Cancer vaccine targeting mesothelin and use thereof

Abstract

To provide nucleic acid molecules comprising one or more nucleic acid sequences that encode a modified consensus mesothelin antigen; to provide vectors, compositions, and vaccines comprising one or more nucleic acid sequences that encode a modified consensus mesothelin antigen; and further, to provide methods of treating a subject with a mesothelin-expressing tumor and methods of preventing a mesothelin-expressing tumor.SOLUTION: Nucleic acid molecules contain one or more nucleic acid sequences selected from the group consisting of (a) a nucleic acid sequence that encodes amino acids 19-607 of a specific sequence, (b) a nucleic acid sequence that encodes a fragment containing at least 90% of the full length of a protein containing the amino acids 19-607 of the specific sequence, (c) a nucleic acid sequence that encodes a protein that is at least 96% identical to the amino acids 19-607 of the specific sequence, and (d) a nucleic acid sequence that encodes a fragment containing at least 90% of the full length of a protein that is at least 96% identical to the amino acids 19-607 of the specific sequence.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 62 / 598,289, filed December 13, 2017, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on December 11, 2018, is named 104409_000444_sequence_listing.txt, and is 8,223 bytes in size.

[0003] The present invention relates to mesothelin antigens and nucleic acid molecules encoding same. The present invention also relates to vaccines comprising the mesothelin antigens and / or nucleic acid molecules. The present invention further relates to methods of using the vaccines to induce an immune response and prevent and / or treat subjects having cancerous cells that express mesothelin. [Background technology]

[0004] Cancer remains the leading cause of death in the United States and worldwide. The cancer vaccine market is growing rapidly. Effective tumor vaccines can be useful to prevent tumor growth and / or serve as a more effective and less toxic alternative to standard treatments for patients with advanced cancer. An antigen associated with cancer and therefore a target for antitumor vaccines is mesothelin.

[0005] Mesothelin is a 71 kD protein that is cleaved into two products, the 30 kD megakaryocyte potentiating factor and the 41 kD GPI-anchored membrane-bound mature mesothelin. The function of mesothelin is unknown, but recent studies suggest that mesothelin may play a role in ovarian cancer metastasis by binding to MUC16, and it is also highly expressed on the surface of ovarian cancer cells. Mesothelin expression is observed in 82-100% of serous ovarian cancers by IHC. Hassan, R. European journal of cancer 44, 46-53 (2008), Ordonez, NG The American journal of surgical pathology 27, 1418-1428 (2003). Mesothelin is an attractive target for cancer therapy vaccines due to its high expression in ovarian cancer and its association with tumor invasion.

[0006] Vaccines for the treatment and prevention of cancer are of great interest.Existing vaccines that target tumor cell antigens are often limited by poor antigen expression in vivo.Therefore, there remains a need in the art to develop safe and effective vaccines that can be applied to tumors that express mesothelin, thereby providing treatment and promoting survival of subjects suffering from such cancer. Summary of the Invention

[0007] Provided herein are nucleic acid molecules comprising one or more nucleic acid sequences selected from the group consisting of: (a) a nucleic acid sequence encoding amino acids 19-607 of SEQ ID NO:2; (b) a nucleic acid sequence encoding a fragment comprising at least 90% of the full length of a protein comprising amino acids 19-607 of SEQ ID NO:2; (c) a nucleic acid sequence encoding a protein that is at least 96% identical to amino acids 19-607 of SEQ ID NO:2; and (d) a nucleic acid sequence encoding a fragment comprising at least 90% of the full length of a protein that is at least 96% identical to amino acids 19-607 of SEQ ID NO:2.

[0008] Further provided herein is a nucleic acid molecule comprising one or more nucleic acid sequences selected from the group consisting of: (a) nucleotides 55-1821 of SEQ ID NO:1; (b) a fragment comprising at least 90% of the entire length of a nucleic acid molecule comprising nucleotides 55-1821 of SEQ ID NO:1; (c) a fragment that is at least 96% identical to nucleotides 55-1821 of SEQ ID NO:1; and (d) a fragment that comprises at least 90% of the entire length of a nucleic acid sequence that is at least 96% identical to nucleotides 55-1821 of SEQ ID NO:1.

[0009] Provided herein are nucleic acid molecules comprising one or more nucleic acid sequences selected from the group consisting of: (a) a nucleic acid sequence encoding SEQ ID NO:2; (b) a nucleic acid sequence encoding a fragment comprising at least 90% of the full length of SEQ ID NO:2; (c) a nucleic acid sequence encoding a protein that is at least 96% identical to SEQ ID NO:2; and (d) a nucleic acid sequence encoding a fragment comprising at least 90% of the full length of a protein that is at least 96% identical to SEQ ID NO:2.

[0010] Further provided herein are nucleic acid molecules comprising one or more nucleic acid sequences selected from the group consisting of: (a) SEQ ID NO:1, (b) a fragment comprising at least 90% of the entire length of SEQ ID NO:1, (c) a fragment that is at least 96% identical to SEQ ID NO:1, and (d) a fragment that comprises at least 90% of the entire length of a nucleic acid sequence that is at least 96% identical to SEQ ID NO:1. In some embodiments, the nucleic acid molecule comprises the nucleic acid sequence set forth in SEQ ID NO:1.

[0011] The nucleic acid molecules described herein can be incorporated into a vector, such as a plasmid or viral vector. The nucleic acid molecules described herein can be incorporated into a vector, such as a plasmid or viral vector. In some embodiments, the vector comprises one or more nucleic acid sequences selected from the group consisting of: (a) a nucleic acid sequence encoding amino acids 19-607 of SEQ ID NO:2; (b) a nucleic acid sequence encoding a fragment comprising at least 90% of the full length of a protein comprising amino acids 19-607 of SEQ ID NO:2; (c) a nucleic acid sequence encoding a protein that is at least 96% identical to amino acids 19-607 of SEQ ID NO:2; and (d) a nucleic acid sequence encoding a fragment comprising at least 90% of the full length of a protein that is at least 96% identical to amino acids 19-607 of SEQ ID NO:2. In a further embodiment, the vector comprises one or more nucleic acid sequences selected from the group consisting of nucleotides 55-1821 of SEQ ID NO:1, (b) a fragment comprising at least 90% of the entire length of a nucleic acid molecule comprising nucleotides 55-1821 of SEQ ID NO:1, (c) a fragment that is at least 96% identical to nucleotides 55-1821 of SEQ ID NO:1, and (d) a fragment comprising at least 90% of the entire length of a nucleic acid sequence that is at least 96% identical to nucleotides 55-1821 of SEQ ID NO:1. In yet a further embodiment, the vector comprises one or more nucleic acid sequences selected from the group consisting of (a) a nucleic acid sequence encoding SEQ ID NO:2, (b) a nucleic acid sequence encoding a fragment comprising at least 90% of the entire length of SEQ ID NO:2, (c) a nucleic acid sequence encoding a protein that is at least 96% identical to SEQ ID NO:2, and (d) a nucleic acid sequence encoding a fragment comprising at least 90% of the entire length of a protein that is at least 96% identical to SEQ ID NO:2. In a further embodiment, the vector comprises one or more nucleic acid sequences selected from the group consisting of (a) SEQ ID NO:1, (b) a fragment comprising at least 90% of the full length of SEQ ID NO:1, (c) a fragment that is at least 96% identical to SEQ ID NO:1, and (d) a fragment that comprises at least 90% of the full length of a nucleic acid sequence that is at least 96% identical to SEQ ID NO:1. In yet a further embodiment, the vector comprises the nucleic acid sequence set forth in SEQ ID NO:1.

[0012] In some embodiments, the nucleic acid described herein is operably linked to a regulatory element. In some embodiments, the regulatory element is a promoter and / or a poly-adenylation signal. In further embodiments, the promoter is human cytomegalovirus immediate early promoter (hCMV promoter). In yet further embodiments, the poly-adenylation signal is bovine growth hormone poly-adenylation signal (bGH polyA).

[0013] Also provided herein are compositions comprising one or more of the nucleic acid molecules described herein. In some embodiments, the composition comprises a pharma- ceutically acceptable carrier.

[0014] Further provided is a mesothelin antigen protein comprising an amino acid sequence selected from the group consisting of: (a) amino acids 19 to 607 of SEQ ID NO:2; (b) a fragment comprising at least 90% of the full length of a protein comprising amino acids 19 to 607 of SEQ ID NO:2; (c) an amino acid sequence that is at least 95% identical to amino acids 19 to 607 of SEQ ID NO:2; and (d) a fragment comprising at least 90% of the full length of an amino acid sequence that is at least 95% identical to amino acids 19 to 607 of SEQ ID NO:2.

[0015] Further provided is a mesothelin antigen protein comprising an amino acid sequence selected from the group consisting of: (a) SEQ ID NO:2, (b) a fragment comprising at least 90% of the full length of SEQ ID NO:2, (c) an amino acid sequence that is at least 96% identical to SEQ ID NO:2, and (c) a fragment comprising at least 90% of the full length of an amino acid sequence that is at least 96% identical to SEQ ID NO:2. In some embodiments, the protein comprises the amino acid sequence set forth in SEQ ID NO:2.

[0016] Further provided is a vaccine comprising a mesothelin antigen, wherein the antigen comprises the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the antigen is encoded by SEQ ID NO: 1.

[0017] Further provided is a vaccine comprising a nucleic acid molecule, the nucleic acid molecule comprising a nucleic acid sequence having at least about 96% identity over the entire length of the nucleic acid sequence set forth in SEQ ID NO:1. Further disclosed herein is a vaccine comprising a nucleic acid molecule, the nucleic acid molecule encoding a mesothelin antigen comprising an amino acid sequence having at least about 96% identity over the entire length of the amino acid sequence set forth in SEQ ID NO:2. In some embodiments, the nucleic acid molecule may comprise an expression vector. The vaccine may further comprise a pharma- ceutically acceptable excipient. In some embodiments, the vaccine may further comprise an adjuvant. In certain embodiments, the adjuvant is IL-12, IL-15, IL-28, or RANTES.

[0018] Further provided herein is a vaccine comprising a mesothelin antigen, wherein the antigen comprises an amino acid sequence having at least about 90% identity over the entire length of the amino acid sequence set forth in SEQ ID NO:2.

[0019] Further provided is a method of treating a subject having cancerous cells expressing mesothelin, comprising administering a therapeutically effective amount of a vaccine described herein. In some embodiments, the administration comprises an electroporation step. In other embodiments, the administration occurs at one or more sites in the subject.

[0020] Also provided is a method of vaccinating a subject against cancerous cells expressing mesothelin, comprising administering an amount of a vaccine described herein effective to induce a humoral immune response. In some embodiments, the administration comprises an electroporation step. In other embodiments, the administration occurs at one or more sites in the subject.

[0021] Further provided herein is a nucleic acid molecule comprising one or more nucleic acid sequences selected from the group consisting of: (a) a nucleic acid sequence encoding SEQ ID NO:2; (b) a nucleic acid sequence encoding a fragment comprising at least 90% of the full length of SEQ ID NO:2; (c) a nucleic acid sequence encoding a protein that is at least 96% identical to SEQ ID NO:2; and (d) a nucleic acid sequence encoding a fragment comprising at least 90% of the full length of a protein that is at least 96% identical to SEQ ID NO:2 for use as a medicament. In some embodiments, the nucleic acid molecule described herein is for use as a medicament in the treatment of cancer. In some embodiments, the nucleic acid molecule described herein is for use in the preparation of a medicament. In some embodiments, the nucleic acid molecule described herein is for use in the preparation of a medicament for the treatment of cancer.

[0022] Further provided herein is a nucleic acid molecule comprising one or more nucleic acid sequences selected from the group consisting of: (a) SEQ ID NO:1, (b) a fragment comprising at least 90% of the full length of SEQ ID NO:1, (c) a fragment that is at least 96% identical to SEQ ID NO:1, and (d) a fragment that comprises at least 90% of the full length of a nucleic acid sequence that is at least 96% identical to SEQ ID NO:1. In some embodiments, the nucleic acid molecule comprises the nucleic acid sequence set forth in SEQ ID NO:1 for use as a medicament. In some embodiments, the nucleic acid molecule described herein is for use as a medicament in the treatment of cancer. In some embodiments, the nucleic acid molecule described herein is for use in the preparation of a medicament. In some embodiments, the nucleic acid molecule described herein is for use in the preparation of a medicament for the treatment of cancer. [Brief description of the drawings]

[0023] [Figure 1] 1 shows the generation of mesothelin mutants. [Diagram 2] FIG. 2 is a schematic diagram of the structure of the GPI-anchored mesothelin protein precursor. [Diagram 3] A schematic diagram of synthetic consensus mesothelin is shown. [Figure 4] FIG. 1 is a diagrammatic representation of the modified pVAX1 backbone (pGX0001). [Diagram 5] FIG. 1 is a diagrammatic representation of plasmid pGX1438. [Figure 6] 1 shows a Western blot of synthetic consensus mesothelin antigen expression in human rhabdomyosarcoma cells. [Figure 7A] 1 shows the immunogenicity of synthetic consensus mesothelin using embodiments of the present disclosure. [Figure 7B] 1 shows the immunogenicity of synthetic consensus mesothelin using embodiments of the present disclosure. [Figure 8] 1 illustrates a flow cytometry gating strategy using an embodiment of the present disclosure. [Figure 9A] Figure 9 shows the cellular immune response induced by pGX1430. Figure 9A shows the frequency of synthetic consensus mesothelin induction of antigen-specific CD4+ T cell responses. [Figure 9B] Figure 9B shows the frequency of synthetic consensus mesothelin induction of antigen-specific CD8+ T cell responses. [Figure 9C] Figure 9C shows the cytokine profile of synthetic consensus mesothelin-specific CD4+ T cells. [Figure 9D] Figure 9D shows the cytokine profile of synthetic consensus mesothelin-specific CD8+ T cells induced by pGX1430. [Figure 10A] Figure 10 shows the cytolytic potential of synthetic consensus mesothelin-specific T cells. Figure 10A shows the frequency of antigen-specific CD4+CD107a+ T cells. [Figure 10B] Figure 10B shows the cytolytic potential of synthetic consensus mesothelin-specific T cells. Figure 10B shows the frequency of antigen-specific CD8+CD107a+ T cells. [Figure 10C]Figure 10C shows the cytolytic potential of synthetic consensus mesothelin-specific T cells, and Figure 10D shows the cytokine profile of synthetic consensus mesothelin-specific CD4+CD107a+ T cells. [Figure 10D] Figure 10D shows the cytolytic potential of synthetic consensus mesothelin-specific T cells. Figure 10D shows the cytokine profile of synthetic consensus mesothelin-specific CD8+CD107a+ T cells. [Figure 11A] 11A-11C show mesothelin-specific IFNγ SFU / 106 PBMCs from an individual NHP using embodiments of the present disclosure. [Figure 11B] Figure 11B shows mesothelin-specific IFNγ SFU / 106 PBMC from an individual NHP using embodiments of the present disclosure.Figure 11B shows mesothelin with low dose IL-12 (0.04 mg). [Figure 11C] Figure 11C shows mesothelin-specific IFNγ SFU / 106 PBMC from an individual NHP using embodiments of the present disclosure.Figure 11C shows mesothelin with high dose IL-12 (0.2 mg). [Figure 12A] 12A shows averaged mesothelin-specific IFNγ SFU / 106 PBMCs using embodiments of the present disclosure. FIG. 12A shows mesothelin alone. [Figure 12B] Figure 12B shows averaged mesothelin-specific IFNγ SFU / 106 PBMCs using embodiments of the present disclosure.Figure 12B shows mesothelin with low dose IL-12 (0.04 mg). [Figure 12C] Figure 12C shows averaged mesothelin-specific IFNγ SFU / 106 PBMCs using embodiments of the present disclosure.Figure 12C shows mesothelin with high dose IL-12 (0.2 mg). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] The present invention relates to a vaccine comprising a mesothelin antigen. Mesothelin is expressed in many tumors. Thus, the vaccine provides treatment for cancer or tumors that express mesothelin. The vaccine of the present invention can provide any combination of specific cancer antigens for specific prevention or treatment of cancer in a subject in need of treatment.

