RU2799786C2 - Boris-targeted anti-cancer vaccines and methods for application thereof - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology and can be used in medicine for preventing and/or treating a subject with a BORIS-expressing tumour. Disclosed are nucleic acid molecules containing one or more nucleic acid sequences encoding the synthetic consensus antigen BORIS. Disclosed are a vector, a vaccine, containing one or more nucleic acid sequences encoding the synthetic consensus antigen BORIS. Disclosed are a method for treating a subject with a BORIS-expressing tumour and a method for vaccinating a subject against one expressing an antigen - Brother of the Regulator of an Imprinted Site (BORIS) of the cancer cell. Disclosed is the synthetic consensus antigen BORIS.

EFFECT: invention proposes a safe and effective vaccine and methods for application thereof for preventing and/or treating cancer and reducing the mortality in subjects suffering from cancer.

17 cl, 16 dwg, 8 tbl, 3 ex

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority over U.S. Provisional Application No. 62/598,274, filed December 13, 2017, the disclosure of which is incorporated herein by reference in its entirety.

SEQUENCE LIST

[0002] The application contains a sequence listing, which is presented in electronic ASCII format and is fully incorporated herein by reference. The specified ASCII copy, created on December 11, 2018, is named 104409_000447_sequence_listing.txt and is 9,179 bytes in size.

FIELD OF TECHNOLOGY

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

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, certain 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] CCCTC binding factor (CTCF) is an 11-zinc finger factor involved in gene regulation. The 11 zinc fingers of CTCF bind to various DNA target sites and act as transcriptional repressors. The imprinted site regulator sibling ("BORIS") or CTCF-like ("CTCFL") is a paralog of CTCF as well as a transcriptional regulator. (Loukinov, D. I. et al. BORIS, a novel male germ-line-specific protein associated with epigenetic reprogramming events, shares the same 11-zinc-finger domain with CTCF, the insulator protein involved in reading imprinting marks in the soma. Proceedings of the National Academy of Sciences of the United States of America 99, 6806-6811, doi:10.1073/pnas. 092123699 (2002)). CTCF and BORIS have mutually exclusive expression patterns in normal tissue but are co-expressed in cancerous tissues. Although BORIS mRNA expression is very low or not found in normal ovarian tissue, it is highly expressed in many ovarian epithelial carcinoma ("ECOS") cells. Aberrant BORIS expression was found in 67% of primary ECO tumors. (Link, P.A., et al. BORIS/CTCFL mRNA isoform expression and epigenetic regulation in epithelial ovarian cancer. Cancer Immunity 13, 6 (2013)).

[0007] There are 23 different isoforms of BORIS mRNA formed by alternative splicing, each with canonical exon-intron junctions and poly-A signals that are conserved in primates. Six different families of BORIS isoforms (sf1 to sf6) encode 17 different BORIS proteins. BORIS zinc finger domains show homology to CTCF domains; however, different flanking regions between the two proteins indicate different functional implications of DNA binding. (Cm. Ohlsson, R., Renkawitz, R. & Lobanenkov, V. CTCF is a uniquely versatile transcription regulator linked to epigenetics and disease. Trends in genetics: TIG 17, 520-527 (2001).) The BORIS sf1 isoform is most differentially expressed among normal ovarian tissue and ECO tumor samples.

[0008] Vaccines for the treatment and prevention of cancer, in particular ECO, are of interest. 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 19-680 of SEQ ID NO:2; (b) a nucleic acid sequence that encodes a fragment containing at least 90% of the total length of amino acids 19-680 of SEQ ID NO:2; (c) a nucleic acid sequence that encodes a protein that is at least 95% identical to amino acids 19-680 of SEQ ID NO:2; and (d) a nucleic acid sequence that encodes a fragment containing at least 90% of the total length of the protein, which is at least 95% identical to amino acids 19-680 of SEQ ID NO:2.

[0011] Nucleic acid molecules containing one or more nucleic acid sequences selected from the group consisting of: (a) nucleotides 55-2040 of SEQ ID NO:1; (b) a fragment containing at least 90% of the total length of nucleotides 55-2040 of SEQ ID NO:1; (c) a fragment that is at least 95% identical to nucleotides 55-2040 of SEQ ID NO:1; and (d) a fragment containing at least 90% of the entire length of the nucleic acid sequence, which is at least 95% identical to nucleotides 55-2040 of SEQ ID NO:1.

[0012] Nucleic acid molecules comprising one or more nucleic acid sequences selected from the group consisting of: (a) a nucleic acid sequence that encodes the entire length of SEQ ID NO:2; (b) a nucleic acid sequence that encodes a fragment containing at least 90% of the entire length of SEQ ID NO:2; (c) a nucleic acid sequence that encodes a protein that is at least 95% identical to SEQ ID NO:2; and (d) 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.

[0013] Nucleic acid molecules containing one or more nucleic acid sequences selected from the group consisting of: (a) SEQ ID NO:1; (b) a fragment containing at least 90% of the entire length of SEQ ID NO:1; (c) a fragment that is at least 95% identical to SEQ ID NO:1; and (d) 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.

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

[0015] Nucleic acid molecules provided herein for use as a drug.

[0016] Nucleic acid molecules provided herein for use as a drug in the treatment of cancer.

[0017] Nucleic acid molecules proposed by the document for use in the preparation of a drug.

[0018] Nucleic acid molecules provided herein for use in the preparation of a medicament for the treatment of cancer.

[0019] Vectors containing the nucleic acid molecule provided herein, wherein said vector may be a plasmid or a viral vector.

[0020] Compositions containing one or more nucleic acid molecules provided herein.

[0021] the Compositions proposed herein, containing a pharmaceutically acceptable carrier, and these compositions may contain one or more of the vectors proposed in this document.

[0022] Proteins containing an amino acid sequence selected from the group consisting of: (a) amino acids 19-680 of SEQ ID NO:2; (b) a fragment containing at least 90% of the total length of amino acids 19-680 of SEQ ID NO:2; (c) an amino acid sequence that is at least 95% identical to amino acids 19-680 of SEQ ID NO:2; and (d) a fragment containing at least 90% of the entire length of the amino acid sequence, which is at least 95% identical to amino acids 19-680 of SEQ ID NO:2.

[0023] Proteins containing an amino acid sequence selected from the group consisting of: (a) SEQ ID NO:2; (b) a fragment containing at least 90% of the entire length of SEQ ID NO:2; (c) an amino acid sequence that is at least 95% identical to SEQ ID NO:2; and (d) 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.

[0024] Proteins containing the amino acid sequence shown in SEQ ID NO:2.

[0025] Vaccines containing the nucleic acid molecule provided herein.

[0026] Vaccines containing the vector provided herein.

[0027] Vaccines as described herein, additionally containing a pharmaceutically acceptable excipient, and this vaccine may additionally contain an adjuvant, and the adjuvant may be IL-12, IL-15, IL-28 or RANTES.

[0028] Methods of treating a subject with a BORIS-expressing cancer cell, comprising administering a therapeutically effective amount of the vaccine provided herein, wherein the administration may include an electroporation step, and wherein the administration may occur at one or more sites in the subject.

[0029] Methods for vaccinating a subject against a BORIS-expressing cancer cell, comprising administering an amount of the vaccine provided herein effective to induce a humoral or cellular immune response.

BRIEF DESCRIPTION OF THE DRAWINGS

[0030] 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:

[0031] In FIG. 1 shows a schematic diagram of the amino acid sequence of the synthetic BORIS consensus antigen. Asterisks denote mutations in 11 zinc finger domains and nuclear localization signal.

[0032] FIG. 2 shows the general structure of the BORIS protein. The spheres indicate changes relative to native BORIS.

[0033] Figure 3 shows a comparison of a native zinc finger with the zinc finger structure of the synthetic BORIS consensus antigen. The zinc finger structure of the synthetic BORIS consensus antigen disrupts DNA binding.

[0034] FIG. 4 shows a pGX1440 construct containing a synthetic BORIS antigen consensus sequence insert.

[0035] FIG. 5 shows a flow cytometry gate strategy.

[0036] Figure 6 shows in vitro expression of the synthetic BORIS consensus antigen in human rhabdomyosarcoma (RD) cells transfected with pGX1440 as determined by human anti-BORIS immunoblotting.

[0037] FIG. 7A and 7B show the immunogenicity of the synthetic BORIS consensus antigen. Female CB6F1 mice were immunized 3 times 3 weeks apart with the indicated doses of synthetic BORIS consensus antigen (pGX1440, n=8/group) or pGX0001 (empty vector, n=4). Antigen-specific IFNγ responses to the BORIS consensus synthetic antigen were assessed by ELISpot at the indicated doses of pGX1440. On figa shows the responses of individual animals, and figa shows group responses.

[0038] FIG. 8A, 8B, 8C and 8D show the relative frequency of CD4+ and CD8+ T cells. Cellular immune responses induced by pGX1440 were predominantly in the CD8+T cell compartment relative to the CD4+T cell compartment. The synthetic BORIS consensus antigen induced frequencies of antigen-specific CD4 ⁺ T cell responses that were significantly more robust than naive across all dose groups (FIG. 8A). The frequency of antigen-specific CD8 ⁺ T cells induced by the BORIS synthetic consensus antigen significantly increased compared to controls in all dose groups (FIG. 8B). The cytokine profile of antigen-specific responses of CD4 ⁺ T cells (FIG. 8C) and CD8 ⁺ T cells to the BORIS synthetic consensus antigen is shown in (FIG. 8D).

