5 A, 3 pulses, 52 ms, 0.2 s between pulses. The immunogenicity of survivin was assessed at 2, 6, 10 and 14 weeks. Survivin-specific IFN-gamma responses are shown in FIG. 12 and 13. The homology between native rhesus monkey Survivin and pGX1429 is shown in Table 10. As shown in FIG. 13 and 14, increased Survivin-specific responses were observed in animals immunized with Survivin synthetic consensus antigen 1T3 plus 0.20 mg IL-12 as an adjuvant.

Isolation of PBMCs

[00237] Non-human primate whole blood was collected in sodium citrate cell preparation tubes (CPT CPT's BD Biosciences) containing an anticoagulant and a gel barrier. Before overnight delivery, whole blood was centrifuged shortly after collection (within 2 hours) to separate and concentrate the PMBC. Erythrocytes and neutrophils settle to the bottom of the tubes and are held in place by a gel barrier. Plasma and lymphocytes remain above the gel barrier. Each CPT can contain approximately 8 ml of blood and is supplied at room temperature. CPT centrifuge tubes were processed to isolate PBMCs. Following lysis of erythrocytes with ammonium chloride potassium (ACK) buffer, viable cells were counted using an Invitrogen Countess™ automatic cell counter and resuspended in complete culture medium (RPMI 1640 supplemented with 10% FBS, antibiotics and β-mercaptoethanol). After completion of the assays as described herein, the remaining PBMCs were frozen in freezing medium (10% DMSO from Sigma in 90% FBS from Seradigm) in cryovials and stored long-term in liquid nitrogen.

IFNγ ELISpot

[00238] To evaluate vaccine-induced antigen-specific cellular responses, monkey IFNγ ELISpot assays were performed at each time point on isolated PMBCs using a kit (MabTech IFNγ ELISpotPro, no. 3421M-2APW-10). Briefly, 96-well plates pre-coated with anti-Monkey IFN antibody (mAb MT126L) were washed in PBS and blocked for 2 hours at room temperature with complete culture medium (RPMI 1640 supplemented with 10% FBS, antibiotics and β-mercaptoethanol) NHP PBMC was resuspended in R10 medium (and then added in triplicate at 2 x 10 ⁵ cells per well injected. A set of peptides (GenScript) was synthesized, each containing 15 amino acid residues overlapping with 11 amino acids representing the complete protein sequences of the synthetic These peptide sets were resuspended in DMSO (Sigma) and pooled at a concentration of approximately 2 μg/ml of each respective peptide in pools All antigen-specific pooled peptides were used at a 1:100 dilution resulting in a final 1:200 dilution in each well of the pool with PBMC Differences in the size of each antigenic protein resulted in the formation of 2 peptide pools for Survivin.

[00239] Anti-CD3 (mAb CD-2 Mabtech) and/or PMA (Sigma) with ionomycin (Sigma) was used as a positive control. Complete culture medium R10 was used as a negative control. The plates were incubated for approximately 18 hours at 37° C. in a 5% CO2 incubator. After removing the cells and adding ALP-conjugated anti-monkey IFNγ antibody (MabTech Ab 7-B6-1-ALP), the plates were incubated for 2 hours at room temperature. The sandwich enzyme immunoassay was then performed using the BCIP/NBT substrate solution according to the kit manufacturer's instructions (MabTech). The blue-black stained precipitate forms spots to identify each individual cell producing IFNγ. The spots are then scanned and counted by the analyzer and CTL ImmunoSpot® (Cellular Technology) software and quality controlled by a trained operator. IFNγ responses are reported as Spot Forming Units (SFU) per 1×10 ⁶ PBMC exceeding the SFU in the medium-only control.

Table 10 Characteristics of Survivin's synthetic 1T3 consensus antigen compared to native rhesus monkey Survivin.