[0025] One method for designing the nucleic acid of a recombinant cancer antigen and its encoded amino acid sequence is by introducing mutations that change specific amino acids in the overall amino acid sequence of a natural cancer antigen. Although the introduction of mutations does not modify the cancer antigen much and cannot be universally applied across mammalian subjects, preferably human or canine subjects, the resulting amino acid sequence changes enough to break tolerance or be considered a foreign antigen to generate an immune response. Another method can be to create a consensus recombinant cancer antigen that has at least 85% and up to 99% amino acid sequence identity to its corresponding natural 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 cases, the recombinant cancer antigen has 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to its corresponding natural cancer antigen. A natural cancer antigen is an antigen that is normally associated with a particular cancer or cancer tumor. Depending on the cancer antigen, the consensus sequence of the cancer antigen can be across the entire mammalian species, or within a subtype of a species, or across virus strains, or serotypes. Some cancer antigens do not differ significantly from the wild-type amino acid sequence of the cancer antigen. Some cancer antigens have nucleic acid / amino acid sequences that are so different across species that a consensus sequence cannot be generated. In these cases, a recombinant cancer antigen that breaks tolerance and generates an immune response is generated that has at least 85% and up to 99% amino acid sequence identity 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 cases, the recombinant cancer antigen is 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to its corresponding native cancer antigen. The aforementioned approaches can be combined to make the final recombinant cancer antigen have the percentage similarity to the native cancer antigen amino acid sequence described above.

[0026] The mesothelin antigen may be, in part, a consensus mesothelin antigen derived from sequences of mesothelin from different species or different isoforms within a species, and thus the consensus mesothelin antigen is non-naturally occurring. Modifications may include mutations in the MUC16 binding domain and / or GPI anchoring signal, and addition of upstream Kozak and IgE leader sequences to the N-terminus of the mesothelin antigen.

[0027] Synthetic mesothelin can induce antigen-specific T cell and / or high titer antibody responses, thereby inducing or eliciting an immune response directed to or reactive against a cancer or tumor expressing the antigen. In some embodiments, the induced or elicited immune response can be a cellular, humoral, or both a cellular and humoral immune response. In some embodiments, the induced or elicited cellular immune response can include induction or secretion of interferon-gamma (IFN-γ) and / or tumor necrosis factor alpha (TNF-α). In other embodiments, the induced or elicited immune response can reduce or inhibit one or more immunosuppressive factors that promote the growth of the antigen-expressing tumor or cancer, including, but not limited to, factors that downregulate MHC presentation, factors that upregulate antigen-specific regulatory T cells (Tregs), cytokines such as PD-L1, FasL, IL-10 and TFG-β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by immunosuppressive cells, CTLA-4, PD-1, MDSC, MCP-1, and immune checkpoint molecules.

[0028] The vaccines can be further combined with antibodies against checkpoint inhibitors such as PD-1 and PDL-1 to enhance the stimulation of both cellular and humoral immune responses. By using anti-PD-1 or anti-PDL-1 antibodies, PD-1 or PDL-1 are prevented from suppressing T cell and / or B cell responses. In general, designing cancer antigens that are recognized by the immune system helps overcome other forms of immune suppression by tumor cells, and these vaccines can be used in combination with suppressive or inhibitory therapies (such as anti-PD-1 and anti-PDL-1 antibody therapy) to further increase T cell and / or B cell responses.

[0029] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, but methods and materials similar or equivalent to those described herein can be used to practice or test the present invention. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are merely illustrative and are not intended to be limiting. The terms used herein are for the purpose of describing specific embodiments only and are not intended to be limiting.

[0030] As used herein, the terms "comprise," "include," "having," "has," "can," "contain," and variations thereof are intended to be open-ended transitional phrases, terms, or words that do not exclude the possibility of additional acts or structures. The singular forms "a," "and," and "the" include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments that "comprising," "consisting of," and "consisting essentially of" the embodiments or elements presented herein, whether or not expressly stated.

[0031] For the recitation of numerical ranges herein, each intervening value is expressly contemplated with the same degree of precision as the minimum and maximum values ​​of the recited range. For example, in the range of 6 to 9, the values ​​7 and 8 are contemplated in addition to 6 and 9, and in the range of 6.0 to 7.0, the values ​​6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are expressly contemplated.

[0032] As used herein, "adjuvant" means any molecule added to a vaccine described herein to enhance the immunogenicity of a mesothelin antigen and / or a nucleic acid molecule encoding an antigen described herein.

[0033] As used herein, "antibody" refers to an antibody of class IgG, IgM, IgA, IgD, or IgE, or a fragment or derivative thereof, including Fab, F(ab')2, Fd, and single chain antibodies, diabodies, bispecific antibodies, bifunctional antibodies, and derivatives thereof. The antibody may be an antibody isolated from a mammalian serum sample, a polyclonal antibody, an affinity purified antibody, or any mixture thereof, and exhibits sufficient binding specificity for the desired epitope or a sequence derived therefrom.

[0034] "Mesothelin antigen" refers to a mutated mesothelin amino acid sequence comprising amino acids 19-607 of SEQ ID NO:2, SEQ ID NO:2, fragments thereof of lengths as described herein, proteins having variants, i.e., fragments of SEQ ID NO:2, ... and combinations thereof. Antigens may optionally include signal peptides, such as those from other proteins.

[0035] As used herein, a "coding sequence" or "encoding nucleic acid" refers to a nucleic acid (RNA or DNA molecule) that comprises a nucleotide sequence that codes for a protein. The coding sequence may further comprise initiation and termination signals operably linked to regulatory elements, including a promoter and polyadenylation signal, capable of directing expression in the cells of a subject or mammal to which the nucleic acid is administered.

[0036] As used herein, "complement" or "complementary" means that a nucleic acid can refer to Watson-Crick (e.g., AT / U and CG) or Hoogsteen base pairing between nucleotides or nucleotide analogs of a nucleic acid molecule.

[0037] As used herein, "consensus" or "consensus sequence" refers to a polypeptide sequence based on the analysis of the alignment of multiple sequences of the same gene from different organisms. Nucleic acid sequences encoding consensus polypeptide sequences can be prepared. Vaccines containing proteins containing consensus sequences and / or nucleic acid molecules encoding such proteins can be used to induce broad immunity against antigens.

[0038] "Constant current" as used herein refers to the current received or experienced by a tissue or cells defining the tissue over the duration of an electrical pulse delivered to the same tissue. The electrical pulse is delivered from an electroporation device as described herein. The electroporation device provided herein has a feedback element, preferably an immediate feedback, so that this current remains at a constant amperage in the tissue over the life of the electrical pulse. The feedback element measures the resistance of the tissue (or cells) throughout the duration of the pulse and causes the electroporation device to change its electrical energy output (e.g., increase the voltage) so that the current in the same tissue remains constant throughout the electrical pulse (on the order of microseconds) and between pulses. In some embodiments, the feedback element includes a controller.

[0039] As used herein, "current feedback" or "feedback" may be used interchangeably and may refer to the active response of the electroporation device provided, including measuring the current in the tissue between the electrodes and appropriately modifying the energy output delivered by the EP device to maintain the current at a constant level. This constant level is pre-adjusted by the user before the start of the pulse sequence or electrical therapy. The feedback may be achieved by the electroporation components of the electroporation device, e.g., a controller, as the electrical circuitry continuously monitors the current in the tissue between the electrodes, compares the monitored current (or the current in the tissue) to a pre-adjusted current, and continuously makes energy output adjustments to maintain the monitored current at the pre-adjusted level. The feedback loop may be instantaneous, as it is an analog closed-loop feedback.

[0040] As used herein, "distributed current" refers to a pattern of current delivered from the various needle electrode arrays of the electroporation devices described herein that minimizes or preferably eliminates the occurrence of electroporation-related thermal stress in any region of the tissue being electroporated.

[0041] "Electroporation," "electropermeabilization," or "electrokinetic enhancement" ("EP"), as used interchangeably herein, refers to the use of transmembrane electric field pulses to induce microscopic pathways (pores) in biological membranes, the presence of which allows biomolecules such as plasmids, oligonucleotides, siRNA, drugs, ions, and water to pass from one side of the cell membrane to the other.

[0042] A "fragment" as used herein with respect to a nucleic acid sequence refers to a nucleic acid sequence or a portion thereof that encodes a polypeptide capable of eliciting an immune response in a mammal that cross-reacts with an antigen disclosed herein. The fragment may be a DNA fragment selected from at least one of the various nucleotide sequences encoding protein fragments described below. The fragment may comprise at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the full length of one or more of the nucleic acid sequences described herein, excluding any added heterologous signal peptide. In some embodiments, the fragment may comprise at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the full length of one or more of the nucleic acid sequences described below, excluding any added heterologous signal peptide.

[0043] In some embodiments, the fragments may be at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identical to one or more of the nucleic acid sequences described herein excluding any heterologous signal peptide. In some embodiments, the fragments may be at least 96%, at least 97%, at least 98%, or at least 99% identical to one or more of the nucleic acid sequences described below excluding any heterologous signal peptide.

[0044] In further embodiments, the fragment may additionally, optionally, include a sequence encoding a heterologous signal peptide that is not included when calculating percent identity. The fragment may also include a coding sequence for a signal peptide, such as an immunoglobulin signal peptide, e.g., an IgE or IgG signal peptide. A coding sequence encoding an N-terminal methionine and / or a signal peptide may be linked to the fragment of the coding sequence.

[0045] In some embodiments, the fragment is at least 1500 nucleotides or more, 1510 nucleotides or more, 1520 nucleotides or more, 1530 nucleotides or more, 1540 nucleotides or more, 1550 nucleotides or more, 1560 nucleotides or more, 1570 nucleotides or more, 1580 nucleotides or more, 1590 nucleotides or more, 1600 nucleotides or more, 1610 nucleotides or more, 1620 nucleotides or more, or 1630 nucleotides or more, 1640 nucleotides or more, 1650 nucleotides or more, 1660 nucleotides or more, 1670 nucleotides or more, 1680 nucleotides or more, 1690 nucleotides or more, 1610 nucleotides or more, 1620 nucleotides or more, 1630 nucleotides or more, 1640 nucleotides or more, 1650 nucleotides or more, 1660 nucleotides or more, 1670 nucleotides or more, 1680 nucleotides or more, 1690 nucleotides or more, 1690 nucleotides or more, 1692 nucleotides or more, 1694 nucleotides or more, 1696 nucleotides or more, 1698 nucleotides or more, 1699 nucleotides or more, 20 ... It can comprise 650 or more nucleotides, 1660 or more nucleotides, 1670 or more nucleotides, 1680 or more nucleotides, 1690 or more nucleotides, 1700 or more nucleotides, 1710 or more nucleotides, 1720 or more nucleotides, 1730 or more nucleotides, 1740 or more nucleotides, 1750 or more nucleotides, 1760 or more nucleotides, 1770 or more nucleotides, 1780 or more nucleotides, 1790 or more nucleotides, 1800 or more nucleotides, 1810 or more nucleotides, or 1820 or more nucleotides.

[0046] "Fragment" or "immunogenic fragment" with respect to a polypeptide sequence refers to a polypeptide capable of eliciting an immune response in a mammal that cross-reacts with an antigen disclosed herein. The fragment may be a polypeptide fragment selected from at least one of the various amino acid sequences described herein. A fragment of a consensus protein may comprise at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the full length of the consensus protein, excluding any added heterologous signal peptide. In some embodiments, a fragment may comprise at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the full length of one or more of the amino sequences set forth below, excluding any added heterologous signal peptide.

[0047] In some embodiments, the fragments may be at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identical to one or more of the amino acid sequences set forth herein, excluding any heterologous signal peptide. In some embodiments, the fragments may be at least 96%, at least 97%, at least 98%, or at least 99% identical to one or more of the amino acid sequences set forth below, excluding any added heterologous signal peptide.

[0048] In further embodiments, the fragment may additionally, optionally, include a sequence encoding a heterologous signal peptide, which is not included when calculating percent identity. The fragment may further include a signal peptide, such as an immunoglobulin signal peptide, e.g., an IgE or IgG signal peptide.

[0049] In some embodiments, a consensus protein fragment comprises at least 546 or more, 547 or more, 548 or more, 549 or more, 550 or more, 551 or more, 552 or more, 553 or more, 554 or more, 555 or more, 556 or more, 557 or more, 558 or more, 559 or more, 560 or more, 561 or more, 562 or more, 563 or more, 564 or more, 565 or more, 566 or more, 567 or more, 568 or more, 569 or more, 570 or more, 571 or more, 572 or more, 573 or more, 574 or more amino acids of a protein sequence disclosed herein. and may include 575 or more amino acids, 576 or more amino acids, 577 or more amino acids, 578 or more amino acids, 579 or more amino acids, 580 or more amino acids, 581 or more amino acids, 582 or more amino acids, 583 or more amino acids, 584 or more amino acids, 585 or more amino acids, 586 or more amino acids, 587 or more amino acids, 588 or more amino acids, 589 or more amino acids, 590 or more amino acids, 591 or more amino acids, 592 or more amino acids, 593 or more amino acids, 594 or more amino acids, 595 or more amino acids, 596 or more amino acids, 597 or more amino acids, 598 or more amino acids, 599 or more amino acids, 600 or more amino acids, 601 or more amino acids, 602 or more amino acids, 603 or more amino acids, 604 or more amino acids, 605 or more amino acids, 606 or more amino acids, or 607 or more amino acids.

[0050] The term "genetic construct" as used herein refers to a DNA or RNA molecule that comprises a nucleotide sequence that codes for a protein. The coding sequence comprises an initiation and termination signal that is operably linked to a regulatory element, including a promoter and a polyadenylation signal that can direct expression in the cells of a subject to which the nucleic acid molecule is administered. The term "expressible form" as used herein refers to a genetic construct that contains the necessary regulatory elements that are operably linked to a coding sequence that codes for a protein, so that the coding sequence is expressed when present in the cells of a subject.

[0051] The term "homology" as used herein refers to the degree of complementarity. There may be partial or complete homology (i.e., identity). A partial homologous sequence that at least partially inhibits a complete complementary sequence from hybridizing to a target nucleic acid is referred to using the functional term "substantially homologous". When used in relation to a double-stranded nucleic acid sequence, such as a cDNA or genomic clone, the term "substantially homologous" as used herein refers to a probe that can hybridize to a strand of a double-stranded nucleic acid sequence under low stringency conditions. 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 to a single-stranded nucleic acid template sequence under low stringency conditions (i.e., is its complement).

[0052] As used herein, "identical" or "identity" in the context of two or more nucleic acid or polypeptide sequences means that the sequences have a certain percentage of residues that are the same over a certain region. The percentage can be calculated by optimally aligning the two sequences, comparing the two sequences over a certain region, determining the number of positions where identical residues occur in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the certain region, and multiplying the result by 100 to obtain the percentage of sequence identity. If the two sequences are of different lengths or the alignment results in one or more sticky ends and the certain region of comparison contains only a single sequence, the residues of the single sequence are included in the denominator of the calculation but not in the numerator. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. Identity can be performed manually or by using computer sequence algorithms such as BLAST or BLAST 2.0.