[0039] FIG. 9A, 9B, 9C and 9D show the cytolytic potential of antigen-specific T cells for the synthetic BORIS consensus antigen. Cytolytic immune responses induced by pGX1440 were predominantly in the CD8+T cell compartment relative to the CD4+T cell compartment. The frequency of antigen-specific CD4 ⁺ CD107a ⁺ T cells was increased in all dose groups (Fig. 9A). Similarly, the frequency of antigen-specific CD8 ⁺ CD107a ⁺ T cells was increased in all dose groups (Fig. 9B). The cytokine profile of CD4+CD107a+T cells (FIG. 9C) and CD8+CD107a+T cells is shown in (FIG. 9D).

DETAILED DESCRIPTION OF THE INVENTION

[0040] The present invention relates to a vaccine containing a synthetic BORIS consensus antigen. BORIS is expressed in many tumors. Accordingly, the vaccine provides treatment for cancer or cancers expressing BORIS.

[0041] A synthetic BORIS consensus antigen may be a BORIS consensus antigen derived from BORIS sequences from different species or from different isoforms within a species, and thus the synthetic BORIS consensus antigen is not native. The synthetic BORIS consensus antigen can be further modified by introducing one or more mutations in the consensus sequence to create a synthetic BORIS consensus antigen sequence. Mutations can destroy or modify specific functional domains of the native BORIS sequence, thereby disrupting or enhancing the structure or function of the functional domains. In one embodiment, mutations are introduced into the BORIS consensus sequence to disrupt each of the native BORIS zinc finger domains. In other embodiments of the synthetic BORIS antigen consensus sequence, the mutation is introduced into the native BORIS nuclear localization signal sequence. In other embodiments, mutations are introduced into the BORIS consensus sequence to disrupt each zinc finger domain and nuclear localization sequence.

[0042] The synthetic BORIS 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 the 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 associated with tumor fibroblasts, soluble factors produced by immunosuppressive cells, CTLA-4, PD-1, MDSC, MCP-1, and an immune checkpoint molecule.

[0043] 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.

[0044] One way to construct a nucleic acid and its encoded amino acid sequence of a recombinant cancer antigen is to introduce mutations that change specific amino acids in the overall amino acid sequence of the native cancer antigen. The introduction of mutations does not 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 becomes 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 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. The above approaches can be combined such that the final recombinant cancer antigen has a percentage similarity to the amino acid sequence of the native cancer antigen, as discussed above.

[0045] 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 (for example, anti-PD-1 and anti-PDL-1 antibody therapy) to further enhance T-cell and/or B-cell responses.

[0046] 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, 5 7, 58, 59% and 60%.

[0047] The vaccine can enhance a 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-fold to about 4500-fold, from about 100-fold to about 6000-fold, from about 1 50 times to about 6,000 times, about 200 times to about 6,000 times, 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, a multiple of approximately 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 95 0, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000 3100 3200 3300 3400 3500 3600 3700 3800 3900 4000 4100 4200 4300 4400 4500 4600 4700 4800 4900 500 0.5100, 5200, 5300, 5400, 5500, 5600, 5700, 5800, 5900 times, or 6000 times the cellular immune response in a subject who was not vaccinated.

[0048] The vaccine can increase levels of interferon gamma (IFN-γ) in a subject who is administered the vaccine by about 50-fold to about 6000-fold, about 50-fold to about 5500-fold, about 50-fold to about 5000-fold, from about 50-fold to about 4500-fold, from about 100-fold to about 6000-fold, 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 levels of IFN-γ in a subject who is administered the vaccine by about 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 95 multiples 0, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000 3100 3200 3300 3400 3500 3600 3700 3800 3900 4000 4100 4200 4300 4400 4500 4600 4700 4800 4900 500 0, 5100, 5200, 5300, 5400, 5500, 5600, 5700, 5800, 5900 times, or 6000 times compared to the levels of IFN-γ in the subject who did not receive the vaccine.

[0049] 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 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 immune cell proliferation and/or differentiation signaling. 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

[0050] Unless otherwise indicated, all technical and scientific terms used in this document have the same meaning as commonly known to one 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.

[0051] The terms "include(includes)", "having", "has", "may", "comprises(contains)" and variations thereof used herein are intended to be non-limiting translatable phrases, terms or words that do not exclude the possibility of additional acts or structures. The singular noun forms and "and" include plural forms unless the context clearly specifies otherwise. The present specification also discusses other embodiments "comprising", "consisting of", and "consisting essentially of" the embodiments or elements presented herein, whether explicitly stated or not.

[0052] 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 provided explicitly.

[0053] The term "adjuvant" as used herein means any molecule added to the vaccines described herein to enhance the immunogenicity of an antigen.

[0054] "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 derivatives thereof. The antibody may be an antibody isolated from a mammalian serum sample, a polyclonal antibody, an affinity-purified antibody, or any mixture thereof, that exhibits sufficient binding specificity for the desired epitope or sequence derived from it.

[0055] "Antigen" refers to proteins having the amino acid sequences of the BORIS antigen, including: (a) amino acids 19-680 of SEQ ID NO:2; (b) fragments containing at least 90% of amino acids 19-680 of SEQ ID NO:2; (c) amino acid sequences that are at least 96% identical to amino acids 19-680 of SEQ ID NO:2; and (d) fragments containing at least 90% of an amino acid sequence that is at least 96% identical to amino acids 19-680 of SEQ ID NO:2; and proteins having the amino acid sequences of the BORIS antigen, including: (a) SEQ ID NO:2; (b) fragments containing at least 90% of the total length of SEQ ID NO:2; (c) amino acid sequences that are at least 96% identical to SEQ ID NO:2; and (d) fragments containing at least 90% of the entire length of the amino acid sequence, which is at least 95% identical to SEQ ID NO:2; as well as BORIS antigens containing the amino acid sequence specified in SEQ ID NO:2. Antigens may optionally include signal peptides such as peptides from other proteins.

[0056] The terms "coding sequence" or "coding nucleic acid" as used herein 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 a polyadenylation signal, capable of directing expression in cells of the subject or mammal receiving the nucleic acid.

[0057] 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.

[0058] The terms "consensus", "consensus sequence" or "BORIS consensus sequence" in the context of the present description means a polypeptide sequence based on the analysis of the alignment of multiple sequences for the same gene from different organisms or from different isoforms in the body. Can be obtained sequences of nucleic acids that encode the consensus polypeptide sequence.

[0059] As used herein, the term "direct current" describes 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) in such a way that the current density in the same tissue remains constant throughout the entire duration of the electrical pulse (on the order of microseconds) and from pulse to pulse. In some embodiments, the feedback element comprises a controller.

[0060] The terms "current feedback" or "feedback" as used herein can be used interchangeably and can refer to the active response of the provided electroporation devices, which includes measuring the current in the tissue between the electrodes and correspondingly changing the output power delivered 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 electrical action. Feedback can be provided by an electroporation component, such as an electroporation device controller, since the electrical circuit in it is capable of continuously monitoring the tissue current between the electrodes and comparing this controlled current (or tissue current) with a target current and continuously adjusting the output power to maintain the controlled current at target levels. The feedback loop can be instantaneous because it is analog closed loop.

[0061] The term "decentralized current" as used herein can mean a pattern of electrical currents supplied from the various sets of needle electrodes of the electroporation devices described herein, the circuits minimizing or preferably eliminating the occurrence of thermal stress associated with electroporation in any area of tissue that is electroporated.

[0062] "Electroporation", "electropermeabilization" or "electrokinetic enhancement" ("EP"), used interchangeably herein, refers to the effect of a transmembrane electric field pulse to induce microscopic pathways (pores) in a biomembrane; their presence allows biomolecules such as plasmids and vectors, oligonucleotides, miRNAs, drugs, ions, and water to pass from one side of the cell membrane to the other.

[0063] 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 the 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, which 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, for example an IgE or IgG signal peptide. A coding sequence encoding an N-terminal methionine and/or a signal peptide may be linked to a fragment of a coding sequence.

[0064] 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 than, at least 150 nucleotides or more, at least 200 nucleotides or more, at least 250 nucleotides or more, at least 300 nucleotides or more, at least 350 nucleotides or more, at least 400 nucleotides or more, at least 450 nucleotides or more, at least 500 nucleotides or more, at least 550 nucleotides or more, at least 600 nucleotides or more, at least 650 nucleotides or more, at least 700 nucleotides or more, at least 750 nucleotides or more, at least 800 nucleotides or more, at least 850 nucleotides or more, at least 900 nucleotides or more, at least 950 nucleotides or more, at least 1000 nucleotides or more, at least 1100 nucleotides or more, at least 1200 nucleotides or more, at least 1300 nucleotides or more, at least 1400 nucleotides or more, at least 1500 nucleotides or more, at least 1600 nucleotides or more, at least 1700 nucleotides or more, at least 1800 nucleotides or more, at least 1900 nucleotides or more, or at least 2 000 nucleotides or more of at least one of the nucleic acid sequences listed below.