Region Native part of the rhesus monkey pGX1429 Full length 95.8% (NP_001253110.1) pGX1429 Isoform 1 region 95.9% (NP_001253110.1) pGX1429 area T3 9.4% (NP_001253110.1)

Table 11. Construct, Antigen, Dose

Design ID Antigen Dose (mg) pGX1429 Synthetic Consensus Survivin 1T3 3 pGX6006 opt rIL-12 0.04 or 0.2

[00240] Groups 1, 2 and 3 received the following:

Group 1-3.0 mg pGX1429 (Synthetic Consensus Survivin 1T3). Formulated in SSC, 1.0 ml injection volume, IM.

Group 2-3.0 mg pGX1429 (Synthetic Consensus Survivin 1T3) + 0.04 pGX6006 (opt. rIL-12). Formulated in SSC, 1.0 ml injection volume, IM.

Group 3-3.0 mg pGX1429 (Synthetic Consensus Survivin 1T3) + 0.20 pGX6006 (opt. rIL-12). Formulated in SSC, 1.0 ml injection volume, IM.

[00241] All groups were immunized according to the following schedule:

Immunization 1 (Week 0)

Immunization 2 (Week 4)

Immunization 3 (Week 8)

Immunization 4 (Week 12)

Immunization 5 (optional)

Results

[00242] Survivin-specific IFNγ responses are shown in FIG. 12-14 for groups 1-3, respectively. The results show the response at each time point 2 weeks post-dose. In general, all groups and individual animals had an increase in response by the end of the study 2 weeks after dose 4 compared with the initial pre-blood collection. The addition of a higher dose of IL-12 (0.2 mg) resulted in larger and more consistent responses at each time point compared to Survivin alone or Survivin plus 0.04 mg IL-12. Higher responses already at PD2 were also noted for Survivin plus IL-12 0.2 mg.

[00243] There were no differences in any of the physiological parameters measured due to immunization, as shown in tables 12-14. There were no significant differences for erythrocytes, HCT, neutrophils, lymphocytes, monocytes, eosinophils (results not shown). These values are within the expected ranges for animals of this species, sex and age, subjected to similar experimental procedures. Any deviations from the stated normal ranges are sporadic, present in only one sex, and are not related to dose levels or timing.

Table 12. Assessment of physiological parameters in group 1

Group 1 Prevaccination Post-vaccination normal range Week -2 Week 6 Week 14 WBC count (#/10 ³ /ml) 7.8-14.3 4.8-9.6 4.9-11.2 4.0-15.0 Creatinine (mg/dl) 0.4-0.8 0.4-0.7 0.4-0.8 0.3-1.4 BUN (mg/dl) 11-17 13-18 13-18 9-29 ALK P (units/l) 232-491 184-491 212-505 65-641 AST (units/l) 17*-24
(#6867, 6859, 6892, 6873) 10*-24
(#6867, 6859, 6892, 6873, 6879) 20*-36
(#6859) 23-175 ALT (units/l) 20-40 17-38 12-31 18-204 TBIL (mg/dl) 0.1-0.2 0.1-0.2 0.1-0.2 0.1-0.6

Note: Out of normal range *

Table 13. Assessment of physiological parameters in group 2

Group 2 Prevaccination Post-vaccination normal range Week -2 Week 6 Week 14 WBC count (#/10 ³ /ml) 6.3-10.8 5.7-8.5 6.2-12.1 4.0-15.0 Creatinine (mg/dl) 0.6-0.7 0.5-0.6 0.5-0.7 0.3-1.4 BUN (mg/dl) 10-19 12-21 10-23 9-29 ALK P (units/l) 269-508 279-521 295-455 65-641 AST (units/l) 15*-32
(#6868, 6874, 6880, 6887, 6893) 12*-26
(#6874, 6868, 6861) 21*-38
(#6893) 23-175 ALT (units/l) 19-33 16*-28
(#6861) 19-30 18-204 TBIL (mg/dl) 0.1-0.2 0.2 0.1-0.2 0.1-0.6