[0053] As used herein, "impedance" may be used when discussing feedback mechanisms and is converted to a current value that follows Ohm's law and can therefore be compared to a pre-adjusted current.

[0054] As used herein, "immune response" refers to activation of a host's immune system, e.g., a mammal's immune system, in response to the introduction of an antigen. The immune response can be in the form of a cellular or humoral response, or both.

[0055] As used herein, "nucleic acid" or "oligonucleotide" or "polynucleotide" refers to at least two nucleotides covalently linked together. A description of a single strand also defines the sequence of the complementary strand. Thus, a nucleic acid also encompasses the complementary strand of a 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 encompasses substantially identical nucleic acids and their complements. A single strand provides a probe that can hybridize to a target sequence under stringent hybridization conditions. Thus, a nucleic acid also encompasses a probe that hybridizes under stringent hybridization conditions.

[0056] Nucleic acids may be single-stranded or double-stranded, or may contain portions of both double-stranded and single-stranded sequences. Nucleic acids are defined as any combination of deoxyribo- and ribo-nucleotides, as well as any of the following amino acids: uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, The nucleic acid may be DNA, both genomic and cDNA, RNA, or a hybrid, where it may contain combinations of bases including hypoxanthine, isocytosine, and isoguanine. The nucleic acid may be obtained by chemical synthesis or recombinant methods.

[0057] As used herein, "operably linked" means that the expression of a gene is under the control of a promoter that is spatially connected. The promoter can be located 5' (upstream) or 3' (downstream) of the gene under its control. The distance between the promoter and the gene can be approximately the same as the distance between the promoter and the gene that it controls in the gene from which the promoter is derived. As is known in the art, this difference in distance can be adjusted without losing the function of the promoter.

[0058] As used herein, "peptide," "protein," or "polypeptide" may mean a linked sequence of amino acids, which may be natural, synthetic, or a modified or combination of natural and synthetic.

[0059] "Promoter" as used herein means a synthetic or naturally derived molecule that can confer, activate or enhance the expression of a nucleic acid in a cell. A promoter can contain one or more specific transcription control sequences to further enhance the expression of a nucleic acid in a cell and / or to alter the spatial and / or temporal expression. A promoter can also contain distal enhancer or repressor elements that can be located as far as several thousand base pairs from the start site of transcription. Promoters can be derived from sources including viruses, bacteria, fungi, plants, insects, and animals. A promoter can constitutively or differentially control the expression of gene components with respect to the cell, tissue, or organ in which expression occurs, or with respect to the developmental stage in which expression occurs, or in response to external stimuli such as physiological stress, pathogens, metal ions, or inducing agents. Representative examples of promoters include bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operator-promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, SV40 early promoter, or SV40 late promoter, CMV IE promoter, and human cytomegalovirus immediate early promoter (hCMV). In a specific embodiment, the promoter is a hCMV promoter.

[0060] "Signal peptide" and "leader sequence" are used interchangeably herein and refer to an amino acid sequence that may be linked at the amino terminus of a protein described herein. A signal peptide / leader sequence typically directs the localization of a protein. A signal peptide / leader sequence as used herein preferably facilitates the secretion of a protein from the cell in which it is produced. A signal peptide / leader sequence is often cleaved from the remainder of a protein, and is often referred to as a mature protein once secreted from a cell. A signal peptide / leader sequence is linked at the amino terminus (i.e., N-terminus) of a protein.

[0061] As used herein, "stringent hybridization conditions" refers to conditions under which a first nucleic acid sequence (e.g., a probe) hybridizes to a second nucleic acid sequence (e.g., a target) in a complex mixture of nucleic acids or the like. Stringent conditions are sequence-dependent and will be different in different circumstances. Stringent conditions may be selected to be about 5-10°C lower than the thermal melting point (Tm) of the specific sequence at a defined ionic strength pH. Tm may be the temperature (under defined ionic strength, pH, and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (the target sequence is present in excess so that at Tm, 50% of the probes are occupied at equilibrium). Stringent conditions can be those in which the salt concentration is less than about 1.0 M sodium ion, for example, about 0.01 to 1.0 M sodium ion concentration (or other salt) at pH 7.0 to 8.3, and the temperature is at least about 30° C. for short probes (e.g., about 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g., more than about 50 nucleotides). Stringent conditions can also be achieved by the addition of a destabilizing agent such as formamide. For selective or specific hybridization, a positive signal can be at least 2 to 10 times background hybridization. Exemplary stringent hybridization conditions include 50% formamide, 5xSSC, and 1% SDS, incubated at 42° C., or 5xSSC, 1% SDS, incubated at 65° C., washed in 0.2xSSC, and 0.1% SDS at 65° C.

[0062] As used herein, "subject" may refer to a mammal that desires or needs to be immunized with a vaccine described herein. The mammal may be a human, a non-human primate such as a chimpanzee, a dog, a cat, a horse, a cow, a mouse, or a rat.

[0063] As used herein, "substantially complementary" refers to a sequence that is substantially complementary to a first sequence over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 180, 270, 360, 450, 540, or more nucleotides or amino acids. By this term, we mean that the two sequences are at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the complement of the two sequences, or that the two sequences hybridize under stringent hybridization conditions.

[0064] As used herein, "substantially identical" refers to a sequence in which the first and second sequences are at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 180, 270, 360, 450, 540 or more nucleotides or amino acids apart. By nucleic acid, we mean 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 across a region of the acid sequence, or where a first sequence is substantially complementary to the complement of a second sequence.

[0065] As used herein, "treat", "treatment" or "treating" can mean protecting an animal from a disease by means of preventing, suppressing, arresting, or completely eliminating the disease. Prevention of the disease involves administering a vaccine of the invention to an animal before the onset of the disease. Suppression of the disease involves administering a vaccine of the invention to an animal after induction of the disease but before its clinical appearance. Prevention of the disease involves administering a vaccine of the invention to an animal after the appearance of the disease.

[0066] "Variant," as used herein with respect to a nucleic acid, means (i) a portion or fragment of a referenced nucleotide sequence, (ii) the complement of a referenced nucleotide sequence or a portion thereof, (iii) a nucleic acid that is substantially identical to a referenced nucleic acid or its complement, or (iv) a nucleic acid that hybridizes under stringent conditions to a referenced nucleic acid, its complement, or a sequence substantially identical thereto.

[0067] "Variant" as used herein with respect to a peptide or polypeptide refers to a peptide or polypeptide that differs in amino acid sequence by insertion, deletion, or conservative substitution of amino acids, but retains at least one biological activity. Variant can also refer to a protein with an amino acid sequence that is substantially identical to the referenced protein with an amino acid sequence that retains at least one biological activity. Conservative substitution of amino acids, i.e., replacing an amino acid with an amino acid with a different amino acid with similar properties (e.g., hydrophilicity, degree and distribution of charged regions), is recognized in the art as typically involving minor changes. These minor changes can be identified, in part, by considering the hydropathic index of an amino acid, as understood in the art. Kyte et al, J. Mol. Biol. 157:105-132 (1982). The hydropathic index of an amino acid is based on considering its hydrophobicity and charge. It is known in the art that amino acids with similar hydropathic indexes can be substituted and still retain protein function. In one embodiment, amino acids with hydropathic indexes of ±2 are substituted. The hydrophilicity of amino acids can also be used to identify substitutions that may result in proteins that retain biological function. In the context of peptides, consideration of the hydrophilicity of amino acids allows calculation of the greatest local average hydrophilicity of the peptide, a useful tool that has been reported to correlate well with antigenicity and immunogenicity. U.S. Patent No. 4,554,101, fully incorporated herein by reference. Substitution of amino acids with similar hydrophilicity values ​​may result in peptides that retain biological activity, such as immunogenicity, as understood in the art. Substitutions may be made with amino acids that have hydrophilicity values ​​within ±2 of each other. Both the hydrophobicity index and hydrophilicity value of an amino acid are influenced by the particular side chain of that amino acid. Consistent with that observation, it is understood that amino acid substitutions that are compatible with biological function depend on the relative similarity of amino acids and particularly their side chains, as revealed by hydrophobicity, hydrophilicity, charge, size, and other properties.

[0068] A variant may be a nucleic acid sequence that is substantially identical over the full length of the complete gene sequence or a fragment thereof. The nucleic acid sequence may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the full length of the gene sequence or a fragment thereof. A variant may be an amino acid sequence that is substantially identical over the full length of the amino acid sequence or a fragment thereof. The amino acid sequence may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the full length of the amino acid sequence or a fragment thereof.

[0069] As used herein, "vector" refers to a nucleic acid sequence that includes a replication origin. A vector can be a virus vector, a bacteriophage, a bacterial artificial chromosome, or a yeast artificial chromosome. A vector can be a DNA or RNA vector. A vector can be a self-replicating extrachromosomal vector, preferably a DNA plasmid. A vector can contain or include one or more heterologous nucleic acid sequences.

[0070] vaccine Provided herein are vaccines comprising a mesothelin antigen described herein or a nucleic acid encoding a mesothelin antigen. In some embodiments, the vaccine comprises one or more nucleic acid molecules encoding a mesothelin antigen described herein. In some embodiments, the vaccine comprises one or more nucleic acid molecules comprising a nucleic acid sequence encoding amino acids 19-607 of SEQ ID NO:2, a nucleic acid sequence encoding a fragment comprising at least 90% of the full length of a protein comprising amino acids 19-607 of SEQ ID NO:2, a nucleic acid sequence encoding a protein that is at least 96% identical to amino acids 19-607 of SEQ ID NO:2, and / or a nucleic acid sequence encoding a fragment comprising at least 90% of the full length of a protein that is at least 96% identical to amino acids 19-607 of SEQ ID NO:2.

[0071] In some embodiments, the vaccine comprises one or more nucleic acid molecules comprising a nucleic acid sequence encoding SEQ ID NO:2, a nucleic acid sequence encoding a fragment comprising at least 90% of the length of SEQ ID NO:2, a nucleic acid sequence encoding a protein that is at least 96% identical to SEQ ID NO:2, and / or a nucleic acid sequence encoding a fragment that comprises at least 90% of the full length of a protein that is at least 96% identical to SEQ ID NO:2.

[0072] In some embodiments, the vaccine comprises one or more nucleic acid molecules comprising nucleotides 55-1821 of SEQ ID NO:1, a fragment comprising at least 90% of the full length of a nucleic acid molecule comprising nucleotides 55-1821 of SEQ ID NO:1, a fragment that is at least 96% identical to nucleotides 55-1821 of SEQ ID NO:1, and / or a fragment that comprises at least 90% of the full length of a nucleic acid sequence that is at least 96% identical to nucleotides 55-1821 of SEQ ID NO:1.

[0073] In some embodiments, the vaccine comprises one or more nucleic acid molecules comprising SEQ ID NO:1, a fragment comprising at least 90% of the full length of SEQ ID NO:1, a fragment that is at least 96% identical to SEQ ID NO:1, and / or a fragment that comprises at least 90% of the full length of a nucleic acid sequence that is at least 96% identical to SEQ ID NO:1.

[0074] In some embodiments, the vaccine comprises a MUC16 antigen, wherein the antigen comprises amino acids 19-607 of SEQ ID NO:2, a fragment comprising at least 90% of the full length of a protein comprising amino acids 19-607 of SEQ ID NO:2, an amino acid sequence that is at least 95% identical to amino acids 19-607 of SEQ ID NO:2, and / or a fragment that comprises at least 90% of the full length of an amino acid sequence that is at least 95% identical to amino acids 19-607 of SEQ ID NO:2.

[0075] In some embodiments, the vaccine comprises a MUC16 antigen, wherein the antigen comprises SEQ ID NO:2, a fragment comprising at least 90% of the length of SEQ ID NO:2, an amino acid sequence that is at least 96% identical to SEQ ID NO:2, and / or a fragment that comprises at least 90% of the full length of a protein that is at least 96% identical to SEQ ID NO:2.

[0076] The vaccine may be capable of generating an immune response against an antigen in a subject. The immune response may be a therapeutic or prophylactic immune response. The vaccine may be used to prevent cancer, for example, cancer or tumor expressing mesothelin. The vaccine may be used in a subject in need thereof to prevent and / or treat tumors expressing mesothelin. The vaccine may induce a cellular and / or antibody response against mesothelin and against tumors expressing mesothelin. In one embodiment, the vaccine may be used to protect, prevent and / or treat, or induce a cellular and / or antibody response against ovarian cancer cells expressing mesothelin, particularly epithelial ovarian cancer cells expressing mesothelin, more particularly serous ovarian cancer cells expressing mesothelin.

[0077] The development of the cancer vaccine described herein includes identifying cancer antigens that are not recognized by immune system and are abnormally expressed self-antigens, such as mesothelin. The identified cancer antigens are modified from self-antigens to foreign antigens so that they can be recognized by immune system. Redesigning the nucleic acid and amino acid sequence of recombinant cancer antigens from self to foreign antigens breaks the tolerance of antigens by immune system. To break tolerance, some redesigning measures can be applied to cancer antigens, as described below.

[0078] The recombinant cancer antigens of the vaccine are not recognized as self, thereby breaking tolerance. Breaking tolerance can induce antigen-specific T cell and / or high titer antibody responses, thereby inducing or eliciting an immune response directed to or reactive against the cancer or tumor expressing the antigen. In some embodiments, the induced or elicited immune response can be a cellular, humoral, or both cellular and humoral immune response. In some embodiments, the induced or elicited cellular immune response can include the induction or secretion of interferon-gamma (IFN-γ) and / or tumor necrosis factor alpha (TNF-α). In other embodiments, the induced or elicited immune response can reduce or inhibit one or more immunosuppressive factors that promote the growth of the antigen-expressing tumor or cancer, including, but not limited to, factors that downregulate MHC presentation, factors that upregulate antigen-specific regulatory T cells (Tregs), cytokines such as PD-L1, FasL, IL-10 and TFG-β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by immunosuppressive cells, CTLA-4, PD-1, MDSC, MCP-1, and immune checkpoint molecules.

[0079] The vaccine can be a DNA vaccine.DNA vaccines are disclosed in U.S. Patent Nos. 5,593,972, 5,739,118, 5,817,637, 5,830,876, 5,962,428, 5,981,505, 5,580,859, 5,703,055 and 5,676,594 (incorporated herein in their entirety by reference).In some embodiments, the nucleic acid molecule can comprise an expression vector.The DNA vaccine can further comprise an element or reagent that inhibits it from being integrated into chromosome.

[0080] The vaccine may comprise RNA encoding a cancer antigen. The RNA vaccine may be introduced into a cell.

[0081] The vaccines can be live attenuated vaccines, vaccines using recombinant vectors to deliver antigens, subunit vaccines, and glycoprotein vaccines, for example, but not limited to, vaccines described in U.S. Pat. Nos. 4,510,245; 4,797,368; 4,722,848; 4,790,987; 4,920,209; 5,017,487; 5,077,044; 5,110,587; 5,112,749; 5,174,993; 5,223,424; 5,225,336; 5,240,703; 5,242,829; 5,294,441; No. 5,294,548, No. 5,310,668, No. 5,387,744, No. 5,389,368, No. 5,424,065, No. 5,451,4 No. 99, No. 5,453,364, No. 5,462,734, No. 5,470,734, No. 5,474,935, No. 5,482,713, No. 5, Nos. 5,955,088, 6,034,298, 6,042,836, 6,156,319, and 6,589,529, each of which is incorporated herein by reference.