[0065] "Fragment" or "immunogenic fragment" in relation to polypeptide sequences means a polypeptide capable of eliciting an immune response in a mammal that cross-reacts with an antigen disclosed herein. Fragments may be polypeptide fragments selected from at least one of the various amino acid sequences listed below. The consensus protein fragments may comprise at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the consensus protein, 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, for example, an IgE or IgG signal peptide.

[0066] In some embodiments, consensus protein fragments may comprise at least 20 amino acids or more, at least 30 amino acids or more, at least 40 amino acids or more, at least 50 amino acids or more, at least 60 amino acids or more, at least 70 amino acids or more, at least 80 amino acids or more, at least 90 amino acids or more, at least 100 amino acids or more, at least 110 amino acids or more , at least 120 amino acids or more, at least 130 amino acids or more, at least 140 amino acids or more, at least 150 amino acids or more, at least 160 amino acids or more, at least 170 amino acids or more, at least 180 amino acids or more, at least 200 amino acids or more, at least 220 amino acids or more, at least 240 amino acids or more, at least 260 amino acids or more, at least 280 amino acids or more, at least 300 amino acids or more, at least 320 amino acids or more, at least 360 amino acids or more, at least 380 amino acids or more, at least 400 amino acids or more, at least 420 amino acids or more, at least 440 amino acids or more, at least 460 amino acids or more, at least 480 amino acids or more, at least 500 amino acids or more, at least 520 amino acids or more, at least 540 amino acids or more, at least 560 amino acids or more, at least 580 amino acids or more, at least 600 amino acids or more, at least 620 amino acids or more, at least 640 amino acids or more, or at least 660 amino acids or more of the protein sequence disclosed herein.

[0067] 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. As used herein, 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.

[0068] As used herein, 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 in relation to a single stranded nucleic acid sequence, the term "substantially homologous " as used herein refers to a probe that can hybridize (i.e., is complementary) to a single stranded nucleic acid sequence template under conditions of low stringency.

[0069] The term "identical" or "identity" as used herein in the context of two or more nucleic acid or polypeptide sequences means that the sequences have a certain percentage of residues that are the same in a specified region. The percentage can be calculated by optimally aligning two sequences, comparing the two sequences in a specified region, determining the number of positions where the same residue 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 the percent sequence identity. In cases where the two sequences are of different lengths or the alignment results in one or more staggered ends and the specified area of comparison 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.

[0070] The "impedance" used herein 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.

[0071] The term "immune response" as used herein refers to the activation of the immune system of a host, such as 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.

[0072] The terms "nucleic acid" or "oligonucleotide" or "polynucleotide" as used herein 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 variants of a nucleic acid 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.

[0073] 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.

[0074] "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.

[0075] As used herein, the terms "peptide", "protein", or "polypeptide" may refer to an associated sequence of amino acids and may be natural, synthetic, or a modification or combination of natural and synthetic.

[0076] 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.

[0077] 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 protein residue, 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.

[0078] "Stringent hybridization conditions", as used herein, means the conditions under which a first nucleic acid sequence (eg, a probe) will hybridize to a second nucleic acid sequence (eg, a target), such as in a complex mixture of nucleic acids. Stringent conditions are sequence dependent and will be different in different circumstances. Stringent conditions can be chosen to be approximately 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 concentration of sodium ion (or other salts) is about 0.01-1.0 M at pH 7.0-8.3, and the temperature is at least about 30°C for short probes (for example, about 10-50 nucleotides) and at least about 60°C for long probes (for example, more than about 50 nucleotides). Harsh conditions can also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal can be at least 2-10 times greater than 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.

[0079] The term "subject" as used herein can mean a mammal that needs or requires immunization with the vaccines described herein. The mammal may be a human, chimpanzee, dog, cat, horse, cow, mouse, or rat.

[0080] 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.

[0081] 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 in the region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 180, 270, 360, 450, 540 or more nucleotides or amino acids or, for amino acids, if the first sequence is substantially complementary to the complement of the second sequence.

[0082] The terms "treat", "treating" or "treating" as used herein can 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. Disease suppression 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.

[0083] The term "variant" as used herein in relation to a nucleic acid means (i) 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.

[0084] The term "variant" as used herein 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. 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), as known in the art, usually involves 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 of ±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 Pat. 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.

[0085] 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 may be 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, or 100% identical over the entire length of the gene sequence or fragment thereof. A variant may be an amino acid sequence that is substantially identical throughout the amino acid sequence or fragment thereof. The amino acid sequence may be 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, or 100% identical over the entire length of the amino acid sequence or fragment thereof.

[0086] The term "vector" as used herein means a nucleic acid sequence containing an origin of replication. The vector may be a viral vector, a bacteriophage, a bacterial artificial chromosome, or a yeast artificial chromosome. The vector may be a DNA or RNA vector. The vector may be a self-replicating extrachromosomal vector and is preferably a DNA plasmid. The vector may contain or include one or more heterologous nucleic acid sequences.

Vaccines

[0087] Provided herein are vaccines comprising a synthetic BORIS consensus antigen as described herein, a nucleic acid molecule encoding a synthetic BORIS consensus antigen, a nucleic acid molecule encoding a fragment of a synthetic BORIS consensus antigen, a nucleic acid molecule encoding a variant synthetic BORIS consensus 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.

[0088] In some embodiments, the vaccine comprises a nucleic acid molecule. In some embodiments, the nucleic acid molecule encodes a synthetic BORIS consensus antigen. In some embodiments, the nucleic acid molecule contains a nucleic acid sequence that encodes SEQ ID NO: 2; a nucleic acid sequence that encodes a fragment containing at least 90% of the length of SEQ ID NO 2; a nucleic acid sequence that encodes a protein that is at least 95% identical to SEQ ID NO: 2; 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: 2. 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. In some embodiments, the vaccine contains a synthetic BORIS consensus antigen, where the antigen contains SEQ ID NO: 2; a fragment containing at least 90% of the length of SEQ ID NO 2; an amino acid sequence that is at least 95% identical to SEQ ID NO: 2; or a fragment containing at least 90% of the entire length of the protein, which is at least 95% identical to SEQ ID NO: 2.

[0089] Vaccines can be used to protect against cancer, such as cancer or tumor expressing BORIS. The vaccines can be used to prevent and/or treat a BORIS expressing tumor in a subject in need thereof. Vaccines can induce cellular and/or antibody responses against BORIS and against tumors expressing BORIS.

[0090] In one embodiment, vaccines can be used to protect, prevent and/or treat, or to induce a cellular and/or antibody response against BORIS-expressing ovarian cancer cells, in particular BORIS-expressing ovarian epithelial cells, more specifically BORIS-expressing serous ovarian cancer cells.

[0091] The development of a cancer vaccine as described herein involves the identification of a cancer antigen, such as BORIS, which is not recognized by the immune system and is a tumor associated antigen ("cancer/testicle", "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, several redesign measures can be applied to the cancer antigen, as described below.

[0092] 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 eliciting 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 a cellular, humoral, or both cellular and humoral immune response. In some embodiments, the induced or evoked cellular immune response may include the induction or secretion of interferon-gamma (IFN-γ) and/or tumor necrosis factor alpha (TNF-α) 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 associated with tumor fibroblasts, soluble factors produced by immunosuppressive cells, CTLA-4, PD-1, MDSC, MCP-1, and an immune checkpoint molecule.

[0093] In a specific embodiment, the vaccine may 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.

[0094] 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.

[0095] The vaccine may contain RNA encoding a cancer antigen. The RNA vaccine can be introduced into the cell.

[0096] The vaccine can 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; 5.453364; 5462734; 5470734; 5474935; 5482713; 5591439; 5643579; 5650309; 5698202; 5955088; 6034298; 6042836; 6156319 and 6589529, each of which is incorporated herein by reference.

[0097] 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 cell antigen 4 (CTLA-4), against the 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 can 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 can be included in separate nucleic acid molecules, such as a separate plasmid or vector.

[0098] 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

[0099] 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 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 immune cell proliferation and/or differentiation signaling.

[00100] 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, eg, less than 800 Daltons, organic or inorganic compound that can serve as an enzyme substrate, a ligand (or equivalent) bound to 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.

[00101] 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

[00102] 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, eg, less than 800 Daltons, organic or inorganic compound that can serve as an enzyme substrate, a ligand (or equivalent) bound to 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

[00103] 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.

[00104] 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 by 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

[00105] 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 can be encoded by a nucleic acid sequence contained in

[00106] 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.

[00107] 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.

[00108] A 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.

[00109] 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.

[00110] An antibody may contain a set of complementarity determining regions ("CDRs") of a heavy chain and a light chain, and, accordingly, be placed between a set of scaffolding heavy chain and light chain ("FR"), which provide support for the CDRs and determine the spatial relationship of the CDRs 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 V region: heavy and light chain.

[00111] 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.

[00112] In addition, the proteolytic enzyme papain preferentially cleaves IgG molecules to form multiple fragments, two of which (F(ab) fragments) each contain a covalent heterodimer that 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 Fab light chain may include a VL region and a CL region.

[00113] The antibody may be a polyclonal or monoclonal antibody. The antibody may be a chimeric antibody, a single chain antibody, an affinity matured antibody, a human antibody, a humanized antibody, or a fully human antibody. 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

[00114] 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 PD-1 activity, thereby inducing, eliciting or enhancing an immune response against a tumor or cancer and reducing tumor growth.