Note: Out of normal range *

Table 14. Assessment of physiological parameters in group 3

Group 3 Prevaccination Post-vaccination normal range Week -2 Week 6 Week 14 WBC count (#/10 ³ /ml) 6.0-12.8 5.6-9.2 5.8-9.8 4.0-15.0 Creatinine (mg/dl) 0.5-0.7 0.4-0.7 0.5-0.7 0.3-1.4 BUN (mg/dl) 10-16 14-18 9-20 9-29 ALK P (units/l) 186-345 156-406 198-476 65-641 AST (units/l) 12*-47
(#6894, 6869) 12*-68
(#6894, 6869) 23-43 23-175 ALT (units/l) 20-44 16*-56
(#6863) 15*-55
(#6894) 18-204 TBIL (mg/dl) 0.1-0.2 0.2-0.4 0.1-0.2 0.1-0.6

Note: Out of normal range *

[00244] For all groups, there was no significant weight change during the study (data not shown).

[00245] In general, the results show that synthetic consensus Survivin administered alone is capable of inducing an immune response in 100% NHP. The addition of an adjuvant to IL-12 improved the response noted for synthetic consensus Survivin, resulting in a much earlier, greater PD2 response, but only with a higher dose of IL-12.

[00246] It is understood that the foregoing detailed description and accompanying examples are merely illustrative and should not be construed as limiting the scope of the invention, which is defined solely by the appended claims and their equivalents.

[00247] Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications to the disclosed embodiments, including, without limitation, changes relating to the chemical structures, substituents, derivatives, intermediates, syntheses, compositions, compositions, or uses of the invention, may be made without departing from its spirit and scope.

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SEQUENCE LIST

<110> INOVIO PHARMACEUTICALS, INC.