[0082] In some embodiments, the nucleic acid vaccine may further comprise a molecular adjuvant, which in some cases 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 anti-cytotoxic T-lymphocyte antigen 4 (CTLA-4), anti-programmed death receptor-1 (PD-1), and anti-lymphocyte activation gene (LAG-3). The coding sequence for IL-12, IL-15, IL-28, and / or RANTES may be included in one or more nucleic acid molecules that include the coding sequence for one or more antigens. The coding sequence for IL-12, IL-15, IL-28, IL-31, IL-33, and / or RANTES may be encoded by the nucleic acid vaccine, for example, in the same plasmid, or they may be included in separate nucleic acid molecules, such as separate plasmids.

[0083] The vaccine of the present invention can have the characteristics required for an effective vaccine, such as being safe so that the vaccine itself does not cause illness or death, preventing illness, inducing neutralizing antibodies, inducing protective T cell responses, and providing ease of administration, few side effects, biological stability, and low cost per dose.The vaccine can achieve some or all of these characteristics by containing the cancer antigens discussed below.

[0084] The vaccine may further comprise one or more inhibitors of one or more immune checkpoint molecules (i.e., immune checkpoint inhibitors). Immune checkpoint molecules are described in more detail below. An immune checkpoint inhibitor is any nucleic acid or protein that prevents the suppression of any component 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 signaling for immune cell proliferation and / or differentiation. As described in more detail below, the vaccine can be further combined with antibodies against checkpoint inhibitors, such as PD-1 and PDL-1, to enhance the stimulation of both cellular and humoral immune responses. By using anti-PD-1 or anti-PDL-1 antibodies, PD-1 or PDL-1 is prevented from suppressing T cell and / or B cell responses. antigen

[0085] As described above, the vaccine can include an antigen or a nucleic acid encoding the antigen. The antigen can be mesothelin, a fragment thereof, a variant thereof, or a combination thereof.

[0086] Mesothelin is a 71 kD protein that is cleaved into two products: megakaryocyte potentiating factor (30 kD) and mature GPI-anchored membrane-bound mesothelin (41 kD). The function of mesothelin is unknown, but recent studies suggest that mesothelin may play a role in ovarian cancer metastasis by binding to MUC16, and it is also highly expressed on the surface of ovarian cancer cells. Mesothelin expression is observed by IHC in 82-100% of serous ovarian cancers. Hassan, R. European journal of cancer 44, 46-53 (2008), Ordonez, NG The American journal of surgical pathology 27, 1418-1428 (2003).

[0087] Thus, the vaccine can be used to treat subjects suffering from cancer or tumors that express mesothelin. In some embodiments, the cancer is ovarian cancer. Mesothelin antigens differ from the naturally occurring "normal" mesothelin, and thus can provide therapy or prophylaxis against mesothelin antigen-expressing tumors. Thus, provided herein are mesothelin antigen sequences that differ from the naturally occurring mesothelin gene (i.e., combined or mutated mesothelin genes or sequences).

[0088] Nucleic acid molecules comprising the heterologous sequences described above are provided. Nucleic acid molecules consisting of the heterologous sequences described above are provided. In some embodiments, the nucleic acid molecules comprise one or more nucleic acid sequences selected from the group consisting of: (a) nucleotides 55-1821 of SEQ ID NO:1, (b) a fragment comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the full length of a nucleic acid molecule comprising nucleotides 55-1821 of SEQ ID NO:1, (c) a fragment that is at least 96%, 97%, 98%, or 99% identical to nucleotides 55-1821 of SEQ ID NO:1, and (d) a fragment that comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the full length of a nucleic acid sequence that is at least 96%, 97%, 98%, or 99% identical to nucleotides 55-1821 of SEQ ID NO:1. In some embodiments, the nucleic acid molecule comprises one or more nucleic acid sequences selected from the group consisting of: (a) SEQ ID NO:1, (b) a fragment comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the full length of SEQ ID NO:1, (c) a fragment that is at least 96%, 97%, 98%, or 99% identical to SEQ ID NO:1, and (d) a fragment that comprises at least 90% of the full length of a nucleic acid sequence that is at least 96%, 97%, 98%, or 99% identical to SEQ ID NO:1. In some embodiments, the nucleic acid molecule comprises the nucleic acid sequence set forth in SEQ ID NO:1.

[0089] Nucleic acid molecules encoding mesothelin antigens are provided herein. In some embodiments, the nucleic acid molecule comprises one or more nucleic acids selected from: (a) a nucleic acid sequence encoding amino acids 19-607 of SEQ ID NO:2; (b) a nucleic acid sequence encoding a fragment comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the full length of a protein comprising amino acids 19-607 of SEQ ID NO:2; (c) a nucleic acid sequence encoding a protein that is at least 96%, 97%, 98%, or 99% identical to amino acids 19-607 of SEQ ID NO:2; and (d) a nucleic acid sequence encoding a fragment comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the full length of a protein that is at least 96%, 97%, 98%, or 99% identical to amino acids 19-607 of SEQ ID NO:2. In some embodiments, nucleic acid molecules are provided that comprise one or more nucleic acid sequences selected from the group consisting of: (a) a nucleic acid sequence encoding SEQ ID NO:2; (b) a nucleic acid sequence encoding a fragment comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the full length of SEQ ID NO:2; (c) a nucleic acid sequence encoding a protein that is at least 96%, 97%, 98%, or 99% identical to SEQ ID NO:2; and (d) a nucleic acid sequence encoding a fragment that comprises at least 90% of the full length of a protein that is at least 96%, 97%, 98%, or 99% identical to SEQ ID NO:2.

[0090] The isolated nucleic acid molecule comprising a heterologous sequence as described above can be incorporated into a vector, such as a plasmid, viral vector, and other forms of nucleic acid molecules as described below.

[0091] Protein molecules are provided that include the heterologous amino acid sequences described above. Protein molecules are provided that consist of the heterologous amino acid sequences described above. Proteins and polypeptides having the sequences described above are provided herein. The proteins and polypeptides may be referred to as mesothelin antigens and mesothelin immunogens. Mesothelin antigens can induce an immune response against tumors that express the mesothelin antigen. In some embodiments, a protein comprising an amino acid sequence selected from the group consisting of: (a) amino acids 19 to 607 of SEQ ID NO:2; (b) a fragment comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the full length of a protein comprising amino acids 19 to 607 of SEQ ID NO:2; (c) an amino acid sequence that is at least 95% identical to amino acids 19 to 607 of SEQ ID NO:2; and (d) a fragment that comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the full length of an amino acid sequence that is at least 96%, 97%, 98%, or 99% identical to amino acids 19 to 607 of SEQ ID NO:2. In some embodiments, a protein is provided that comprises an amino acid sequence selected from the group consisting of: (a) SEQ ID NO:2, (b) a fragment comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the full length of SEQ ID NO:2, (c) an amino acid sequence that is at least 96%, 97%, 98%, or 99% identical to SEQ ID NO:2, and (c) a fragment that comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the full length of an amino acid sequence that is at least 96%, 97%, 98%, or 99% identical to SEQ ID NO:2. In some embodiments, the protein comprises the amino acid sequence set forth in SEQ ID NO:2.

[0092] In one aspect, it is desirable for the consensus antigen to provide improved transcription and translation, including one or more of a low GC-content leader sequence to increase transcription, mRNA stability and codon optimization, and, whenever possible, elimination of cis-acting sequence motifs (i.e., internal TATA boxes).

[0093] The mesothelin antigen may be a consensus antigen (or immunogen) sequence derived from two or more species. The mesothelin consensus antigen may include one or more mutations, which may include one or more substitutions of amino acids in the MUC-16 binding domain. The one or more mutations may include a nucleic acid mutation resulting in a tyrosine to alanine substitution. The one or more mutations may include one or more substitutions of amino acids in the GPI anchoring signal. The one or more mutations may include a serine to threonine and / or a threonine to valine substitution. Thus, in some embodiments, the one or more mutations may replace one, two, or three amino acids in the mesothelin MUC16 binding and / or GPI anchoring signal.

[0094] The mesothelin antigen may include modifications to improve expression. Modifications may include codon optimization, RNA optimization, addition of a Kozak sequence (e.g., GCC ACC) to increase translation initiation, and / or addition of an immunoglobulin leader sequence to increase the immunogenicity of the mesothelin antigen. The mesothelin antigen may include 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.

[0095] Vaccines combined with immune checkpoint inhibitors The vaccine may further comprise one or more inhibitors of one or more immune checkpoint molecules (i.e., immune checkpoint inhibitors).Immune checkpoint molecules are described in more detail below.An immune checkpoint inhibitor is any nucleic acid or protein that prevents the suppression of any component 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 signal transduction for immune cell proliferation and / or differentiation.

[0096] Such inhibitors can be nucleic acid sequences, amino acid sequences, small molecules, or combinations thereof. The nucleic acid sequences can be DNA, RNA, cDNA, variants thereof, fragments thereof, or combinations thereof. The nucleic acid can also include additional sequences that code for linker or tag sequences that are linked to the immune checkpoint inhibitor by peptide bonds. The small molecule can be a low molecular weight, e.g., less than 800 daltons, organic or inorganic compound that can function as an enzyme substrate, a ligand (or analog thereof) that is bound by a protein or nucleic acid, or a regulator of a biological process. The amino acid sequence can be a protein, peptide, variants thereof, fragments thereof, or combinations thereof.

[0097] In some embodiments, the immune checkpoint inhibitor may be one or more nucleic acid sequences encoding an antibody, a variant thereof, a fragment thereof, or a combination thereof. In other embodiments, the immune checkpoint inhibitor may be an antibody, a variant thereof, a fragment thereof, or a combination thereof.

[0098] Immune checkpoint molecules The immune checkpoint molecule can be a nucleic acid sequence, an amino acid sequence, a small molecule, or a combination thereof. The nucleic acid sequence can be DNA, RNA, cDNA, a variant thereof, a fragment thereof, or a combination thereof. The nucleic acid can also include an additional sequence that codes for a linker or tag sequence that is linked to the immune checkpoint inhibitor by a peptide bond. The small molecule can be a low molecular weight, e.g., less than 800 daltons, organic or inorganic compound that can function as an enzyme substrate, a ligand (or an analog thereof) that is bound by a protein or nucleic acid, or a regulator of a biological process. The amino acid sequence can be a protein, a peptide, a variant thereof, a fragment thereof, or a combination thereof.

[0099] (1)PD-1 and PD-L1 The immune checkpoint molecule can be programmed cell death protein 1 (PD-1), programmed cell death ligand 1 (PD-L1), their fragments, their variants, or combinations thereof. PD-1 is a cell surface protein encoded by the PDCD1 gene. PD-1 is a member of the immunoglobulin superfamily and is expressed on T cells and pro-B cells, thus contributing to the fate and / or differentiation of these cells. In particular, PD-1 is a type 1 membrane protein of the CD28 / CTLA-4 family of T cell regulators, which negatively regulates T cell receptor (TCR) signals, thereby negatively regulating immune responses. PD-1 can negatively regulate CD8+ T cell responses, thus inhibiting CD8-mediated cytotoxicity and enhancing tumor growth.

[0100] PD-1 has two ligands, PD-L1 and PD-L2, which are members of the B7 family. PD-L1 is upregulated on macrophages and dendritic cells (DCs) in response to LPS and GM-CSF treatment, and on T cells and B cells upon TCR and B cell receptor signaling. PD-L1 is expressed by many tumor cell lines, including myeloma, mast cell tumor, and melanoma.

[0101] b. Anti-immune checkpoint molecule antibody As described above, the immune checkpoint inhibitor can be an antibody. The antibody can bind to or react with an antigen (i.e., the immune checkpoint molecule described above). Thus, the antibody can be considered an anti-immune checkpoint molecule antibody or an immune checkpoint molecule antibody. The antibody can be encoded by a nucleic acid sequence contained in.

[0102] The antibody may comprise a heavy chain polypeptide and a light chain polypeptide. The heavy chain polypeptide may comprise a variable heavy chain (VH) region and / or at least one constant heavy chain (CH) region. The at least one constant heavy chain region may comprise a constant heavy chain region 1 (CH1), a constant heavy chain region 2 (CH2), and a constant heavy chain region 3 (CH3), and / or a hinge region.

[0103] In some embodiments, the heavy chain polypeptide may comprise a VH region and a CH1 region. In other embodiments, the heavy chain polypeptide may comprise a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region.

[0104] The heavy chain polypeptide may comprise a set of complementarity determining regions ("CDRs"). The CDR set may comprise three hypervariable regions of the VH region. From the N-terminus of the heavy chain polypeptide, these CDRs are designated as "CDR1", "CDR2" and "CDR3", respectively. The CDR1, CDR2 and CDR3 of the heavy chain polypeptide may contribute to antigen binding or recognition.

[0105] The light chain polypeptide may comprise a variable light (VL) region and / or a constant light (CL) region. The light chain polypeptide may comprise a set of complementarity determining regions ("CDRs"). The CDR set may comprise three hypervariable regions of the VL region. From the N-terminus of the light chain polypeptide, these CDRs are designated as "CDR1", "CDR2" and "CDR3", respectively. The CDR1, CDR2 and CDR3 of the light chain polypeptide may contribute to antigen binding or recognition.

[0106] An antibody may comprise a set of heavy and light chain complementarity determining regions ("CDRs"), each interposed between a set of heavy and light chain frameworks ("FRs") that provide support for the CDRs and define the spatial relationship of the CDRs to each other. A set of CDRs may comprise three hypervariable regions of a heavy or light chain V region. From the N-terminus of the heavy or light chain, these regions are designated as "CDR1", "CDR2", and "CDR3", respectively. Thus, an antigen-binding site may comprise six CDRs, including a set of CDRs from each of the heavy and light chain V regions.

[0107] The antibody may be an immunoglobulin (Ig). The Ig may be, for example, IgA, IgM, IgD, IgE, and IgG. The immunoglobulin may comprise a heavy chain polypeptide and a light chain polypeptide. The heavy chain polypeptide of the immunoglobulin may comprise a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region. The light chain polypeptide of the immunoglobulin may comprise a VL region and a CL region.

[0108] In addition, the proteolytic enzyme papain preferentially cleaves IgG molecules to provide several fragments, two of which (F(ab) fragments) each contain a covalently linked heterodimer with an intact antigen-binding site. The enzyme pepsin can cleave IgG molecules to provide several fragments, including the F(ab')2 fragment, which contains both antigen-binding sites. Thus, an antibody can be a Fab or F(ab')2. A Fab can contain a heavy chain polypeptide and a light chain polypeptide. The heavy chain polypeptide of a Fab can contain a VH region and a CH1 region. The light chain of a Fab can contain a VL region and a CL region.

[0109] The antibody may be a polyclonal or monoclonal antibody. The antibody may be a chimeric antibody, a single chain antibody, an affinity matured antibody, a human antibody, a humanized antibody, or a fully human antibody. A humanized antibody may be an antibody from a non-human species that binds to a desired antigen having one or more complementarity determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule.

[0110] (1)PD-1 antibody The anti-immune checkpoint molecule antibody can be an anti-PD-1 antibody (also referred to herein as "PD-1 antibody"), a variant thereof, a fragment thereof, or a combination thereof. The PD-1 antibody can be nivolumab. The anti-PD-1 antibody can inhibit PD-1 activity, thereby inducing, eliciting, or increasing an immune response against tumors or cancer, and reducing tumor growth.