Antibody to PD-L1

[00115] 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

[00116] As described above, the vaccine may contain an antigen or a nucleic acid encoding an antigen. The antigen may be BORIS, a fragment thereof, a variant thereof, or a combination of a fragment and a variant thereof.

[00117] Accordingly, the vaccine can be used to treat subjects suffering from cancer or tumors that express BORIS. 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 BORIS to prevent the subjects from developing such tumors. The synthetic BORIS consensus antigen may differ from the native BORIS gene and thus provide therapy or prophylaxis against a tumor expressing the BORIS antigen. Accordingly, provided herein are synthetic BORIS antigen consensus sequences that differ from the native BORIS gene (i.e., mutated or synthetic BORIS genes or sequences).

[00118] Transcripts of the native BORIS gene are processed into a variety of mRNAs. Specific BORIS mRNA isoforms can be selected based on, for example, their expression in cancer cells. In specific embodiments, the BORIS isoform is selected based on its expression in ovarian cancer cells. The synthetic BORIS 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.

[00119] 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 incorporated 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 BORIS consensus antigens. The coding sequences encoding the synthetic BORIS consensus antigens have the sequences as described above.

[00120] Provided are protein molecules comprising the heterologous synthetic consensus amino acid sequences of the BORIS antigen described above. Proposed protein molecules consisting of the heterologous synthetic consensus amino acid sequences of the BORIS antigen described above. Provided herein are proteins and polypeptides having the synthetic BORIS antigen consensus sequences described above. Proteins and polypeptides may be referred to as synthetic BORIS consensus antigens and BORIS immunogens. Consensus synthetic BORIS antigens are capable of eliciting an immune response against BORIS-expressing tumors.

[00121] In one aspect, it is desirable that the synthetic BORIS 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, the elimination of cis-acting sequence motifs (i.e. internal TATA boxes).

[00122] A synthetic BORIS consensus antigen may be a consensus antigen (or immunogen) sequence derived from two or more species. In one embodiment, the consensus sequence is generated from different species of BORIS isoforms. The consensus sequence is derived from BORIS sequences collected from GenBank or other similar DNA or protein sequence database. A synthetic BORIS 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 BORIS consensus antigen. The synthetic BORIS consensus antigen may contain 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 BORIS consensus antigen may contain a hemagglutinin (HA) tag. The BORIS consensus antigen can be engineered to elicit stronger and broader cellular and/or humoral immune responses than the corresponding codon-optimized synthetic BORIS consensus antigen.

[00123] The BORIS consensus sequence can be mutated to destroy and/or enhance certain structures and/or functions of native BORIS to produce a synthetic BORIS antigen consensus sequence. In one embodiment, mutations are introduced into each of the 11 zinc finger domains to disrupt the structure and functionality of the zinc finger. One or more mutations may be a substitution of one or more amino acids that coordinate the zinc ion in one or more zinc fingers. One or more amino acids that coordinate the zinc ion may be a CCHH motif. Accordingly, in some embodiments, one or more mutations may replace 1, 2, 3, or all 4 amino acids of one or more CCHH motifs. In a preferred embodiment, the cysteines in the eleven zinc fingers in BORIS are mutated to glycines to disrupt the structure and binding of the zinc fingers. (See Stoll, R. et al. Structure of the Wilms tumor suppressor protein zinc finger domain bound to DNA. Journal of molecular biology 372, 1227-1245, doi:10.1016/j.jmb.2007.07.017 (2007)).

[00124] The synthetic BORIS consensus antigen may contain mutations or deletions to disrupt, for example, a native localization signal sequence, including, for example, a nuclear localization signal to disrupt a nuclear translocation upon expression. For example, RRRK can be substituted for RKRK to prevent nuclear localization. In a specific embodiment, disruptions occur in each of the 11 zinc finger domains and in the sequence of the nuclear localization signal. Those skilled in the art will appreciate that a recombinant synthetic BORIS consensus antigen having one or more or any combination of the mutations described herein will also have functionality as a non-self antigen for the purposes of this invention, and that each of these options is provided by the present invention.

[00125] In a preferred embodiment, the BORIS synthetic consensus antigen sequence has 95.0% identity with SEQ ID NO:1. In this embodiment, the nucleic acid sequence of SEQ ID NO:1 encodes the amino acid sequence of SEQ ID NO:2. In other embodiments, the BORIS synthetic consensus antigen 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 identity, 96.2% or more identity, 96.4% or more identity, 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 identical, 99.8% or more identical, or 100% identical with SEQ ID NO:1.

Vectors

[00126] The vaccine may contain one or more vectors that include a heterologous nucleic acid encoding a synthetic BORIS consensus antigen. For example, one or more vectors may include a nucleic acid sequence encoding the entire length of the amino acid sequence of SEQ ID NO: 2; a nucleic acid sequence encoding a fragment comprising at least 90% of the entire length of SEQ ID NO: 2; a nucleic acid sequence encoding a protein that is at least 95% identical to SEQ ID NO: 2; or a nucleic acid sequence encoding a fragment comprising at least 90% of the entire length of the protein, which is at least 95% identical to SEQ ID NO:2. One or more vectors may include a nucleic acid sequence encoding amino acids 19-680 of SEQ ID NO: 2; a nucleic acid sequence encoding a fragment comprising at least 90% of the total length of amino acids 19-680 of SEQ ID NO:2; a nucleic acid sequence encoding a protein that is at least 95% identical to amino acids 19-680 of SEQ ID NO:2; 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 amino acids 19-680 of SEQ ID NO:2. One or more vectors may be capable of expressing the synthetic BORIS consensus antigen in an amount effective to elicit an immune response in a mammal. The vector may contain a heterologous nucleic acid encoding a synthetic BORIS consensus 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.

[00127] One or more vectors can be an expression construct, which is usually a plasmid, which is 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.

[00128] 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).

[00129] 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, meaning 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 pGX1440 as described herein, further comprising the nucleic acid sequence of SEQ ID NO:1.

[00130] 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.

[00131] 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 fusion proteins that consist of combinations of consensus proteins and/or fragments of a consensus protein and/or variants of a consensus protein and/or fragments of variants of a consensus dry proteins.

[00132] 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.

[00133] 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.

[00134] The plasmid may further comprise 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.

[00135] The plasmid may also contain a promoter that is operably linked to a coding sequence. The promoter operably linked to the 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 (BIV) long terminal repeat (LTR) promoter, a Moloney virus promoter, an avian leukemia virus (ALV) promoter, a cytomegalovirus (CM) promoter V), such as the CMV immediate-early promoter, the Epstein-Barr virus (EBV) promoter, or the 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.

[00136] 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).

[00137] 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. Amplifications of polynucleotide function are described in US Pat.

[00138] 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 plasmid backbone can be pA V0242. The plasmid may be a replication defective adenovirus type 5 (Ad5) plasmid.

[00139] 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.

[00140] 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.

[00141] The plasmid may be pSE420 (Invitrogen, San Diego, CA), which may 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.

[00142] 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).

[00143] 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.

[00144] 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.

[00145] 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.

[00146] 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.

[00147] 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.

[00148] 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.

[00149] 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.

[00150] 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

[00151] Provided herein are methods for producing vectors that include the DNA vaccines discussed herein. The vectors 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.

[00152] The vector for use with EP devices, which are described in more detail below, may be prepared or manufactured using a combination of known devices and technologies, but preferably they are manufactured using an optimized manufacturing technique as described in pending U.S. Provisional Application Serial No. 60/939792, which was filed May 23, 2007 (see U.S. 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 issued on 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

[00153] 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, poly anions, polycations or nanoparticles or other known means to facilitate transfection.

[00154] 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 the 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. 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.

[00155] The pharmaceutically acceptable excipient may be one or more adjuvants. The adjuvant may be other genes that are expressed in an alternative plasmid or vector or delivered as proteins in combination with the above plasmid or 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), mucosal-associated epithelial 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, IL-18 mutants, CD40, CD40L, vascular growth factor, fibroblast growth factor, IL-7, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Flt, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DRS, 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-I, JNK, interferon response genes, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, Ox40, Ox40 LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAPI, TAP2, IL-15, having a signal sequence or coding sequence that encodes a deleted signal sequence, and optionally 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 may be IL-12, IL-15, IL-28, CTACK, TECK, platelet-derived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermal growth factor (EGF), IL-1, IL-2α, IL-4, IL-5, IL-6, IL-10, IL-12, IL-18, or a combination thereof.

[00156] 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. 08/956865 and US Provisional Application No. 61/569600, filed December 12, 2011, each of which is incorporated herein by reference. 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 PCT Application No. PCT/USI0/048827, each of which is included in this document 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.

[00157] 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, NF kB, 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.

[00158] The vaccine may further comprise a genetic vaccine promoter as described in U.S. Serial No. 021579, filed April 1, 1994, which is incorporated by reference in its entirety.

[00159] The vaccine may contain an antigen-coding vector 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 milligram to about 2 milligrams. 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 approximately 1 milligram to approximately 2 milligrams, from about 5 nanograms to approximately 1000 micrograms, from about 10 nanograms to about 800 micrograms, from approximately 0.1 to approximately 500 micrograms, from about 350 micrograms, about 25 to about 250 micrograms to approximately 200 micrograms of antigen or plasmida, which Digging him.