YAN, Jian

SLAGER, Anna

GARMAN, Bradley

Cook, Neil

<120> ANTI-CANCER VACCINES TARGETING SURVIVIN AND THEIR APPLICATIONS

<130> 104409.000448 / INO-1004 WO

<150> US 62/598,267

<151> 2017-12-13

<160> 7

<170> PatentIn version 3.5

<210> 1

<211> 483

<212> DNA

<213> Artificial sequence

<220>

<223> pGX1428 DNA sequence encoding isoform 1

synthetic consensus Survivin

<400> 1

atggattgga cctggattct gttcctggtg gcagcagcaa cccgggtgca ctccggagcc 60

cccacactgc cccctgcctg gcagctgttt ctgaaggacc acaggatctc tacattcaag 120

aactggccct ttctggaggg atgcgcatgt gcacctgaga ggatggcaga ggcaggcttc 180

atccactgcc ctgccgagaa tgagccagat ctggcccagt gcttcttttg ttttaaggag 240

ctggagggct gggagccaga cgatgacccc atcgaggagc acaagaagca cagctccggc 300

gccgccttcc tgtctgtgaa gaagcagttt gaggagctga ccctgagcga gttcctgaag 360

ctggatcggg aggagccaa gaacaagatc gccaaggaga ccaacaacaa gaagaaggag 420

tttgaggaga cagccaagaa ggtgaggtgt gccatcgagc agctggccgc catggactga 480

taa 483

<210> 2

<211> 159

<212> PROTEIN

<213> Artificial sequence

<220>

<223> pGX1428 sequence encoding isoform 1 protein

synthetic consensus Survivin

<400> 2

Met Asp Trp Thr Trp Ile Leu Phe Leu Val Ala Ala Ala Thr Arg Val

1 5 10 15

His Ser Gly Ala Pro Thr Leu Pro Pro Ala Trp Gln Leu Phe Leu Lys

20 25 30

Asp His Arg Ile Ser Thr Phe Lys Asn Trp Pro Phe Leu Glu Gly Cys

35 40 45

Ala Cys Ala Pro Glu Arg Met Ala Glu Ala Gly Phe Ile His Cys Pro

50 55 60

Ala Glu Asn Glu Pro Asp Leu Ala Gln Cys Phe Phe Cys Phe Lys Glu

65 70 75 80

Leu Glu Gly Trp Glu Pro Asp Asp Asp Pro Ile Glu Glu His Lys Lys

85 90 95

His Ser Ser Gly Ala Ala Phe Leu Ser Val Lys Lys Gln Phe Glu Glu

100 105 110

Leu Thr Leu Ser Glu Phe Leu Lys Leu Asp Arg Glu Arg Ala Lys Asn

115 120 125

Lys Ile Ala Lys Glu Thr Asn Asn Lys Lys Lys Glu Phe Glu Glu Thr

130 135 140

Ala Lys Lys Val Arg Cys Ala Ile Glu Gln Leu Ala Ala Met Asp

145 150 155

<210> 3

<211> 696

<212> DNA

<213> Artificial sequence

<220>

<223> pGX1429 DNA sequence encoding isoform 1 + truncated

isoform 3 of the synthetic consensus Survivin

<400> 3

atggattgga catggattct gttcctggtg gcagcagcaa ccagggtgca ctctggagca 60

ccaacactgc cccctgcatg gcagctgttt ctgaaggacc accggatcag caccttcaag 120

aactggcctt ttctggaggg ctgcgcctgt gccccagaga gaatggcaga ggcaggcttc 180

atccactgcc cagccgagaa tgagcctgat ctggcccagt gcttcttttg ttttaaggag 240

ctggagggct gggagcctga cgatgaccca atcgaggagc acaagaagca cagctccgga 300

gcagccttcc tgagcgtgaa gaagcagttt gaggagctga cactgtccga gttcctgaag 360

ctggataggg agcgcgccaa gaacaagatc gccaaggaga ccaacaacaa gaagaaggag 420

tttgaggaga cagccaagaa ggtgcggtgt gcaatcgagc agctggcagc aatggacagg 480

ggaagaaagc ggagatccat gcagaggaag cctaccatca ggcgcaagaa tctgcgcaag 540

ctgcggagaa agtgcgccgt gccatctagc tcctggctgc cctggacaga ggcctctggc 600

tggagctgtc tggtgcccga gtggctgcac cacttccagg gactgtttcc tggagccacc 660

tccctgccag tgggaccact ggccatgtct tgataa 696

<210> 4

<211> 210

<212> PROTEIN

<213> Artificial sequence

<220>

<223> pGX1429 sequence encoding protein of isoform 1 + truncated