[0111] (2)PD-L1 antibody The anti-immune checkpoint molecule antibody can be an anti-PD-L1 antibody (also referred to herein as a "PD-L1 antibody"), a variant thereof, a fragment thereof, or a combination thereof. The anti-PD-L1 antibody can inhibit PD-L1 activity, thereby inducing, eliciting, or increasing an immune response against tumors or cancer, and reducing tumor growth.

[0112] vector The vaccine may include one or more vectors that contain a heterologous nucleic acid that encodes a mesothelin antigen. The one or more vectors may be capable of expressing the antigen in an amount effective to induce an immune response in a mammal. The vector may include a heterologous nucleic acid that encodes the antigen. The vector may have a nucleic acid sequence that includes an origin of replication. The vector may be a plasmid, a bacteriophage, a bacterial artificial chromosome, or a yeast artificial chromosome. The vector may be either a self-replicating extrachromosomal vector or a vector that integrates into the host genome.

[0113] One or more vectors may be expression constructs, typically plasmids, used to introduce specific genes into target cells. Once the expression vector is inside the cell, the protein encoded by the gene is produced by the cellular transcription and translation machinery ribosome complex. Plasmids are frequently engineered to contain control sequences that function as enhancer and promoter regions and result in efficient transcription of the gene carried in the expression vector. The vectors of the present invention express large amounts of stable messenger RNA and therefore proteins.

[0114] The vector may have expression signals such as a strong promoter, a strong stop codon, adjustment of the distance between the promoter and the cloned gene, and insertion of a transcription termination sequence and a PTIS (portable translation initiation sequence).

[0115] The vector may comprise a nucleic acid sequence operably linked to a regulatory element selected from a promoter and a poly-adenylation signal. In some embodiments, the promoter is the human cytomegalovirus immediate early promoter (hCMV promoter). In some embodiments, the poly-adenylation signal is the bovine growth hormone poly-adenylation signal (bGH polyA).

[0116] The vector may be a circular plasmid or a linear nucleic acid. The circular plasmid and linear nucleic acid can direct the expression of a particular nucleotide sequence in a suitable target cell. The vector may have a promoter operably linked to the antigen-encoding nucleotide sequence, which may be operably linked to a termination signal. The vector may also include sequences required for proper translation of the nucleotide sequence, as well as sequences for cloning and subcloning the vector and its fragments. The vector containing the nucleotide sequence of interest may be chimeric, that is, at least one of its components is heterologous to at least one of the other components. The expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive or inducible promoter that initiates transcription only when the host cell is exposed to a certain external stimulus. In the case of a multicellular organism, the promoter may also be specific to a particular tissue or organ or developmental stage.

[0117] The vector may be a plasmid, which may be useful for transfecting cells with a nucleic acid encoding the antigen, and the transformed host cells are cultured and maintained under conditions in which expression of the antigen occurs.

[0118] The plasmid may contain a nucleic acid sequence encoding one or more of the various antigens disclosed above, including coding sequences encoding synthetic consensus antigens capable of eliciting an immune response against the antigen, fragments of such proteins, variants of such proteins, fragments of variants or fusion proteins, which are composed of combinations of consensus proteins and / or fragments of consensus proteins and / or variants of consensus proteins and / or fragments of variants of consensus proteins.

[0119] In some embodiments, the plasmids may further comprise coding sequences encoding CCR20, either alone or as part of these plasmids. Similarly, the plasmids may further comprise coding sequences for IL-12, IL-15, and / or IL-28.

[0120] The plasmid may further comprise a start codon, which may be upstream of the coding sequence, and a stop codon, which may be downstream of the coding sequence. The start and stop codons may be in frame with the coding sequence.

[0121] The plasmid may also include a promoter operably linked to the coding sequence. The promoter operably linked to the coding sequence may be a promoter from a human immunodeficiency virus (HIV) promoter, such as a simian virus 40 (SV40), mouse mammary tumor virus (MMTV) promoter, a bovine immunodeficiency virus (BIV) long terminal repeat (LTR) promoter, a Moloney virus promoter, an avian leukosis virus (ALV) promoter, a cytomegalovirus (CMV) promoter, such as a CMV immediate early promoter (hCMV promoter), an Epstein-Barr virus (EBV) promoter, or a Rous sarcoma virus (RSV) promoter. The promoter may also be a promoter from a human gene, such as human actin, human myosin, human hemoglobin, human muscle creatine, or human metalothionein. The promoter may also be a tissue-specific promoter, such as a muscle or skin specific promoter, natural or synthetic. Examples of such promoters are described in U.S. Patent Application Publication No. US2004 / 0175727, the contents of which are incorporated herein in their entirety.

[0122] The plasmid may also include a polyadenylation signal, which may be downstream of the coding sequence. The polyadenylation signal may be an SV40 polyadenylation signal, an LTR polyadenylation signal, a bovine growth hormone (bGH) polyadenylation signal, a human growth hormone (hGH) polyadenylation signal, a human β-globin polyadenylation signal, or a bovine growth hormone polyadenylation signal (bGH polyA). The SV40 polyadenylation signal may be the polyadenylation signal from pCEP4 plasmid (Invitrogen, San Diego, CA).

[0123] The plasmid may also contain an enhancer upstream of the coding sequence.The enhancer may be human actin, human myosin, human hemoglobin, human muscle creatine, or a viral enhancer such as that from CMV, FMDV, RSV, or EBV.Polynucleotide function enhancer is described in U.S. Patent No. 5,593,972, U.S. Patent No. 5,962,428, and WO94 / 016737, the contents of each of which are fully incorporated by reference.

[0124] The plasmid may also contain a mammalian origin of replication to maintain the plasmid extrachromosomally and produce multiple copies of the plasmid in the cell. The plasmid may be pVAXI, pCEP4, or pREP4 from Invitrogen (San Diego, CA), which may contain the Epstein-Barr virus origin of replication and the nuclear antigen EBNA-1 coding region to produce high copy number episomal replicatives without integration. The backbone of the plasmid may be pA V0242. The plasmid may be a replication-deficient adenovirus type 5 (Ad5) plasmid.

[0125] The plasmid may also contain control sequences that may be well suited for gene expression in a cell into which the plasmid is administered. The coding sequence may contain codons that may allow for more efficient transcription of the coding sequence in the host cell.

[0126] The coding sequence may also include an immunoglobulin (Ig) leader sequence. The leader sequence may be 5'' of the coding sequence. The consensus antigen encoded by this sequence may include an N-terminal Ig leader followed by the consensus antigen protein. The N-terminal Ig leader may be IgE or IgG.

[0127] The plasmid may be pSE420 (Invitrogen, San Diego, Calif.), which may be used for protein production in Escherichia coli. The plasmid may also be p-YES2 (Invitrogen, San Diego, Calif.), which may be used for protein production in Saccharomyces cerevisiae strains of yeast. The plasmid may also be of the MAXBAC™ Complete Baculovirus Expression System (Invitrogen, San Diego, Calif.), which may be used for protein production in insect cells. The plasmid may also be pcDNA I or pcDNA3 (Invitrogen, San Diego, Calif.), which may be used for protein production in mammalian cells such as Chinese Hamster Ovary (CHO) cells. The plasmid may also be pGX001 (Inovio), which is modified from pVAX1 (Thermo Fisher Scientific, Waltham, Mass.).

[0128] Vectors can be circular plasmids (eg, autonomously replicating plasmids having an origin of replication) which transform target cells by integrating into the cellular genome, or which can exist extrachromosomally.

[0129] The vector can be pVAX, pcDNA3.0, or provax, or any other expression vector capable of expressing DNA encoding an antigen and allowing the cell to translate the sequence into an antigen recognized by the immune system.

[0130] Also provided herein is a linear nucleic acid vaccine or linear expression cassette ("LEC") that can be efficiently delivered to a subject by electroporation and express one or more desired antigens. The LEC can be any linear DNA that lacks any phosphate backbone. The DNA can encode one or more antigens. The LEC can contain a promoter, an intron, a stop codon, and / or a polyadenylation signal. The expression of the antigen can be controlled by a promoter. The LEC may not contain any antibiotic resistance gene and / or a phosphate backbone. The LEC may not contain other nucleic acid sequences unrelated to the desired antigen gene expression.

[0131] The LEC can be derived from any plasmid that can be localized. The plasmid can be capable of expressing the antigen. The plasmid can be pNP (Puerto Rico / 34) or pM2 (New Caledonia / 99). The plasmid can be WLV009, pVAX, pcDNA3.0, or provax, or any other expression vector that can express DNA encoding an antigen and allow the cell to translate the sequence into an antigen that is recognized by the immune system.

[0132] The LEC can be pcrM2. The LEC can be pcrNP. The pcrNP and pcrMR can be derived from pNP (Puerto Rico / 34) and pM2 (New Caledonia / 99), respectively.

[0133] The vector may have a promoter. The promoter may be any promoter that can drive gene expression and control the expression of the isolated nucleic acid. Such a promoter is a cis-acting sequence element that is required for transcription via DNA-dependent RNA polymerase and transcribes the antigen sequence described herein. The choice of promoter used to direct the expression of heterologous nucleic acid depends on the specific application. The promoter may be located at approximately the same distance from the transcription start of the vector as it is from the transcription start site in its natural setting. However, this difference in distance may be accommodated without losing the function of the promoter.

[0134] The promoter can be operably linked to nucleic acid sequences encoding antigens and signals required for efficient polyadenylation of the transcript, a ribosome binding site, and translation termination.

[0135] The promoter can be the CMV promoter, the SV40 early promoter, the SV40 late promoter, the metallothionein promoter, the mouse mammary tumor virus promoter, the Rous sarcoma virus promoter, the polyhedrin promoter, or another promoter shown to be effective for expression in eukaryotic cells.

[0136] The vector may contain an enhancer and an intron with functional splice donor and acceptor sites. The vector may contain a transcription termination region downstream of the structural gene to provide efficient termination. The termination region may be obtained from the same gene as the promoter sequence or may be obtained from a different gene. Methods for preparing vectors

[0137] Provided herein are methods for preparing vectors containing nucleic acid molecules encoding the mesothelin antigens discussed herein. After a final subcloning step into a mammalian expression plasmid, the vectors can be used to inoculate cell cultures in large-scale fermentation tanks using methods known in the art.

[0138] Vectors for use in the electroporation device described in more detail below can be formulated or manufactured using a combination of known devices and techniques, but are preferably manufactured using the optimized plasmid manufacturing techniques described in U.S. Patent Publication No. 2009 / 004716, filed May 23, 2008. In some examples, the DNA plasmids used in these studies can be formulated at concentrations of 10 mg / mL or higher. The manufacturing techniques also include or incorporate a variety of devices and protocols commonly known to those skilled in the art, in addition to those described in U.S. Patent Publication No. 60 / 939792, including those described in U.S. Patent No. 7,238,522, issued July 3, 2007. The above-referenced applications and patents, U.S. Patent Publication No. 2009 / 004716 and U.S. Patent No. 7,238,522, are each incorporated herein in their entirety.

[0139] Excipients and other components of the vaccine The vaccine may further comprise a pharmaceutically acceptable excipient.The pharmaceutically acceptable excipient may be a functional molecule such as vehicle, carrier or diluent.The pharmaceutically acceptable excipient may be a transfection-facilitating agent, which may include immune stimulating complexes (ISCOMS), Freund's incomplete adjuvant, LPS analogues containing monophosphoryl lipid A, muramyl peptides, quinone analogues, vesicles such as squalene and squalene, hyaluronic acid, lipids, liposomes, calcium ions, viral proteins, polyanions, polycations, or surfactants such as nanoparticles, or other known transfection-facilitating agents.

[0140] The transfection facilitating agent is a polyanion, a polycation (including poly-L-glutamate (LGS)), or a lipid. The transfection facilitating agent is poly-L-glutamate, and poly-L-glutamate may be present in the vaccine at a concentration of less than 6 mg / ml. The transfection facilitating agent may also include surfactants such as immune stimulating complexes (ISCOMS), Freund's incomplete adjuvant, LPS analogs containing monophosphoryl lipid A, muramyl peptides, quinone analogs, and vesicles such as squalene and squalene, and hyaluronic acid administered with the gene construct may also be used. The DNA plasmid vaccine may also include transfection facilitating agents such as lipids, liposomes (including lecithin liposomes or other liposomes known in the art as DNA-liposome mixtures) (see, for example, WO9324640), calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other known transfection facilitating agents. The transfection facilitating agent is a polyanion, polycation (including poly-L-glutamate (LGS)), or lipid. The concentration of the transfection agent in the vaccine is less than 4 mg / ml, less than 2 mg / ml, less than 1 mg / ml, less than 0.750 mg / ml, less than 0.500 mg / ml, less than 0.250 mg / ml, less than 0.100 mg / ml, less than 0.050 mg / ml, or less than 0.010 mg / ml.

[0141] The pharma- ceutically acceptable excipient may be one or more adjuvants, which may be other genes expressed in alternative plasmids or delivered as proteins in combination with the above plasmids in the vaccine.The one or more adjuvants may be selected from the group consisting of CCL20, alpha-interferon (IFN-α), beta-interferon (IFN-β), gamma-interferon, platelet-derived growth factor (PDGF), tumor necrosis factor-like peptide (TNFα), tumor necrosis factor-beta (TNFβ), GM-CSF, epidermal growth factor (EGF), cutaneous T cell-attracting chemokine (CTACK), epithelial thymus-expressed chemokine (TECK), mucosa-associated epithelial chemokine (MEC), IL-12, IL-15, IL-28, IL-31, IL-33, MHC, CD80, CD86, IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-18, MCP-1, MIP-1a, MIP-1β, IL-8, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, Ma dCAM-1, LFA-1, VLA-1, Mac-1, pl50.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, variant of IL-18, CD40, CD40L, vascular growth factor, fibroblast growth factor, IL-7, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Flt, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DRS, KILLER, TRAIL-R2, TRICK2, DR6, caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, inactive NIK, SAP The signal sequence or coding sequence encoding a deleted signal sequence may be selected from the group consisting of K, SAP-I, JNK, interferon response genes, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, Ox40, Ox40 LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAPI, TAP2, IL-15 with a signal sequence or coding sequence encoding a deleted signal sequence and optionally including a different signal peptide such as from IgE, or a coding sequence encoding a different signal peptide such as from IgE, and functional fragments thereof, or combinations thereof.The adjuvant can be IL-12, IL-15, IL-28, CTACK, TECK, platelet-derived growth factor (PDGF), TNFα, TNRβ, GM-CSF, epidermal growth factor (EGF), IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-18, or a combination thereof.