[00160] 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 stability to the composition at room temperature or ambient temperature for extended periods of time, including LGS or polycations or polyanions.

Pharmaceutical compositions of the vaccine

[00161] 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 micrograms to about 100 micrograms of vaccine nucleic acid molecule(s). In some embodiments, the pharmaceutical compositions contain from about 20 micrograms to about 80 micrograms of vaccine nucleic acid molecule(s). In some embodiments, the pharmaceutical compositions contain from about 25 micrograms 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).

[00162] 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 of a 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, 1 30, 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 37 5, 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, 8 45 850 855 860 865 870 875 880 885 890 895 900 905 910 915 920 925 930 935 940 945 950 955 960 965 970, 975, 980, 985, 990, 995 or 1000 micrograms of vaccine nucleic acid molecule(s). In some embodiments, pharmaceutical compositions may contain at least 1.5; 2; 2.5; 3; 3.5; 4; 4.5; 5; 5.5; 6; 6.5; 7; 7.5; 8; 8.5; 9; 9.5 or 10 mg or more vaccine nucleic acid molecule(s).

[00163] 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 of 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, 1 30, 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 37 5, 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, 8 45 850 855 860 865 870 875 880 885 890 895 900 905 910 915 920 925 930 935 940 945 950 955 960 965 970, 975, 980, 985, 990, 995 or 1000 micrograms of vaccine nucleic acid molecule(s). In some embodiments, pharmaceutical compositions may contain up to and including 1.5; 2; 2.5; 3; 3.5; 4; 4.5; 5; 5.5; 6; 6.5; 7; 7.5; 8; 8.5; 9; 9.5 or 10 mg of vaccine nucleic acid molecule(s).

[00164] The pharmaceutical composition may further contain other agents for formulation purposes in accordance with the route of administration used. Where the pharmaceutical compositions are injectable pharmaceutical compositions, they are sterile, 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.

[00165] 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

[00166] Vaccines and pharmaceutical compositions containing the vaccines provided herein can be used to treat or prevent cancer cells and cancers expressing BORIS. 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

[00167] 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 the 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 by transfection of cancer antigens, as described herein, in the form of a nucleic acid molecule that is expressed in the cell and delivered to the cell surface, after which the immune system recognizes and induces a cellular, humoral or cellular and humoral response. The administration of a vaccine can be used to induce or elicit an immune response in a subject against one or more cancer antigens as described herein by administering the vaccine to the subject as described herein.

[00168] 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 through a humoral, cellular, or both cellular and humoral immune response.

[00169] 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.

[00170] A vaccine may be administered to a mammal to elicit an immune response in the mammal. The mammal can be a human, non-human primate, cow, pig, sheep, goat, antelope, bison, water buffalo, bull, deer, hedgehog, elephant, llama, alpaca, mouse, rat, and preferably human, cow, or pig. The vaccine can also be administered to a non-mammalian subject, such as a chicken, to elicit an immune response.

[00171] 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 day. 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

[00172] 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.

[00173] 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 can be routinely performed prior to vaccine administration.

Ways to treat cancer with a vaccine

[00174] The vaccine can be used to generate or induce an immune response in a mammal that is reactive or directed against a BORIS-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.

[00175] The elicited immune response can prevent and/or reduce the metastasis of cancer or tumor cells. Accordingly, the vaccine can 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

[00176] The vaccine or pharmaceutical composition may be administered by various routes, including oral, parenteral, sublingual, transdermal, rectal, transmucosal, topical, inhaled, buccal, intrapleural, intravenous, intraarterial, intraperitoneal, subcutaneous, intramuscular, intranasal, intrathecal and/or intraarticular, 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.

[00177] 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 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.

[00178] 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 an incoming current specified 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 plasmid.

[00179] 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. Other electroporation devices and electroporation methods that can be used to facilitate the administration of DNA vaccines include those disclosed in co-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 U.S. Ser. No. 60/852149, filed October 17, 2006, and 60/978982, filed October 10, 2007, all of which are incorporated herein in their entirety.

[00180] 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.

[00181] US Patent Publication 2005/0052630 submitted by Smith et al. describes an electroporation device that can be used to efficiently ensure 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. The entire contents of US Patent Publication 2005/0052630 are incorporated herein by reference in their entirety.

[00182] The electrode arrays and methods described in US Patent 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 array of electrodes, 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 21 mm wide.

[00183] In addition, in some embodiments that include electroporation devices and their use, there are electroporation devices that are described in the following patents: U.S. Patent 5,273,525 issued December 28, 1993; U.S. Patents 6,110,161 issued August 29, 2000; and US Pat. No. 6,958,060, issued Oct. 25, 2005, and US Pat. 64, issued February 5, 2008, which relates to a method for introducing DNA. The above patents are incorporated by reference in their entirety.

Ways to get a vaccine

[00184] Provided herein are methods for producing vectors that contain nucleic acid molecule(s) encoding the synthetic BORIS consensus antigen discussed herein. DNA vectors after the final step of subcloning into a mammalian expression plasmid can be used to inoculate a cell culture in a large scale fermentation tank using methods known in the art.

[00185] DNA vectors for use with the EP devices of the present invention can be designed or manufactured using a combination of known devices and technologies, but are preferably manufactured using the optimized plasmid production technology as described in U.S. Published Application No. 20090004716, filed May 23, 2007. 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 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.

[00186] The present invention has many aspects, illustrated by the following non-limiting examples.

Examples

[00187] The present invention is further illustrated in the following examples. It should be understood that these examples, while indicating 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 Synthetic BORIS Consensus Antigen

[00188] To obtain the BORIS human consensus, 7 BORIS sequences were collected from GenBank (https://www.ncbi.nlm.nih.gov/genbank/). GenBank access numbers for selected BORIS subfamily 1 sequences: NP_542185.2, XP_009435727.1, XP_004062465.1, XP_002830505.1, XP_003806212.1, XP_011833550.1 and XP_003253023.1.

[00189] The consensus sequence was generated using the DNASTAR® Lasergene software package (version 13.0.0.357). The seven sequences listed above were imported into MegAlign and aligned using the ClustalW multiple sequence alignment program. The resulting BORIS consensus sequence has 98.6% identity with native human BORIS.

[00190] To eliminate the potential biological function of the BORIS consensus sequence, 22 mutations (2 mutations in each of the 11 zinc fingers) were introduced to disrupt the zinc finger structure and DNA binding potential. In addition, since BORIS is a transcription factor, one mutation was introduced to prevent nuclear localization. The rationale for introducing these mutations is described below.

Zinc finger mutations

[00191] CTCF and CTCFL (BORIS) are paralogous genes that have the same exons encoding the 11 zinc finger domain (and the same DNA binding potential). The cysteines in eleven zinc fingers in the BORIS consensus were mutated into glycines to disrupt the structure and binding of the zinc fingers.

Nuclear localization signal (NLS) mutations

[00192] Since BORIS is a transcription factor, the predicted nuclear localization signal was identified using the NucPred program of the Stockholm Bioinformatics Center (www.sbc.su.se/~maccallr/nucpred/cgi-bin/single.cgi). The predicted nuclear localization signal in BORIS is of class 1 NLS type with four consecutive basic amino acids. To prevent nuclear localization, the nuclear localization signal was mutated from RRRK to RRRK.

[00193] After obtaining the BORIS consensus sequence and subsequent mutations in the zinc finger and NLS DNA sequences, the resulting synthetic BORIS antigen consensus protein sequence has 95.2% identity with the human native isoform of the BORIS protein "sf1" (i.e., NP_542185.2).

[00194] Once a synthetic BORIS antigen DNA consensus sequence 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. 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, 10991-1 1001, 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. (Muthumani, K. et al. Novel engineered HIV-1 East African Clade-A gp160 plasmid construct induces strong humoral and cell-mediated immune responses in vivo. Virology 314, 134-146 (2003); Schneider, R., Campbell, M., Nasioulas, G., Felber, BK & Pavlakis, GN Inactivation of the human immunodeficiency virus type 1 inhibitory elements allows Rev-independent expression of Gag and Gag/protease and particle formation Journal of virology 71, 4892-4903 (1997)). The BORIS synthetic consensus antigen DNA sequence 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 pGX1440 and full length sequencing was performed and then analyzed and confirmed to be correct. A schematic representation of the DNA construct of the synthetic BORIS consensus antigen is shown in FIG. The nucleotide and amino acid sequences of the synthetic BORIS consensus antigen of the invention are shown in SEQ ID NO:1 and SEQ ID NO:2, respectively. The characteristics of the DNA sequences and protein sequences of the synthetic BORIS consensus antigen are shown in Table 2 below.

Table 1.

Features SEQ ID NO:2 Peculiarity Amino acid position IgE leader sequence 1-18 BORIS coding sequence 19-680 Mutations that disrupt zinc finger binding C276G, C279G, C304G, C307G
C332G, C335G, C361G, C364G
C389G, C392G, C417G, C420G
C447G, C450G, C477G, C480G
C505G, C508G, C533G, C536G
C565G, C568G Mutation that violates GPI attachment K603R

Table 2. Characteristics of the synthetic BORIS consensus antigen

Characteristics Synthetic BORIS consensus antigen (SEQ ID NO:2) Identity to native human BORIS 95.2% Identity to native BORIS rhesus monkey 81.5-91.8% Identity to native BORIS mouse 56.5-57.4% Number of amino acid mutations (compared to native human) 32 Number of built-in mutations (non-consensus origin) 23 Molecular mass 680 aa (75 kDa) Coding sequence length (bp) 2046

Example 2 - Construction of BORIS expression vectors pGX1440.