isoform 3 of the synthetic consensus Survivin

<400> 4

Met Asp Trp Thr Trp Ile Leu Phe Leu Val Ala Ala Ala Thr Arg Val

1 5 10 15

His Ser Gly Ala Pro Thr Leu Pro Pro Ala Trp Gln Leu Phe Leu Lys

20 25 30

Asp His Arg Ile Ser Thr Phe Lys Asn Trp Pro Phe Leu Glu Gly Cys

35 40 45

Ala Cys Ala Pro Glu Arg Met Ala Glu Ala Gly Phe Ile His Cys Pro

50 55 60

Ala Glu Asn Glu Pro Asp Leu Ala Gln Cys Phe Phe Cys Phe Lys Glu

65 70 75 80

Leu Glu Gly Trp Glu Pro Asp Asp Asp Pro Ile Glu Glu His Lys Lys

85 90 95

His Ser Ser Gly Ala Ala Phe Leu Ser Val Lys Lys Gln Phe Glu Glu

100 105 110

Leu Thr Leu Ser Glu Phe Leu Lys Leu Asp Arg Glu Arg Ala Lys Asn

115 120 125

Lys Ile Ala Lys Glu Thr Asn Asn Lys Lys Lys Glu Phe Glu Glu Thr

130 135 140

Ala Lys Lys Val Arg Cys Ala Ile Glu Gln Leu Ala Ala Met Asp Arg

145 150 155 160

Gly Arg Lys Arg Arg Ser Met Gln Arg Lys Pro Thr Ile Arg Arg Lys

165 170 175

Asn Leu Arg Lys Leu Arg Arg Lys Cys Ala Val Pro Ser Ser Ser Ser Trp

180 185 190

Leu Pro Trp Thr Glu Ala Ser Gly Trp Ser Cys Leu Val Pro Glu Trp

195 200 205

Leu His

210

<210> 5

<211> 19

<212> PROTEIN

<213> Artificial sequence

<220>

<223> matrix pools 1, 2, 3, 4 and 7

<400> 5

Leu Pro Pro Ala Trp Gln Leu Phe Leu Lys Asp His Arg Ile Ser Thr

1 5 10 15

Phe Lys Asn

<210> 6

<211> 19

<212> PROTEIN

<213> Artificial sequence

<220>

<223> matrix pools 1, 2, 3, 4 and 11

<400> 6

Leu Lys Leu Asp Arg Glu Arg Ala Lys Asn Lys Ile Ala Lys Glu Thr

1 5 10 15

Asn Asn Lys

<210> 7

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> matrix pools 3, 4 and 7

<400> 7

Glu Trp Leu His His Phe Gln Gly Leu Phe Pro

1 5 10

<210> 8

<211> 232

<212> PROTEIN

<213> Artificial sequence

<220>

<223> pGX1429 full-length protein coding sequence

isoform 1 + shortened isoform 3 of synthetic consensus

Survivina

<400> 8

Met Asp Trp Thr Trp Ile Leu Phe Leu Val Ala Ala Ala Thr Arg Val

1 5 10 15

His Ser Gly Ala Pro Thr Leu Pro Pro Ala Trp Gln Leu Phe Leu Lys

20 25 30

Asp His Arg Ile Ser Thr Phe Lys Asn Trp Pro Phe Leu Glu Gly Cys

35 40 45

Ala Cys Ala Pro Glu Arg Met Ala Glu Ala Gly Phe Ile His Cys Pro

50 55 60

Ala Glu Asn Glu Pro Asp Leu Ala Gln Cys Phe Phe Cys Phe Lys Glu

65 70 75 80

Leu Glu Gly Trp Glu Pro Asp Asp Asp Pro Ile Glu Glu His Lys Lys

85 90 95

His Ser Ser Gly Ala Ala Phe Leu Ser Val Lys Lys Gln Phe Glu Glu

100 105 110

Leu Thr Leu Ser Glu Phe Leu Lys Leu Asp Arg Glu Arg Ala Lys Asn

115 120 125

Lys Ile Ala Lys Glu Thr Asn Asn Lys Lys Lys Glu Phe Glu Glu Thr

130 135 140

Ala Lys Lys Val Arg Cys Ala Ile Glu Gln Leu Ala Ala Met Asp Arg

145 150 155 160

Gly Arg Lys Arg Arg Ser Met Gln Arg Lys Pro Thr Ile Arg Arg Lys

165 170 175

Asn Leu Arg Lys Leu Arg Arg Lys Cys Ala Val Pro Ser Ser Ser Ser Trp

180 185 190

Leu Pro Trp Thr Glu Ala Ser Gly Trp Ser Cys Leu Val Pro Glu Trp

195 200 205

Leu His His Phe Gln Gly Leu Phe Pro Gly Ala Thr Ser Leu Pro Val

210 215 220

Gly Pro Leu Ala Met Glu Thr Ser

225 230

<---

Claims (26)