[0142] In some embodiments, the adjuvant may be one or more proteins and / or nucleic acid sequences encoding a protein selected from the group consisting of CCL-20, IL-12, IL-15, IL-28, CTACK, TECK, MEC, or RANTES. Examples of IL-12 constructs and sequences are disclosed in PCT Application No. PCT / US1997 / 019502 and corresponding U.S. Application No. 08 / 956,865, and U.S. Provisional Application No. 61 / 569600, filed December 12, 2011, each of which is incorporated herein by reference. Examples of IL-15 constructs and sequences are disclosed in PCT Application No. PCT / US04 / 18962 and corresponding US Application No. 10 / 560,650, and PCT Application No. PCT / US07 / 00886 and corresponding US Application No. 12 / 160,766, and PCT Application No. PCT / USI0 / 048827, each of which is incorporated herein by reference. Examples of IL-28 constructs and sequences are disclosed in PCT Application No. PCT / US09 / 039648 and corresponding US Application No. 12 / 936,192, each of which is incorporated herein by reference. Examples of RANTES and other constructs and sequences are disclosed in PCT Application No. PCT / US1999 / 004332 and corresponding US Application No. 09 / 622452, each of which is incorporated herein by reference. Other examples of RANTES constructs and sequences are disclosed in PCT Application No. PCT / US11 / 024098, which is incorporated herein by reference. Examples of RANTES and other constructs and sequences are disclosed in PCT Application No. PCT / US1999 / 004332 and corresponding US Application No. 09 / 622452, each of which is incorporated herein by reference. Other examples of RANTES constructs and sequences are disclosed in PCT Application No. PCT / US11 / 024098, which is incorporated herein by reference. Examples of chemokine CTACK, TECK, and MEC constructs and sequences are disclosed in PCT Application No. PCT / US2005 / 042231 and corresponding US Application No. 11 / 719,646, each of which is incorporated herein by reference.Examples of OX40 and other immunomodulators are disclosed in U.S. Application No. 10 / 560,653, which is incorporated herein by reference. Examples of DR5 and other immunomodulators are disclosed in U.S. Application No. 09 / 622452, which is incorporated herein by reference.

[0143] Other genes that may be useful as adjuvants include MCP-1, MIP-1a, MIP-1p, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, pl50.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, mutated forms of IL-18, CD40, CD40L, vascular growth factor. , fibroblast growth factor, IL-7, IL-22, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Flt, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR 5, KILLER, TRAIL-R2, TRICK2, DR6, caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, inactive NIK, SAP These include those encoding K, SAP-1, JNK, interferon response genes, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, Ox40, Ox40 LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1, TAP2, and functional fragments thereof.

[0144] The vaccine may further comprise a genetic vaccine facilitator as described in US Ser. No. 021,579, filed Apr. 1, 1994, which is incorporated by reference in its entirety.

[0145] The vaccine may contain the antigen-encoding vector in an amount of about 1 nanogram to about 100 milligrams, about 1 microgram to about 10 milligrams, or preferably about 0.1 microgram to about 10 milligrams, or more preferably about 1 milligram to about 2 milligrams. In some preferred embodiments, the vaccine according to the present invention contains about 5 nanograms to about 1000 micrograms of nucleic acid. In some preferred embodiments, the vaccine may contain about 10 nanograms to about 800 micrograms of nucleic acid. In some preferred embodiments, the vaccine may contain about 0.1 to about 500 micrograms of nucleic acid. In some preferred embodiments, the vaccine may contain about 1 to about 350 micrograms of nucleic acid. In some preferred embodiments, the vaccine may contain about 25 to about 250 micrograms, about 100 to about 200 micrograms, about 1 nanogram to 100 milligrams, about 1 microgram to about 10 milligrams, about 0.1 micrograms to about 10 milligrams, about 1 milligram to about 2 milligrams, about 5 nanograms to about 1000 micrograms, about 10 nanograms to about 800 micrograms, about 0.1 to about 500 micrograms, about 1 to about 350 micrograms, about 25 to about 250 micrograms, or about 100 to about 200 micrograms of the antigen or its plasmid.

[0146] The vaccine may be formulated according to the mode of administration to be used. The injectable vaccine pharmaceutical composition may be sterile, pyrogen-free, and particulate free. An isotonic formulation or solution may be used. Additives for isotonicity may include sodium chloride, dextrose, mannitol, sorbitol, and lactose. The vaccine may include a vasoconstriction agent. The isotonic solution may include phosphate buffered saline. The vaccine may further include a stabilizer including gelatin and albumin. The stabilizer including LGS or a polycation or polyanion may make the formulation stable at room or ambient temperature for an extended period of time.

[0147] Pharmaceutical Compositions of Vaccines The vaccine may be in the form of a pharmaceutical composition. The pharmaceutical composition may comprise the vaccine. The pharmaceutical composition may comprise about 5 nanograms (ng) to about 10 milligrams (mg) of the vaccine nucleic acid molecule. In some embodiments, the pharmaceutical composition according to the present invention comprises about 25 ng to about 5 mg of the vaccine nucleic acid molecule. In some embodiments, the pharmaceutical composition comprises about 50 ng to about 1 mg of the vaccine nucleic acid molecule. In some embodiments, the pharmaceutical composition comprises about 0.1 to about 500 micrograms of the vaccine nucleic acid molecule. In some embodiments, the pharmaceutical composition comprises about 1 to about 350 micrograms of the vaccine nucleic acid molecule. In some embodiments, the pharmaceutical composition comprises about 5 to about 250 micrograms of the vaccine nucleic acid molecule. In some embodiments, the pharmaceutical composition comprises about 10 to about 200 micrograms of the vaccine nucleic acid molecule. In some embodiments, the pharmaceutical composition comprises about 15 to about 150 micrograms of the vaccine nucleic acid molecule. In some embodiments, the pharmaceutical composition comprises about 20 to about 100 micrograms of the vaccine nucleic acid molecule. In some embodiments, the pharmaceutical composition comprises about 25 to about 75 micrograms of the vaccine nucleic acid molecule. In some embodiments, the pharmaceutical composition comprises about 30 to about 50 micrograms of the vaccine nucleic acid molecule. In some embodiments, the pharmaceutical composition comprises about 35 to about 40 micrograms of the vaccine nucleic acid molecule. In some embodiments, the pharmaceutical composition comprises about 100 to about 200 micrograms of the vaccine nucleic acid molecule. In some embodiments, the pharmaceutical composition comprises about 10 micrograms to about 100 micrograms of the vaccine nucleic acid molecule. In some embodiments, the pharmaceutical composition comprises about 20 micrograms to about 80 micrograms of the vaccine nucleic acid molecule. In some embodiments, the pharmaceutical composition comprises about 25 micrograms to about 60 micrograms of the vaccine nucleic acid molecule. In some embodiments, the pharmaceutical composition comprises about 30 ng to about 50 micrograms of the vaccine nucleic acid molecule. In some embodiments, the pharmaceutical composition comprises about 35 ng to about 45 micrograms of the vaccine nucleic acid molecule.In some preferred embodiments, the pharmaceutical composition comprises about 0.1 to about 500 micrograms of the vaccine nucleic acid molecule. In some preferred embodiments, the pharmaceutical composition comprises about 1 to about 350 micrograms of the vaccine nucleic acid molecule. In some preferred embodiments, the pharmaceutical composition comprises about 25 to about 250 micrograms of the vaccine nucleic acid molecule. In some preferred embodiments, the pharmaceutical composition comprises about 100 to about 200 micrograms of the vaccine nucleic acid molecule.

[0148] In some embodiments, a pharmaceutical composition according to the invention comprises at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 ng of a vaccine nucleic acid molecule. , 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 605, 610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695, 700, 705, 710, 715, 720, 725, 730, 735, 740, 745, 750, 755, 760, 765, 770, 775, 780, 785, 790, 795, 8 In one embodiment, the vaccine may comprise 00, 805, 810, 815, 820, 825, 830, 835, 840, 845, 850, 855, 860, 865, 870, 875, 880, 885, 890, 895, 900, 905, 910, 915, 920, 925, 930, 935, 940, 945, 950, 955, 960, 965, 970, 975, 980, 985, 990, 995, or 1000 micrograms of the vaccine nucleic acid molecule. In some embodiments, a pharmaceutical composition may comprise at least 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 mg or more of a nucleic acid molecule of a vaccine.

[0149] In other embodiments, the pharmaceutical composition may contain up to 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 ng (inclusive) of a vaccine nucleic acid molecule. In some embodiments, the pharmaceutical composition comprises up to 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470 , 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, 8 The vaccine may contain 10, 815, 820, 825, 830, 835, 840, 845, 850, 855, 860, 865, 870, 875, 880, 885, 890, 895, 900, 905, 910, 915, 920, 925, 930, 935, 940, 945, 950, 955, 960, 965, 970, 975, 980, 985, 990, 995, or 1000 micrograms (inclusive) of the nucleic acid molecule of the vaccine. In some embodiments, a pharmaceutical composition may contain up to 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 mg (inclusive) of a nucleic acid molecule of the vaccine.

[0150] The pharmaceutical composition may further include other agents for formulation purposes depending on the mode of administration used. When the pharmaceutical composition is an injectable pharmaceutical composition, it is sterile, pyrogen-free, and particulate-free. It is preferable to use an isotonic formulation. In general, additives for isotonicity may include sodium chloride, dextrose, mannitol, sorbitol, and lactose. In some cases, an isotonic solution such as phosphate buffered saline is preferred. Stabilizers include gelatin and albumin. In some embodiments, a vasoconstrictor is added to the formulation.

[0151] The pharmaceutical composition may further comprise a pharma- ceutically acceptable excipient as provided above. For example, the pharma- ceutically acceptable excipient may comprise a functional molecule, a vehicle, an adjuvant, a carrier, a diluent, or a transfection-facilitating agent as provided above.

[0152] Vaccination Method Provided herein are methods for treating and / or preventing mesothelin-expressing cancer, such as, but not limited to, ovarian cancer, using the pharmaceutical formulations described above. Also described herein are methods for using the pharmaceutical formulations described above to treat and / or prevent mesothelin-expressing cancer, such as, but not limited to, ovarian cancer, in a subject. Also described herein are methods for vaccinating a subject. Also described herein are methods for administering a pharmaceutical formulation described herein to a subject in need thereof. The methods described herein, collectively referred to as therapeutic methods using the pharmaceutical formulations described herein, may include administering one or more vaccines described herein to a subject in need thereof to induce a therapeutic and / or prophylactic immune response. The vaccine 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 the transfection of a cancer antigen disclosed herein as a nucleic acid molecule that is expressed in a cell and delivered to the surface of the cell upon recognition by the immune system and induction of a cellular, humoral, or cellular and humoral response. Administration of a vaccine can be used to induce or elicit an immune response in a subject against mesothelin by administering to the subject a vaccine discussed herein.

[0153] The vaccine may be administered to a subject to modulate the activity of the subject's immune system, thereby enhancing the immune response. In some embodiments, the subject is a mammal. When the vaccine is administered to a mammal, thereby introducing the vector into the mammal's cells, the transfected cells express and secrete one or more of the cancer antigens disclosed herein. These secreted proteins or synthetic antigens are recognized as foreign by the immune system, which initiates an immune response that may include antibodies directed against one or more cancer antigens, and a T cell response specifically against one or more cancer antigens. In some examples, a mammal vaccinated with a vaccine discussed herein has a primed immune system, and when inoculated with one or more cancer antigens disclosed herein, the primed immune system allows for rapid clearance of the cancer antigens thereafter, whether by humoral, cellular, or both cellular and humoral immune responses, as disclosed herein.

[0154] Methods for administering DNA vaccines are described in US Pat. Nos. 4,945,050 and 5,036,006, both of which are incorporated herein by reference in their entireties.

[0155] The vaccine can be administered to mammals to induce immune response in mammals.The mammals can be human, non-human primates, cows, pigs, sheep, goats, antelopes, bison, buffalo, cattle, deer, hedgehogs, elephants, llamas, alpacas, mice, rats, preferably human, cattle, or pigs.The vaccine can also be administered to non-mammalian subjects, such as chickens, to induce immune response.

[0156] The dose of the vaccine can be from 1 microgram to 10 mg of active component per kilogram (kg) body weight over time (component / kg body weight / hour), and can be from 20 micrograms to 10 mg of component / kg body weight / hour. The vaccine can be administered every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days. The number of vaccine doses for effective treatment can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more doses.

[0157] Methods for generating an immune response with a vaccine Vaccine can be used to generate immune response in mammalian or non-mammalian subjects, including therapeutic or preventive immune response.Immune response can generate antibodies and / or killer T cells directed to one or more cancer antigens disclosed herein.Such antibodies and T cells can be isolated.

[0158] Some embodiments provide a method of generating an immune response against one or more of the cancer antigens disclosed herein, the embodiment of which comprises administering a vaccine to a subject. Some embodiments provide a method of prophylactically vaccinating a subject against a cancer or tumor expressing one or more of the above mesothelin antigens, the embodiment of which comprises administering a vaccine. Some embodiments provide a method of therapeutically vaccinating a subject suffering from an ovarian cancer or tumor expressing mesothelin, the embodiment of which comprises administering a vaccine. Diagnosis of ovarian cancer or tumor expressing one or more mesothelin antigens disclosed herein prior to administration of a vaccine can be routinely performed.

[0159] How vaccines can treat cancer The vaccine can be used to generate or elicit an immune response in a mammal that is reactive or directed against cancers that express mesothelin, such as, but not limited to, ovarian cancer, more particularly epithelial ovarian cancer, and most particularly serous ovarian cancer. The elicited immune response can prevent the growth of ovarian cancer or tumors.

[0160] The induced immune response can prevent and / or reduce metastasis of cancerous or tumor cells in subjects suffering from ovarian cancer. Thus, the vaccine can be used in methods for treating and / or preventing cancer or tumors in mammals or subjects suffering from cancer to which the vaccine is administered.

[0161] In some embodiments, the administered vaccine induces (1) humoral immunity through a B cell response to generate antibodies that block the production of monocyte chemotactic protein-1 (MCP-1), thereby slowing down myeloid-derived suppressor cells (MDSCs) and suppressing tumor growth; and (2) CD8 to attack and kill tumor cells. +(2) increase in cytotoxic T lymphocytes (CTLs) such as IL-1, IL-2, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-36, IL-37, IL-38, IL-39, IL-40, IL-41, IL-42, IL-43, IL-44, IL-45, IL-16, IL-17, IL-18, IL-19, IL-21, IL-22, IL-23, IL-24, IL-25, IL-39, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, IL-37, IL-38, IL-39, IL-40, IL-41, IL-42, IL-43, IL-44, IL-18, IL-19 ... The vaccine can reduce tumor burden 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 or block the increase in monocyte chemoattractant protein 1 (MCP-1), a cytokine secreted by myeloid-derived suppressor cells. The vaccine can enhance tumor survival by 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, and 60%.

[0162] In some embodiments, the immune response is capable of generating a humoral immune response and / or an antigen-specific cytotoxic T lymphocyte (CTL) response in a subject receiving the vaccine that does not damage or result in inflammation of various tissues or systems (e.g., the brain or nervous system, etc.).

[0163] In some embodiments, the vaccine may be administered peripherally to establish an antigen-specific immune response that targets cancerous or tumor cells or tissues to eliminate or eliminate cancers or tumors expressing one or more cancer antigens without causing damage or illness or death in the subject administered the vaccine (described in more detail below).