[00195] 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.

[00196] 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.

base pair modification

Description:

C>G 241 in CMV promoter

C>T 1158 main chain, to the right of bovine growth hormone

polyadenylation signal (bGH polyA)

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

[00197] pGX1440 is a DNA plasmid encoding the BORIS synthetic consensus antigen protein. 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. pGX1440 was generated by cloning the BORIS synthetic consensus antigen DNA sequence into pGX0001 at the BamHI and XhoI sites as shown in FIG. 4.

Example 3 - Immunogenicity of BORIS Synthetic Consensus Antigen Constructs

[00198] The immunogenicity of a vaccine construct designed to target human BORIS, the synthetic BORIS consensus antigen (pGX1440), was evaluated in mice. Construct antigenic protein expression was also assessed in vitro by Western blotting.

Materials and methods

Plasmids

[00199] For in vitro and in vivo studies, a plasmid (10 mg) was ordered from GenScript for pGX1440 (lot # U0638BC040S-3/G61425). The antigen sequence from the 10 mg plasmid starting material was confirmed by Sanger sequencing and validated for accuracy.

Expression of the antigen in vitro

[00200] Protein expression of the pGX1440 antigen was confirmed by Western blotting. Human rhabdomyosarcoma (RD) cells (ATCC, CCL-136) maintained in DMEM with 10% FBS (ThermoFisher) were transfected with pGX1440 or pGX0001 (6 μg/10 cm ² plate) using Turbofectin 8 (Origene). 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 in 4-12% SDS-PAGE gel (ThermoFisher) and detected with anti-BORIS monoclonal antibody (CTCFL) (AbCam, clone 126778), then visualized with horseradish peroxidase (HRP) conjugated anti-mouse IgG analysis (Santa Cruz Biotech, sc-2005) using an ECL western blot system (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).

Animals and immunizations

[00201] 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 five groups as shown in Table 3.

Table 3. Groups of studies

Group n Design Construct Dose (mcg) Injection volume (µl) 1 4 pGX0001 thirty thirty 2 8 pGX1440 10 thirty 3 8 pGX1440 20 thirty 4 8 pGX1440 thirty thirty 5 8 pGX1440 50 thirty

[00202] Mice in the immunized groups were vaccinated with the indicated doses of pGX0001 or pGX1440. 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 a CELLECTRA® 2000 adaptive DC electroporation device with a 3P grid (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 cellular immune counts. No other tissue was collected.

Isolation of spleen lymphocytes

[00203] 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

[00204] Mouse IFNγ ELISpot assay (MabTech) was performed to evaluate antigen-specific cellular responses. Briefly, 96-well plates pre-coated with anti-mouse IFNγ antibody were washed in PBS and blocked for 2 hours at room temperature with complete culture medium (RPMI 1640 supplemented with 10% FBS and antibiotics). Лимфоциты селезенки ресуспендировали в среде R10 (а затем добавляли в трех экземплярах при количестве введенных клеток 2×10 ⁵ клеток на лунку. Был синтезирован набор пептидов (GenScript), каждый из которых содержал 15 аминокислотных остатков, перекрывающихся 11 аминокислотами, представляющими полную белковую последовательность синтетического консенсусного антигена BORIS. Эти наборы пептидов ресуспендировали в ДМСО (Sigma) и объединяли в концентрации пептида ~2 мкг/мл в три пептидных пула (P1, P2 и P3 на фиг. 7B). Пептидный пул содержал пептиды, соответствующие синтетическому консенсусному антигену BORIS. Конкавалин А (Sigma) в концентрации 5 мкг/мл использовали в качестве положительного контроля, а полную культуральную среду использовали в качестве отрицательного контроля. Планшеты инкубировали в течение 18 часов при 37°С в инкубаторе с 5% CO ₂ . Затем добавляли биотинилированное анти-мышиное антитело для определения IFNγ (MabTech) и планшеты инкубировали в течение 2 часов при комнатной температуре. The plates were washed and anti-streptavidin-ALP antibody (MabTech) was added and the plates were incubated for 1 hour at room temperature. Spot detection was performed 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 1×10 ⁶ splenocytes for display purposes.

[00205] Antigen-specific IFNγ ELISpot responses are presented as the number of IFNγ spotting units (SFU) per 1×10 ⁶ splenocytes, greater than the SFU in the medium-only control.

flow cytometry

[00206] Cellular immune responses induced by the synthetic BORIS consensus antigen were further characterized by flow cytometry. Briefly, 2×10 ⁶ splenocytes from vaccinated and naive mice were immediately stimulated after peptide isolation of the synthetic BORIS consensus antigen for 6 hours in the presence of Brefeldin A (BD Biosciences), Monensin (BD Biosciences) and FITC against mouse anti-CD107a antibody (BD Biosciences). 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, Thermo-Fisher) and the following extracellular antibodies: APC-Cy7 anti-mouse CD3e, PerCP-Cy5.5 anti-mouse CD4, and APC-anti-mouse CD8a (BD Biosciences). Intracellular cytokines were then stained with the following antibodies: BV605 anti-mouse IFNγ, APC-R700 anti-mouse IL-2, and PE anti-mouse TNF-α (BD Biosciences). ICS data were collected on 10-color FACS CANTO (BD Biosciences) and analysis completed using FlowJo. The flow cytometry gating strategy is shown in FIG. 5.

[00207] 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

[00208] 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 the entire statistical analysis, a p value of 0.050 was considered significant.

results

Expression of the synthetic BORIS consensus antigen

[00209] Expression of the synthetic BORIS consensus antigen pGX1440 was confirmed by Western blotting. Briefly, human rhabdomyosarcoma (RD) cells were transfected with plasmids pGX1440 or pGX0001 (empty vector, negative control). Cell lysates were tested for expression of the synthetic anti-human BORIS consensus antigen BORIS (CTCFL). A protein band of the expected molecular weight for the synthetic BORIS consensus antigen (76.75 kDa) was found (FIG. 6). A weak band was found in the negative control (pGX0001), which is most likely due to low expression of the endogenous BORIS 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, pGX1440 was found to express its corresponding antigenic protein.

Immunogenicity of BORIS Synthetic Consensus Antigen Vaccine Constructs

IFNγ ELISpot

[00210] The immunogenicity of the BORIS synthetic consensus antigen construct was assessed at four doses (10 μg, 20 μg, 30 μg, and 50 μg) by IFNγ ELISpot and flow cytometry (n=8/group). Mice were immunized with an empty plasmid backbone (pGX0001) as a negative control (n=4/group). Vaccination with the synthetic BORIS consensus antigen (pGX1440) elicited exceptionally robust cellular immune responses compared to negative control vaccinated mice. The amount of IFNγ production specific for the BORIS synthetic consensus antigen, as determined by ELISpot, was dose-independent (Fig. 7A and Fig. 7B) with a similar maximum response achieved at both 20 and 50 μg dose. In particular, the IFNγ SFU value specific for the BORIS synthetic consensus antigen was 10315±4093, 13725±6151, 8645±2304 and 13600±9894 at 10 µg, 20 µg, 30 µg and 50 µg, respectively. IFNγ responses to the BORIS synthetic consensus antigen were significantly greater than naive at 10 µg (p=0.026), 20 µg (p=0.002), and 50 µg (p=0.003) pGX1440 doses, but not at 30 µg (p=0.071). IFNγ responses are summarized in Table 4.

Table 4. IFNγ responses induced by the BORIS synthetic consensus antigen

Synthetic BORIS consensus antigen (pGX1440) Design Dose Mean SFU ± St. Off p-value pGX0001 30 mcg 50±24 n/a pGX1440 10 mcg 10 315 ± 4 093 0.026 20 mcg 13 725 ± 6 151 0.002 30 mcg 8645±2304 0.071 50 mcg 13600 ± 9894 0.003 Statistical significance was taken at p≤0.05. The reported p-values are close to naive (pGX0001 immunized mice).

flow cytometry

[00211] The BORIS synthetic consensus antigen elicited more robust responses in the CD8 ⁺ T cell compartment compared to those in the CD4 ⁺ T cell compartment (FIGS. 8A, 8B, 8C and 8D). The synthetic BORIS consensus antigen induced frequencies of antigen-specific CD4 ⁺ T cell responses that were significantly more robust than naive (0.11% ± 0.06%) at 10 μg (1.41% ± 0.44%) (p < 0.001), 20 μg (1.36% ± 0.42%) (p < 0.001), 30 μg (1. 50% ± 0.22%) (p < 0.001) and 50 μg (1.62% ± 0.66%) (p < 0.001) in dosing groups (Fig. 8A). CD4 ⁺ T cell responses specific to the BORIS synthetic consensus antigen were also dose-independent and consisted mainly of IFNγ ⁺ IL-2 ⁺ TNFα ⁺ , IFNγ ⁺ IL-2 ^- TNFα ⁺ or IFNγ ⁺ IL-2 ^- TNFα ^- -producing CD4 ⁺ T cells (Fig. 8C). The frequency of antigen-specific CD4 ⁺ T cells is described in more detail in the table 5.