1. A nucleic acid molecule that encodes a synthetic Survivin consensus antigen, wherein said nucleic acid molecule encodes a protein containing SEQ ID NO: 5, SEQ ID NO: 6 and alanine at positions 51, 65 and 101 with respect to SEQ ID NO: 2 . 2. A nucleic acid molecule that encodes a synthetic Survivin consensus antigen, wherein said nucleic acid molecule encodes a protein containing SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and alanine at positions 51, 65 and 101 with respect to to SEQ ID NO: 8. 3. Survivin synthetic consensus antigen containing SEQ ID NO: 5, SEQ ID NO: 6 and alanine at positions 51, 65 and 101 with respect to SEQ ID NO: 2. 4. Survivin synthetic consensus antigen containing SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and alanine at positions 51, 65 and 101 with respect to SEQ ID NO: 8. 5. A nucleic acid molecule that encodes a synthetic Survivin consensus antigen, wherein said nucleic acid molecule encodes a protein comprising SEQ ID NO: 5 and SEQ ID NO: 6 separated by an amino acid sequence corresponding to WPFLEGCACAPERMAEAGFIHCPAENEPDLAQCFFCFKELEGWEPDDDPIEEHKKHSSGAAFLSVKKQFEELTLSEF. 6. A nucleic acid molecule that encodes a synthetic consensus antigen of Survivin, where the specified nucleic acid molecule encodes a protein containing SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7, where SEQ ID NO: 5 and SEQ ID NO : 6 are separated by an amino acid sequence corresponding to WPFLEGCACAPERMAEAGFIHCPAENEPDLAQCFFCFKELEGWEPDDDPIEEHKKHSSGAAFLSVKKQFEELTLSEF, and SEQ ID NO: 6 and SEQ ID NO: 7 are separated by an amino acid sequence corresponding to EFEETAKKVRCAIEQLAAMDRGRKRRSMQRKPTIRRKNLRKLRRKCAVPSSSWLPWTEASGWSCLVP. 7. Synthetic consensus antigen Survivin, containing SEQ ID NO: 5 and SEQ ID NO: 6, separated by an amino acid sequence corresponding to WPFLEGCACAPERMAEAGFIHCPAENEPDLAQCFFCFKELEGWEPDDDPIEEHKKHSSGAAFLSVKKQFEELTLSEF. 8. A synthetic Survivin consensus antigen comprising SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7, wherein SEQ ID NO: 5 and SEQ ID NO: 6 are separated by an amino acid sequence corresponding to WPFLEGCACAPERMAEAGFIHCPAENEPDLAQCFFCFKELEGWEPDDDPIEEHKKHSSGAAFLSVKKQFEELTLSEF, and SEQ ID NO: 6 and SEQ ID NO: 7 are separated by an amino acid sequence corresponding to EFEETAKKVRCAIEQLAAMDRGRKRRSMQRKPTIRRKNLRKLRRKCAVPSSSWLPWTEASGWSCLVP. 9. An expression vector containing a nucleic acid molecule according to any one of claims 1, 2, 5 or 6. 10. An expression vector according to claim 9, wherein said expression vector is a plasmid. 11. An immunogenic composition comprising an effective amount of a nucleic acid molecule according to claim 1, 2, 5 or 6 and a pharmaceutically acceptable carrier. 12. An immunogenic composition containing an effective amount of a vector according to claim 9 or 10 and a pharmaceutically acceptable carrier. 13. A vaccine for the treatment or prevention of Survivin-expressing cancer, containing an effective amount of the vector according to claim 9 or 10. 14. The vaccine of claim 13 further comprising a pharmaceutically acceptable excipient. 15. The vaccine according to claim 13 or 14, additionally containing an adjuvant. 16. The vaccine of claim 15 wherein the adjuvant is IL-12, IL-15, IL-28, or RANTES. 17. A method of treating a subject with a Survivin-expressing cancer cell, comprising administering a therapeutically effective amount of a vaccine according to any one of claims 13-16. 18. The method of claim 17, wherein the administration comprises electroporation. 19. The method of claim 17 wherein administration occurs at one or more sites in the subject. 20. A method of vaccinating a subject against a Survivin-expressing cancer cell, comprising administering an amount of the vaccine according to any one of claims 13-16 effective to induce a humoral or cellular immune response. 21. The method of claim 20, wherein the administration comprises electroporation. 22. The method of claim 20 wherein administration occurs at one or more sites in the subject.

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