[0164] Administration of the vaccine can increase the cellular immune response in a subject by about 50-fold to about 6,000-fold, about 50-fold to about 5,500-fold, about 50-fold to about 5,000-fold, about 50-fold to about 4,500-fold, about 100-fold to about 6,000-fold, about 150-fold to about 6,000-fold, about 200-fold to about 6,000-fold, about 250-fold to about 6,000-fold, or about 300-fold to about 6,000-fold. In some embodiments, administration of the vaccine increases or decreases the cellular immune response in the subject by about 50 fold, 100 fold, 150 fold, 200 fold, 250 fold, 300 fold, 350 fold, 400 fold, 450 fold, 500 fold, 550 fold, 600 fold, 650 fold, 700 fold, 750 fold, 800 fold, 850 fold, 900 fold, 950 fold, 1000 fold, 1100 fold, 1200 fold, 1300 fold, 1400 fold, 1500 fold, 1600 fold, 1700 fold, 1800 fold, 1900 fold, 2000 fold, 2100 fold, 2200 fold, 2300 fold, 2400 fold, 2500 fold, 2600 fold, 2700 fold, 2800 fold, 2900 fold, 3000 fold, 3100 fold, 3200 fold, 3300 fold, 3400 fold, 3500 fold, 3600 fold, 3700 fold, 3800 fold, 3900 fold, 4000 fold, 4100 fold, 4200 fold, 4300 fold, 4400 fold, 4500 fold, 4600 fold, 4700 fold, 4800 fold, 4900 fold, 5000 fold, 5100 fold, 5200 fold, 5300 fold, 5400 fold, 2300x, 2400x, 2500x, 2600x, 2700x, 2800x, 2900x, 3000x, 3100x, 3200x, 3300x, 3400x, 3500x, 3600x, 3700x, 3800x, 3900x, 4000x, 4100x, 4200x, 4300x, 4400x, 4500x, 4600x, 4700x, 4800x, 4900x, 5000x, 5100x, 5200x, 5300x, 5400x, 5500x, 5600x, 5700x, 5800x, 5900x, or 6000x.

[0165] Administration of the vaccine can increase interferon gamma (IFN-γ) levels in a subject by about 50-fold to about 6000-fold, about 50-fold to about 5500-fold, about 50-fold to about 5000-fold, about 50-fold to about 4500-fold, about 100-fold to about 6000-fold, about 150-fold to about 6000-fold, about 200-fold to about 6000-fold, about 250-fold to about 6000-fold, or about 300-fold to about 6000-fold. In some embodiments, administration of the vaccine increases IFN-γ levels in the subject by about 50 fold, 100 fold, 150 fold, 200 fold, 250 fold, 300 fold, 350 fold, 400 fold, 450 fold, 500 fold, 550 fold, 600 fold, 650 fold, 700 fold, 750 fold, 800 fold, 850 fold, 900 fold, 950 fold, 1000 fold, 1100 fold, 1200 fold, 1300 fold, 1400 fold, 1500 fold, 1600 fold, 1700 fold, 1800 fold, 1900 fold, 2000 fold, 2100 fold, 220 fold, 230 fold, 240 fold, 250 fold, 260 fold, 270 fold, 280 fold, 290 fold, 300 fold, 310 fold, 320 fold, 330 fold, 340 fold, 350 fold, 360 fold, 370 fold, 380 fold, 390 fold, 400 fold, 410 fold, 420 fold, 430 fold, 440 fold, 450 fold, 460 fold, 470 fold, 480 fold, 490 fold, 500 fold, 500 fold, 510 fold, 520 fold, 530 fold, 540 fold, 550 fold, 560 fold, 570 fold, 580 The increase can be 0x, 2300x, 2400x, 2500x, 2600x, 2700x, 2800x, 2900x, 3000x, 3100x, 3200x, 3300x, 3400x, 3500x, 3600x, 3700x, 3800x, 3900x, 4000x, 4100x, 4200x, 4300x, 4400x, 4500x, 4600x, 4700x, 4800x, 4900x, 5000x, 5100x, 5200x, 5300x, 5400x, 5500x, 5600x, 5700x, 5800x, 5900x, or 6000x.

[0166] The dose of the vaccine can be from 1 microgram to 10 mg of active component per kilogram (kg) body weight over time (component / kg body weight / hour), and can be from 20 micrograms to 10 mg of component / kg body weight / hour. The vaccine can be administered every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days. The number of vaccine doses for effective treatment can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more doses.

[0167] Route of administration The vaccine or pharmaceutical composition may be administered orally, parenterally, sublingually, transdermally, rectally, transmucosally, topically, by inhalation, by buccal administration, intrapleurally, intravenously, intraarterially, intraperitoneally, subcutaneously, intramuscularly, intranasally, intrathecally, and / or intraarticularly, or combinations thereof. For veterinary use, the composition may be administered in an appropriately tolerated formulation in accordance with normal veterinary practice. A veterinarian can readily determine the administration regimen and route of administration most suitable for a particular animal. The vaccine may be administered by conventional syringes, needleless injection devices, "microprojectile bombardment gene guns," or other physical methods such as electroporation ("EP"), "hydrodynamic methods," or ultrasound.

[0168] The vaccine vector can be administered to a mammal by several well-known techniques, including DNA injection with or without in vivo electroporation (also called DNA vaccination), liposome-mediated transfection, nanoparticle facilitated transfection, and recombinant vectors such as recombinant adenovirus, recombinant adenovirus-associated virus, and recombinant vaccinia. One or more cancer antigens of the vaccine can be administered via DNA injection together with in vivo electroporation.

[0169] Electroporation The vaccine or pharmaceutical composition may be administered by electroporation. The administration of the vaccine by electroporation may be accomplished using an electroporation device that may be configured to deliver a pulse of energy to the desired mammalian tissue that is effective to form reversible pores in the cell membrane, preferably the pulse of energy is a constant current similar to the pre-adjusted current input by the user. The electroporation device may include an electroporation component and an electrode assembly or a handle assembly. The electroporation component may include or incorporate one or more of the various elements of the electroporation device, including a controller, a current waveform generator, an impedance tester, a waveform logger, an input element, a status reporting element, a communication port, a memory component, a power source, and a power switch. Electroporation may be accomplished using an in vivo electroporation device, such as the CELLECTRA® EP system (Inovio Pharmaceuticals, Inc., Blue Bell, PA) or the Elgen electroporator (Inovio Pharmaceuticals, Inc.), to facilitate the transfection of cells with plasmids.

[0170] Examples of electroporation devices and electroporation methods that can facilitate administration of the DNA vaccines of the present invention include those described in U.S. Patent No. 7,245,963 by Draghia-Akli et al., and U.S. Patent Publication No. 2005 / 0052630 by Smith et al., the contents of which are incorporated herein by reference in their entireties. Other electroporation devices and electroporation methods that can be used to facilitate administration of the DNA vaccines include those provided in U.S. Provisional Application No. 60 / 852,149, filed October 17, 2006, and co-pending and co-owned U.S. Patent Application No. 11 / 874,072, filed October 17, 2007, which claims the benefit under 35 USC 119(e) to U.S. Provisional Application No. 60 / 978,982, filed October 10, 2007, all of which are incorporated herein in their entireties.

[0171] U.S. Patent No. 7,245,963 by Draghia-Akli et al. describes modular electrode systems and their use to facilitate the introduction of biomolecules into cells of selected tissues of a body or plant. The modular electrode system may include multiple needle electrodes, a hypodermic needle, an electrical connector providing a conductive link from a programmable constant current pulse controller to the multiple needle electrodes, and a power source. An operator can grasp the multiple needle electrodes mounted on a support structure and firmly insert them into selected tissues of a body or plant. The biomolecules are then administered into the selected tissues by the hypodermic needle. The programmable constant current pulse controller is activated to apply a constant current electrical pulse to the multiple needle electrodes. The applied constant current electrical pulse facilitates the introduction of the biomolecules into cells between the multiple electrodes. The entire contents of U.S. Patent No. 7,245,963 are incorporated herein by reference in their entirety.

[0172] US Patent Publication No. 2005 / 0052630, filed by Smith et al., describes an electroporation device that can be used to effectively facilitate the introduction of biomolecules into cells of selected tissues of the body or plant. The electroporation device includes an electrokinetic device ("EKD device") whose operation is specified by software or firmware. The EKD device creates a series of programmable constant current pulse patterns between a row of electrodes and pulse parameters based on user control, allowing for the storage and retrieval of current waveform data. The electroporation device also includes a replaceable electrode disk with a series of needle electrodes, a central injection channel for an injection needle, and a removable guide disk. The entire contents of US Patent Publication No. 2005 / 0052630 are incorporated herein by reference in their entirety.

[0173] The electrode arrays and methods described in U.S. Patent No. 7,245,963 and U.S. Patent Publication No. 2005 / 0052630 are suitable for deep penetration of tissues such as muscle as well as other tissues or organs. The configuration of the electrode array allows the injection needle to be fully inserted into the target organ and injected perpendicularly into the target tissue in the area pre-delineated by the electrode. The electrodes described in U.S. Patent No. 7,245,963 and U.S. Patent Publication No. 2005 / 005263 are preferably 20 mm long and 21 gauge.

[0174] In addition, there are electroporation devices described in the following patents that are contemplated in some embodiments incorporating electroporation devices and their use: U.S. Patent No. 5,273,525 issued December 28, 1993, U.S. Patent No. 6,110,161 issued August 29, 2000, U.S. Patent No. 6,261,281 issued July 17, 2001, and U.S. Patent No. 6,958,060 issued October 25, 2005, and U.S. Patent No. 6,939,862 issued September 6, 2005. Additionally, patents covering subject matter provided in U.S. Patent No. 6,697,669 issued February 24, 2004, which relates to administration of DNA using any of a variety of devices, and U.S. Patent No. 7,328,064 issued February 5, 2008, which focuses on methods of injecting DNA, are contemplated herein. The above patents are incorporated by reference in their entirety.

[0175] Methods for preparing a vaccine The method for preparing the DNA plasmids discussed herein is provided herein.After the final subcloning step into mammalian expression plasmid, the DNA plasmids can be used to inoculate cell cultures in large-scale fermentation tanks using methods known in the art.

[0176] The DNA plasmids for use in the electroporation device of the present invention can be formulated or manufactured using a combination of known devices and techniques, but are preferably manufactured using the optimized plasmid manufacturing techniques described in U.S. Published Application No. 2009 / 0004716, filed May 23, 2007. In some examples, the DNA plasmids used in these studies can be formulated at a concentration of 10 mg / mL or higher. The manufacturing techniques include or incorporate a variety of devices and protocols commonly known to those skilled in the art, in addition to those described in U.S. 60 / 939792, including those described in U.S. 7,238,522, a licensed patent issued July 3, 2007. The above-referenced applications and patents, U.S. 60 / 939,792 and U.S. 7,238,522, respectively, are incorporated herein in their entirety.

[0177] The present invention has many aspects, which are illustrated by the following non-limiting examples. EXAMPLES

[0178] The present invention is further illustrated in the following examples. It should be understood that these embodiments, while showing preferred embodiments of the present invention, are shown for illustration purposes only. From the above discussion and these examples, those skilled in the art can ascertain the essential features of the present invention, and can make various changes and modifications of the present invention to adapt it to various uses and conditions without departing from its spirit and scope. Thus, in addition to those shown and described herein, various modifications of the present invention will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

[0179] Example 1: Generation of a consensus mesothelin sequence Three mesothelin variants have been reported in GenBank. Variants 1 and 2 are 98% identical at the nucleotide level, and variant 3 is a partial sequence with a C-terminus that splits the GPI anchor region by alternative splicing. All three transcript variants have been reported to be expressed by human cancer cells. Based on sequence analysis, a vaccine targeting variant 1 may also target most of the sequences in variants 2 and 3. In addition, variant 1 is the major transcript expressed in ovarian tumors. Therefore, variant 1 was selected as a vaccine target.

[0180] To generate the human consensus mesothelin, 14 human mesothelin sequences were collected from GenBank (www.ncbi.nlm.nih.gov / genbank / ). The GenBank accession numbers of the selected mesothelin sequences are NR_005814.2, BAA08419.1, AAH09272.1, AAV87530.1, AAH03512.1, XP_008959182.1, AAC50348.1, XP_009428309.1, XP_007978969.1, XP_011822299.1, XP_011817841.1, XP_011822298.1, CR_009193880.1, and XP_011817843.1.

[0181] The consensus sequence was generated using the DNASTAR® Lasergene software package (version 13.0.0.357). Mesothelin sequences from GenBank were imported into MegAlign and aligned using the ClustalW multiple sequence alignment program. The resulting mesothelin consensus sequence (SEQ ID NO: 1) shares 95.8% homology with native human mesothelin.

[0182] Example 2: Introduction of mutations to abolish mesothelin function To abolish potential biological functions of the resulting consensus mesothelin protein, one mutation was introduced to abolish MUC16 / CA125 binding. In addition, two mutations were introduced to disrupt GPI attachment. The rationale for introducing these mutations is described below.

[0183] MUC16 / CA125 binding mutations Deletion mutagenesis was used to identify the binding site in mesothelin for MUC16 / CA125 (Figure 1). As shown in Figure 1, the mesothelin precursor (71 kDa) is cleaved into two products, the 30 kDa megakaryocyte potentiating factor (MPF; residues Ser34-Arg286) and the 41 kDa GPI-anchored membrane-bound mature mesothelin (light grey) starting at Glu296. The proteolytic cleavage region (shaded grey) contains a furin cleavage site at Arg295, and the other protease cleavage site contains a trypsin cleavage site at Arg286. The four predicted N-linked glycans in mesothelin (black lollipop; Asn57, Asn388, Asn488, and Asn515) are shown. Deletion mutants (regions I, II, III, IAB, IBC, IA, IB, and IC) were generated as rabbit Fc fusion proteins to sequentially narrow down the mesothelin CA125-binding domain.

[0184] As shown in Figure 5 of Kaneko et al, J Biol Chem, 2009 Feb 6;284(6):3739-49, substitution of tyrosine at position 318 (Y318A) with alanine completely abolished the interaction with CA125. Alanine mutations at Glu324 (E324A; KD 42.4 nM) and Trp321 (W321A; KD 19.5 nM) reduced mesothelin binding to CA125. Alanine mutation at His354 (H354A) did not change the mesothelin-CA125 interaction (KD 2.71 nM).

[0185] As shown in Figure 4 of Kaneko et al, J Biol Chem, 2009 Feb 6;284(6):3739-49, Western blots of alanine mutations within region IAB(296-359) show differential binding. Alanine mutations at Tyr318 (Y318A) and Glu324 (E324A) abolished mesothelin binding to CA125. Alanine mutations at Trp321 (W321A) partially reduced mesothelin binding to CA125. Alanine mutations at His354 did not alter mesothelin-CA125 interaction.

[0186] Finally, the fluorescence intensity (geometric mean) was used to quantitatively measure the binding of CA125, as shown in Figure 6 of Kaneko et al, J Biol Chem, 2009 Feb 6;284(6):3739-49. The binding of the full-length mature form of mesothelin (intact) to CA125 was determined as 100% binding. Region I (296-390), region IAB (296-359), and the H354A mutation in IAB (296-359) bound to CA125 significantly stronger than any other fragment or mutation listed. * , p_0.05). The Y318A mutation significantly reduced binding compared to the full-length mature form of mesothelin. Based on these findings, the Y318A mutation was introduced into the final synthetic consensus mesothelin sequence.

[0187] GPI attachment site GPI anchoring signals consist of a hydrophobic region separated from the GPI attachment site (ω site) by a hydrophilic spacer region (Figure 2). The attachment site is the first of three adjacent amino acids with short side chains. It is usually followed by a short unstructured spacer sequence, which must then be the carboxy-terminal, hydrophobic signaling sequence. Mayor, S. & Riezman, H. Nature reviews. Molecular cell biology 5 (2004). The covalent site of mesothelin was mutated from serine to threonine. The ω+2 of mesothelin was mutated from threonine to valine. As shown in Table 1, the synthetic consensus mesothelin protein sequence shares 95.2% identity with human native mesothelin. [Table 1]

[0188] Example 3: Characterization of synthetic consensus mesothelin constructs Once the synthetic consensus mesothelin DNA sequence was obtained, an upstream Kozak sequence and an IgE leader sequence were added to the N-terminus in order to have a higher level of expression. Yang, JSet al. The Journal of infectious diseases 184, 809-816 (2001). Furthermore, the codon usage of this gene was adapted to the codon bias of Homo sapiens genes. Andre, S. et al. Journal of virology 72, 1497-1503 (1998), Deml, L. et al. Journal of virology 75, 10991-11001 (2001). RNA optimization was also performed to avoid regions with very high (>80%) or very low (<30%) GC content, as well as cis-acting sequence motifs such as internal TATA boxes, chi sites, and ribosome entry sites. Muthumani, K. et al. Virology 314, 134-146 (2003), Muthumani, K. et al. Virology 314, 134-146 (2003).