Table 5 CD4 ⁺ T cell responses induced by synthetic BORIS consensus antigen

CD4 ⁺ T cells of synthetic BORIS consensus antigen Design Dose %CD4 ⁺ ±
Art. off p-value pGX0001 30 mcg 0.11 ± 0.06 n/a pGX1440 10 mcg 1.41 ± 0.44 <0.001 20 mcg 1.36 ± 0.42 <0.001 30 mcg 1.50±0.22 <0.001 50 mcg 1.62±0.66 <0.001 Statistical significance was taken at p≤0.05. The reported p-values are close to naive (pGX0001 immunized mice).

[00212] The frequency of antigen-specific CD8 ⁺ T cells induced by the BORIS synthetic consensus antigen significantly increased compared to controls in all dose groups (FIG. 8B). In particular, the frequency of antigen-specific CD8 ⁺ T responses in groups immunized with 10 µg (12.45% ± 3.86%) (p = 0.002), 20 µg (15.64% ± 5.63%) (p < 0.001), 30 µg (14.49% ± 3.58%) (p < 0.001) and 50 µg (17, 34 ± 8.17%) pGX1440 was significantly more resistant than naive (0.10% ± 0.05%). CD8+ T cell responses specific to the BORIS synthetic consensus antigen were also dose-independent and consisted mainly of IFNγ ⁺ IL-2 ^- TNFα ^- and IFNγ ⁺ IL-2 ^- TNFα ⁺ -producing CD8 ⁺ T cells (Fig. 8D). The frequency of antigen-specific CD8 ⁺ T cells is described in more detail in Table 6.

Table 6 CD8 ⁺ T cell responses induced by synthetic BORIS consensus antigen

CD8 ⁺ T cells of synthetic BORIS consensus antigen Design Dose %CD8 ⁺
± Art. off p-value pGX0001 30 mcg 0.10±0.05 n/a pGX1440 10 mcg 12.45 ± 3.86 0.002 20 mcg 15.64±5.63 <0.001 30 mcg 14.49 ± 3.58 <0.001 50 mcg 17.34 ± 8.17 <0.001 Statistical significance was taken at p≤0.05. Reported p-values are close to naive (pGX0001 immunized mice)

[00213] All doses of synthetic BORIS consensus antigen induced CD4 rates⁺ CD107a⁺ T cells, which was higher than naive (0.08% ± 0.07%), but only the higher doses of 30 µg and 50 µg were significantly more resistant. In particular, the frequency of antigen-specific CD4⁺ CD107a⁺ T cell counts were 0.37% ± 0.23%, 0.30% ± 0.15%, 0.49% ± 0.20%, and 0.50% ± 0.30% at 10 μg (p=0.097), 20 μg (p=0.256), 30 μg (p=0.012), and 50 μg (p=0.010). ) dose groups, respectively (FIG. 9A). CD4 cytokine profile⁺ CD107a⁺ T cells specific for the BORIS synthetic consensus antigen were similar across dose groups and consisted mainly of IFNγ⁺IL-2⁺TNFα⁺, IFNγ⁺IL-2^-TNFα⁺, IFNγ⁺IL-2^-TNFα^- cells (Fig. 9C). Frequency of antigen-specific CD4⁺ T cells with cytolytic potential are described in more detail in Table 7.

Table 7. Cytolytic potential of antigen-specific CD4 ⁺ T cells induced by synthetic BORIS consensus antigen

CD4 ⁺ CD107a ⁺ T cells of the synthetic BORIS consensus antigen Design Dose %CD4 ⁺ CD107a ⁺ ± St. off p-value pGX0001 30 mcg 0.08 ± 0.07 n/a pGX1440 10 mcg 0.37 ± 0.23 0.097 20 mcg 0.30±0.15 0.256 30 mcg 0.49±0.20 0.012 50 mcg 0.50±0.30 0.010 Statistical significance was taken at p≤0.05. Reported p-values are close to naive (pGX0001 immunized mice)

[00214] Similar to the magnitude of antigen-specific CD8 ⁺ T cells, the synthetic BORIS consensus antigen induced a significant change in the frequency of CD8 ⁺ CD107a ⁺ T cells in all groups compared to naive (0.02% ± 0.01%) (Fig. 9C). In particular, the frequency of antigen-specific CD8 ⁺ CD107a ⁺ T cells was 11.52% ± 3.50%, 14.49% ± 5.22%, 13.57% ± 3.45% and 16.24% ± 7.74% at 10 µg (p=0.002), 20 µg (p<0.001), 30 µg ( p < 0.001) and 50 μg (p < 0.001) dose groups, respectively (Fig. 9B). The cytokine profile of CD8 ⁺ CD107a ⁺ T cells specific for the BORIS synthetic consensus antigen was similar across dose groups and consisted mainly of IFNγ ⁺ IL-2 ^- TNFα ^- with some IFNγ ⁺ IL-2 ^- TNFα ⁺ cells (Fig. 9D). The frequency of antigen-specific CD8 ⁺ T cells with cytolytic potential is described in more detail in Table 8.

Table 8. Cytolytic potential of antigen-specific CD8 ⁺ T cells induced by synthetic BORIS consensus antigen

CD8 ⁺ CD107a ⁺ T cells of the synthetic BORIS consensus antigen Design Dose %CD8 ⁺ CD107a ⁺ ± St. off p-value pGX0001 30 mcg 0.02±0.01 n/a pGX1440 10 mcg 11.52 ± 3.50 0.002 20 mcg 14.49±5.22 <0.001 30 mcg 13.57 ± 3.45 <0.001 50 mcg 16.24 ± 7.74 <0.001 Statistical significance was taken at p≤0.05. The reported p-values are close to naive (pGX0001 immunized mice).

Overall, there were no significant differences in responses between immunized groups for any data presented (i.e., 10 µg was not significantly lower than 50 µg, etc.). The synthetic BORIS consensus antigen significantly increased the frequency of antigen-specific CD4 ⁺ , CD4 ⁺ CD107a ⁺ and CD8 ⁺ , CD8 ⁺ CD107a ⁺ T cells compared to naive, although the magnitude of the response was much more robust in the CD8 ⁺ T cell compartment.

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SEQUENCE LIST

<110> INOVIO PHARMACEUTICALS, INC.

YAN, Jian

SLAGER, Anna

GARMAN, Bradley

Cook, Neil

<120> CANCER VACCINES TARGETING BORIS AND THEIR USES

<130> 104409.000447 / INO-1001 WO

<150> US 62/598,274

<151> 2017-12-13

<160> 2

<170> Patent Version 3.5

<210> 1

<211> 2046

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic consensus BORIS (CTCFL)

pGX1440 DNA coding sequence

<400> 1

atggattgga cttggattct gttcctggtc gcagcagcaa ctagagtgca ttccgcagcc 60

accgagattt ccgtcctgag tgagcagttc accaagatca aggagctgga gctgatgccc 120

gagaagggcc tgaaggagga ggagaaggac ggcgtgtgca gagagaagga tcacaggtcc 180

ccttctgagc tggaggccga gagaacaagc ggagcattcc aggactccgt gctggaggag 240

gaggtggagc tggtgctggc accatctgag gagagcgaga agcacatcct gacactgcag 300

accgtgcact ttacctctga ggccgtggag ctgcaggata tgtccctgct gtctatccag 360

cagcaggagg gagtgcaggt ggtggtgcag cagccaggcc ctggcctgct gtggctggag 420

gagggaccta ggcagtccct gcagcagtat gtggccatct ctatccagca ggagctgtac 480

agcctgcagg agatggaggt gctgcagttt cacgccctgg aggagaatgt gatggtggcc 540

agcgaggact ccaagctggc cgtgagcctg gcagagacag caggcctgat caagctggag 600

gagggccagg agaagaacca gctgctggcc gagcgcacaa aggagcagct gttctttgtg 660

gagacaatgt ctggcgacga gcggagcgat gagatcgtgc tgacagtgag caactccaat 720

gtggaggagc aggaggacca gccaaccgca ggacaggccg atgccgagaa ggccaagtcc 780

acaaagaatc agagaaagac caagggcgcc aagaggacat tccacggcga cgtgggcatg 840

tttacaagct cccgcatgtc tagcttcaac cggcacatga agacccacac aaatgagaag 900

ccacacctgg gccacctggg cctgaagacc tttagaaccg tgacactgct gaggaaccac 960

gtgaataccc acacaggcac cagaccctat aagggcaacg atggcaatat ggccttcgtg 1020

acaagcggcg agctggtgag gcaccggaga tataagcaca cccacgagaa gccttttaag 1080

ggctccatgg gcaagtacgc cagcgtggag gcctccaagc tgaagaggca cgtgcggagc 1140

cacaccggag agcggccctt ccagggctgt cagggctctt acgccagcag ggacacatat 1200

aagctgaaga gacacatgag gacccactct ggcgagaagc cctatgaggg ccacatcggc 1260

cacacacgct ttacccagag cggcacaatg aagatccaca tcctgcagaa gcacggcgag 1320

aatgtgccaa agtaccaggg accacacggga gcaaccatca tcgcacggaa gtccgatctg 1380

cgcgtgcaca tgaggaacct gcacgcatac agcgccgcag agctgaaggg cagatatggc 1440

tccgccgtgt tccacgagag gtacgccctg atccagcacc agaagacaca caagaacgag 1500

aagcggttca agggcaagca cggcagctac gcctgcaagc aggagcgcca catgacagcc 1560

cacatccggga cacacaccgg cgagaagcct ttcaccggcc tgtccggcaa caagtgtttt 1620

1680

accgtgtaca agggctccaa gggcggcaag ggcttctctc gctggatcaa tctgcaccgg 1740

cactccgaga agtgcggctc tggagaggca aagtccgccg catctggcaa gggcaggcgc 1800

acccggagaa ggaagcagac aatcctgaag gaggcaacca agggacagaa ggaggcagca 1860

aagggatgga aggaggcagc aaacggcgat gaggcagcag ccgaggaggc cagcaccaca 1920

aagggcgagc agttccctgg cgagatgttt ccagtggcct gtggcgagac aacagccaga 1980

gtgaaggaag aagtggatga aggggtgacc tgtgagatgc tgctgaacat gatggacaaa 2040

tgataa 2046

<210> 2

<211> 680

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic consensus BORIS (CTCFL)

pGX1440 protein sequence

<400> 2

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

1 5 10 15

His Ser Ala Ala Thr Glu Ile Ser Val Leu Ser Glu Gln Phe Thr Lys

20 25 30

Ile Lys Glu Leu Glu Leu Met Pro Glu Lys Gly Leu Lys Glu Glu Glu

35 40 45

Lys Asp Gly Val Cys Arg Glu Lys Asp His Arg Ser Pro Ser Glu Leu

50 55 60

Glu Ala Glu Arg Thr Ser Gly Ala Phe Gln Asp Ser Val Leu Glu Glu

65 70 75 80

Glu Val Glu Leu Val Leu Ala Pro Ser Glu Glu Ser Glu Lys His Ile

85 90 95

Leu Thr Leu Gln Thr Val His Phe Thr Ser Glu Ala Val Glu Leu Gln

100 105 110

Asp Met Ser Leu Leu Ser Ile Gln Gln Gln Glu Gly Val Gln Val Val

115 120 125

Val Gln Gln Pro Gly Pro Gly Leu Leu Trp Leu Glu Glu Gly Pro Arg

130 135 140

Gln Ser Leu Gln Gln Tyr Val Ala Ile Ser Ile Gln Gln Glu Leu Tyr

145 150 155 160

Ser Leu Gln Glu Met Glu Val Leu Gln Phe His Ala Leu Glu Glu Asn

165 170 175

Val Met Val Ala Ser Glu Asp Ser Lys Leu Ala Val Ser Leu Ala Glu

180 185 190

Thr Ala Gly Leu Ile Lys Leu Glu Glu Gly Gln Glu Lys Asn Gln Leu

195 200 205

Leu Ala Glu Arg Thr Lys Glu Gln Leu Phe Phe Val Glu Thr Met Ser

210 215 220

Gly Asp Glu Arg Ser Asp Glu Ile Val Leu Thr Val Ser Asn Ser Asn

225 230 235 240

Val Glu Glu Gln Glu Asp Gln Pro Thr Ala Gly Gln Ala Asp Ala Glu

245 250 255

Lys Ala Lys Ser Thr Lys Asn Gln Arg Lys Thr Lys Gly Ala Lys Arg

260 265 270

Thr Phe His Gly Asp Val Gly Met Phe Thr Ser Ser Arg Met Ser Ser

275 280 285

Phe Asn Arg His Met Lys Thr His Thr Asn Glu Lys Pro His Leu Gly

290 295 300

His Leu Gly Leu Lys Thr Phe Arg Thr Val Thr Leu Leu Arg Asn His

305 310 315 320

Val Asn Thr His Thr Gly Thr Arg Pro Tyr Lys Gly Asn Asp Gly Asn

325 330 335

Met Ala Phe Val Thr Ser Gly Glu Leu Val Arg His Arg Arg Tyr Lys

340 345 350

His Thr His Glu Lys Pro Phe Lys Gly Ser Met Gly Lys Tyr Ala Ser

355 360 365

Val Glu Ala Ser Lys Leu Lys Arg His Val Arg Ser His Thr Gly Glu

370 375 380

Arg Pro Phe Gln Gly Cys Gln Gly Ser Tyr Ala Ser Arg Asp Thr Tyr

385 390 395 400

Lys Leu Lys Arg His Met Arg Thr His Ser Gly Glu Lys Pro Tyr Glu

405 410 415

Gly His Ile Gly His Thr Arg Phe Thr Gln Ser Gly Thr Met Lys Ile

420 425 430

His Ile Leu Gln Lys His Gly Glu Asn Val Pro Lys Tyr Gln Gly Pro

435 440 445

His Gly Ala Thr Ile Ile Ala Arg Lys Ser Asp Leu Arg Val His Met

450 455 460

Arg Asn Leu His Ala Tyr Ser Ala Ala Glu Leu Lys Gly Arg Tyr Gly

465 470 475 480

Ser Ala Val Phe His Glu Arg Tyr Ala Leu Ile Gln His Gln Lys Thr

485 490 495

His Lys Asn Glu Lys Arg Phe Lys Gly Lys His Gly Ser Tyr Ala Cys

500 505 510

Lys Gln Glu Arg His Met Thr Ala His Ile Arg Thr His Thr Gly Glu

515 520 525

Lys Pro Phe Thr Gly Leu Ser Gly Asn Lys Cys Phe Arg Gln Lys Gln

530 535 540

Leu Leu Asn Ala His Phe Arg Lys Tyr His Asp Ala Asn Phe Ile Pro

545 550 555 560

Thr Val Tyr Lys Gly Ser Lys Gly Gly Lys Gly Phe Ser Arg Trp Ile

565 570 575

Asn Leu His Arg His Ser Glu Lys Cys Gly Ser Gly Glu Ala Lys Ser

580 585 590

Ala Ala Ser Gly Lys Gly Arg Arg Thr Arg Arg Arg Lys Gln Thr Ile

595 600 605

Leu Lys Glu Ala Thr Lys Gly Gln Lys Glu Ala Ala Lys Gly Trp Lys

610 615 620

Glu Ala Ala Asn Gly Asp Glu Ala Ala Ala Glu Glu Ala Ser Thr Thr

625 630 635 640

Lys Gly Glu Gln Phe Pro Gly Glu Met Phe Pro Val Ala Cys Gly Glu

645 650 655

Thr Thr Ala Arg Val Lys Glu Glu Val Asp Glu Gly Val Thr Cys Glu

660 665 670

Met Leu Leu Asn Met Met Asp Lys

675 680

<---

Claims (17)

1. A nucleic acid molecule that encodes an imprinted site regulator antigen brother (BORIS) containing an amino acid sequence containing mutations C276G, C279G, C304G, C307G, C332G, C335G, C361G, C364G, C389G, C392G, C417G, C420G, C447G, C4 50G, C477G, C480G, C505G, C508G, C533G, C536G, C565G, C568G, K603R and having at least 95% amino acid sequence identity as shown in amino acids 19-680 of SEQ ID NO: 2. 2. Use of the nucleic acid molecule according to claim 1 for the treatment of a cancer expressing an imprinted site regulator brother (BORIS). 3. Use of the nucleic acid molecule according to claim 1 for the preparation of a medicament for the treatment of a cancer expressing an imprinted site regulator brother (BORIS). 4. Expression vector containing the nucleic acid molecule according to claim 1. 5. An expression vector according to claim 4, wherein said expression vector is a plasmid. 6. Antigen-brother of the imprinted site regulator (BORIS), containing an amino acid sequence containing mutations C276G, C279G, C304G, C307G, C332G, C335G, C361G, C364G, C389G, C392G, C417G, C420G, C447G, C450G, C477G, C4 80G, C505G, C508G, C533G, C536G, C565G, C568G, K603R and having at least 95% amino acid sequence identity as shown in amino acids 19-680 of SEQ ID NO: 2. 7. A vaccine for the treatment and/or prevention of a malignant tumor expressing the imprinted site regulator sibling antigen (BORIS), containing an effective amount of the nucleic acid molecule according to claim 1. 8. A vaccine for the treatment and/or prevention of cancer expressing the imprinted site regulator sibling antigen (BORIS) containing an effective amount of the vector according to claim 4 or 5. 9. The vaccine according to claim 7 or 8, additionally containing a pharmaceutically acceptable excipient. 10. The vaccine according to claim 7 or 8, additionally containing an adjuvant. 11. The vaccine of claim 10 wherein the adjuvant is IL-12, IL-15, IL-28, or RANTES. 12. A method of treating a subject with an imprinted site regulator antigen-brother (BORIS)-expressing cancer cell, comprising administering a therapeutically effective amount of the vaccine according to any one of paragraphs. 7-11. 13. The method of claim 12, wherein the administration comprises electroporation. 14. The method of claim 12, wherein the administration occurs at one or more sites in the subject. 15. A method of vaccinating a subject against an imprinted site regulator antigen-brother (BORIS) expressing a cancer cell, comprising administering an amount of the vaccine according to any one of paragraphs. 7-11 effective for inducing a humoral or cellular immune response. 16. The method of claim 15, wherein the administration comprises electroporation. 17. The method of claim 16, wherein the administration occurs at one or more sites in the subject.