[0189] A schematic of the synthetic consensus mesothelin construct is shown in FIG. * ) show mutations to abolish MUC16 binding and GPI binding. The nucleotide sequence (SEQ ID NO:1) and amino acid sequence (SEQ ID NO:2) in the synthetic consensus mesothelin are shown in Tables 16 and 17, respectively. Annotation of the elements of SEQ ID NO:2 and the corresponding amino acid positions is provided in Table 2. [Table 2]

[0190] To better understand the possible protein structure effects of the synthetic consensus design process, comparative models of the molecular precursors of MPF and mesothelin were generated. The models were aligned and generated using multiple secondary structure elements. The predicted glycosylation sites are precisely oriented on the surface of the model. The mesothelin portion of the model was extrapolated from a study done by Sathyanarayana et al. and modeled as a series of ARM repeats. Sathyanarayana, BK, Hahn, Y. et al. BMC structural biology 9, 1 (2009). The Sathyanarayana reference did not appear to address the location of the disulfide bonds. This feature is addressed in the improved model, which is precisely oriented locally. In addition, potential N-linked glycosylation sites are accessible on the surface.

[0191] The characteristics of the synthetic consensus mesothelin are summarized in Table 3. [Table 3]

[0192] Example 4: Plasmid constructs and structures The vector backbone is pGX0001, a 2998 bp modified pVAX1 expression vector under the control of the human cytomegalovirus immediate early promoter (hCMV promoter). The original pVAX1 was obtained from Thermo Fisher Scientific. The pGX0001 backbone contains a kanamycin resistance gene (KanR) and a plasmid origin of replication (pUC ori) for production purposes. These elements are not functional in eukaryotic cells. A map and description of the modified expression vector pVAX1 (pGX0001) is shown in Figure 4.

[0193] Modifications were introduced into pVAX1 to generate pGX0001. These modifications are listed in FIG. 4, and no problems were detected with respect to plasmid amplification, and antigen transcription and translation. So far, no further changes have been observed in the sequence of pGX0001 using pGX0001 as the backbone. Base pairs 2, 3, and 4 are changed from ACT to CTG in the backbone upstream of the CMV promoter. [Table 4]

[0194] The synthesized synthetic consensus mesothelin was digested with BamHI and XhoI and cloned into pGX0001 with an expression cassette placed under the transcriptional control of the cytomegalovirus immediate early promoter. The resulting plasmid was named pGX1430. Full-length sequencing was performed and then analyzed by two analytical experts to confirm correctness. A schematic diagram of pGX1430 is presented in Figure 5.

[0195] Example 5: In vitro antigen expression Expression of antigen protein by pGX1430 was confirmed by Western blotting. Human rhabdomyosarcoma (RD) cells (ATCC, CCL-136) maintained in DMEM medium containing 10% FBS (ThermoFisher) were transfected with pGX1430 or pGX0001 (6 μg / 10 cm2 dish) using Turbofectin 8 (Origene). 48 hours after transfection, cells were lysed using RIPA cell lysis buffer (ThermoFisher) and cell lysates were collected. After BCA assay (ThermoFisher) to determine total protein concentration, 15 μg of cell lysate was electroporated on 4-12% SDS-PAGE gels (ThermoFisher) and detection was performed using a commercial anti-mesothelin antibody (Cell Signaling Technology clone D4X7M) and then visualized with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (Santa Cruz Biotech #sc-2004) using an ECL Western blot analysis system (GE Healthcare). As a loading control, blots were reprobed for actin expression using anti-β-actin monoclonal antibody (Santa Cruz Biotech, clone, C4).

[0196] The human mesothelin precursor is 70 kDa with a glycosylated form truncated at 46-48 kDa, whereas the synthetic consensus mesothelin precursor is 64.28 kDa with a glycosylated form truncated at 35-37 kDa. As shown in Figure 6, a protein band of the expected molecular weight was detected for pGX1430 (64.28 kD). No protein band was detected in the pGX0001 lane. Anti-β-actin bands of similar intensity were detected, indicating that equal amounts of protein were loaded in each lane.

[0197] Example 6: Immunogenicity of synthetic consensus mesothelin vaccine constructs Animals and immunization Eight-week-old female CB6F1 mice were purchased from Jackson Laboratories. All animals were housed in a temperature-controlled, light cycle facility at BTS Research (San Diego, CA). Animal care was performed in accordance with the guidelines of the National Institutes of Health and the Animal Care and Use Proposal (ACUP) (BTS ACUP #15-091). Mice were divided into five groups as shown in Table 5. [Table 5]

[0198] Mice in the immunized groups were vaccinated with the indicated doses of pGX0001 or pGX1430 according to SOP R20-003147 CELLECTRA® 3P Mouse Treatment. Briefly, plasmids were formulated in sterile water for injection (VetOne) such that the indicated doses were 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 Constant Current Electroporation Device (Inovio Pharmaceuticals) with a 3P array. The device was configured to deliver two 0.1 Amp pulses with a 52 ms pulse width, separated by a 1 second delay. Mice were immunized three times, 3 weeks apart. Mice were sacrificed 3 weeks after the last immunization, and spleens were harvested for cellular immunity readout. No other tissues were collected.

[0199] Isolation of splenic lymphocytes Splenocytes were isolated aseptically and placed in 5 mL of R10 medium (Rosewell Park Memorial Institute medium 1640 supplemented with 10% fetal bovine serum and 1% antibiotic-antimycotic). Splenocytes were isolated by mechanically disrupting the spleen using a Stomacher instrument (Seward Laboratory Systems Inc.), and the resulting product was filtered using a 40 μm cell strainer (BD Falcon). The resulting product was centrifuged, and the pellet was treated with ACK lysis buffer (Lonza) for 5 min for lysis of RBCs. Splenocytes were then centrifuged, washed with PBS, and then resuspended in R10 medium and used immediately for further analysis. IFNγ ELISPOT

[0200] Mouse IFNγ ELISpot assay (MabTech) was performed to evaluate antigen-specific cellular responses. 96-well plates pre-coated with anti-mouse IFNγ antibodies were washed with PBS and blocked with complete medium (RPMI1640 supplemented with 10% FBS and antibiotics) for 2 hours at room temperature. Splenic lymphocytes were resuspended in R10 medium (then 2 × 10 5 Cells / well input cell counts were added in triplicate. A set of peptides was synthesized (GenScript), each containing 15 amino acid residues with 11 amino acid overlaps representing the entire synthetic consensus mesothelin protein sequence. These sets of peptides were resuspended in DMSO (Sigma) and pooled into two peptide pools at a concentration of approximately 2 μg / ml peptide. The peptide pools contained peptides corresponding to the synthetic consensus mesothelin antigen protein. 5 μg / ml concanavalin A (Sigma) was used as a positive control and complete culture medium was used as a negative control. Plates were incubated for 18 hours at 37°C in an incubator with 5% CO2 atmosphere. Biotinylated anti-mouse IFNγ detection antibody (MabTech) was then added and the plates were incubated for 2 hours at room temperature. Plates were washed and streptavidin-ALP antibody (MabTech) was added and the plates were incubated for 1 hour at room temperature.

[0201] Spot detection was completed according to the kit manufacturer's instructions (MabTech). Plate spots were counted using an automated Elispot reader (Cellular Technology). The average number of spot-forming units (SFU) was calculated using a 1 × 10 count for data presentation. 6 Spleen cells were cultured in 1×10 IFNγ ELISpot-based antigen-specific responses were assessed using 1×10 IFNγ ELISpot-based antibodies, which were greater than the SFU in the medium-only control. 6 Reported as number of IFNγ spot forming units (SFU) per splenocyte.

[0202] The immunogenicity of the synthetic consensus mesothelin construct was evaluated at four doses (10 μg, 20 μg, 30 μg, and 50 μg) by IFNγ ELISpot and flow cytometry (n=8 / group). Mice were immunized with an empty plasmid backbone (pGX0001) as a negative control (n=4 / group). Vaccination with synthetic consensus mesothelin (pGX1430) induced a cellular immune response compared to negative control vaccinated mice. The magnitude of synthetic consensus mesothelin-specific IFNγ production as determined by ELISpot was dose-independent at doses of 10 and 20 μg (Figures 7A and 7B), achieving similar maximum responses at doses of 30 and 50 μg. Specifically, the synthetic consensus mesothelin IFNγ SFUs were 1345 ± 1290, 2241 ± 1721, 2242 ± 1932, and 2004 ± 674 at 10 μg, 20 μg, 30 μg, and 50 μg, respectively. The synthetic consensus mesothelin IFNγ responses were significantly higher than naïve at doses of 10 μg (p = 0.004), 20 μg (p = 0.004), 30 μg (p = 0.004), and 50 μg (p = 0.004) of pGX1430. The IFNγ responses are summarized in Table 6. [Table 6]

[0203] Flow cytometry The cellular immune response induced by synthetic consensus mesothelin was further characterized by flow cytometry. 6 Spleen cells were stimulated immediately after isolation with a synthetic consensus mesothelin peptide for 6 hours in the presence of Brefeldin A (BD Biosciences), monensin (BD Biosciences), and FITC anti-mouse CD107a antibody (BD Biosciences). After peptide stimulation, spleen cells were spun down and resuspended in 20 μL per well of mouse BD Fc Block (BD Biosciences) solution. Fc Block was used at an initial dilution of 1:40 in PBS and incubated for 5 minutes at 4°C.

[0204] After incubation, the remaining extracellular antibodies (in PBS) are added at 30 μL per well and incubated at 4 °C for 30 min. With the addition of the extracellular strains, the final volume of each well is 50 μL, consisting of Fc Block at a final dilution of 1:100 and extracellular antibodies at their appropriate working dilutions. Cells were then stained with a 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).

[0205] ICS data was collected on a 10-color FACS CANTO (BD Biosciences) and analysis was completed using FlowJo. The flow cytometry gating strategy is shown in Figure 8. For cells to be called antigen-specific by flow cytometry, the frequency of the reported parameters must exceed the frequency of the media-only control. For cells to be identified as producing antigen-specific CD107a, cells must be identified as positive for antigen-specific production of IFNγ and / or IL-2 and / or TNFα, as identified by Boolean gating.

[0206] Synthetic consensus mesothelin binds to CD4 + Compared with responses in the T cell compartment, CD8 + Synthetic consensus mesothelin elicited a much stronger response in the T cell compartment than untreated (0.09% ± 0.07%) in the 10 μg (0.29% ± 0.20%) (p < 0.004), 20 μg (0.50% ± 0.30%) (p < 0.004), 30 μg (0.42% ± 0.24%) (p < 0.004), and 50 μg (0.38% ± 0.17%) (p < 0.004) dose groups. + The frequency of T cell responses was induced (Figure 9A). + T cell responses were dose-dependent at the 10 and 20 μg doses, but not at the 30 and 50 μg doses, and were predominantly IFNγ-IL-2-TNFα+, IFNγ+IL-2-TNFα-, IFNγ-IL-2+TNFα+, or IFNγ+IL-2+TNFα+ producing CD4 + T cells (Figure 9C). + The T cell frequencies are further detailed in Table 7. [Table 7]

[0207] Antigen-specific CD8 induced by synthetic consensus mesothelin +The frequency of T cells was significantly increased over the control in all dose groups (Figure 9B). Specifically, the frequency of antigen-specific CD8+ T responses in groups immunized with 10 μg (0.64% ± 0.42%) (p = 0.016), 20 μg (0.70% ± 0.48%) (p = 0.028), 30 μg (0.97% ± 0.96%) (p = 0.008), and 50 μg (1.00% ± 0.22%) (p = 0.004) of pGX1430 was much stronger at the higher doses compared to untreated (0.15% ± 0.04%). Synthetic consensus mesothelin-specific CD8 + T cell responses increased with dose and were predominantly IFNγ+IL-2-TNFα producing CD8 + T cells (Figure 9D). + The T cell frequencies are further detailed in Table 8. [Table 8]

[0208] All doses of synthetic consensus mesothelin inhibited CD4 + CD107a + T cells were induced. Specifically, antigen-specific CD4 + CD107a + The frequencies of T cells were 0.10% ± 0.11%, 0.20% ± 0.13%, 0.16% ± 0.13%, and 0.15% ± 0.11% in the 10 μg (p = 0.004), 20 μg (p = 0.004), 30 μg (p = 0.016), and 50 μg (p = 0.004) dose groups, respectively (Figure 10A). + CD107a + The cytokine profile of T cells was similar across dose groups, primarily IFNγ + IL-2 + TNFα + , IFNγ + IL-2 - TNFα + , IFNγ + IL-2 - TNFα -The cells were composed of antigen-specific CD4 + The T cell frequencies are further detailed in Table 9. [Table 9]

[0209] antigen-specific CD8 + Similar to T cell size, synthetic consensus mesothelin significantly increased CD8 T cell proliferation across all groups compared to untreated (0.05% ± 0.02%). + CD107a + The results showed that the antigen-specific CD8 T cells were significantly increased in frequency (Fig. 10C). + CD107a + The frequencies of T cells were 0.27% ± 0.22%, 0.57% ± 0.43%, 0.83% ± 0.85%, and 0.90% ± 0.24% in the 10 μg (p = 0.008), 20 μg (p = 0.028), 30 μg (p = 0.004), and 50 μg (p = 0.004) dose groups, respectively (Figure 10A). + CD107a + The cytokine profile of T cells was similar across dose groups, predominantly IFNγ - IL-2 - TNFα + and IFNγ + IL-2 + TNFα + Most of the subjects were treated with IFNγ, except for the 10 μg dose group, which consisted of + IL-2 - TNFα - (Figure 10D). Antigen-specific CD8 + The T cell frequencies are further detailed in Table 10. [Table 10]

[0210] Overall, none of the data reported showed significant differences in responses between immunization groups (i.e., 10 μg was not significantly lower than 50 μg, etc.). statistical analysis

[0211] Statistical analyses were completed using IBM SPSS Statistics 22 (IBM Corporation). Data for many comparisons were not normally distributed. For this reason, the Mann-Whitney U test was used for all analyses, with post-hoc Bonferroni correction for many comparisons. Statistical significance is assumed to be p ≤ 0.013 (0.05 / 4 comparisons = 0.0125).

[0212] conclusion Synthetic consensus mesothelin inhibits antigen-specific CD4 + , CD4 + CD107a + , and CD8 + , CD8 + CD107a + Although the frequency of CD8 T cells increased, the magnitude of the response was + It was much more robust in the T cell compartment.

[0213] Example 7: Synthetic Consensus Mesothelin Monovalent Non-Human Primate Study To investigate the potential of synthetic consensus mesothelin alone and in combination with low and high doses of IL-12, 18 adult rhesus macaques, each identified by a unique NHP ID number, were divided into three groups of six and immunized with pGX1430 as follows: six animals were immunized with 3.0 mg pGX1430 (group 1), six with 3.0 mg pGX1430 + 0.04 mg pGX6006 (opt rh IL-12) as an adjuvant (group 2), and six with 3.0 mg pGX1430 + 0.20 mg pGX6006 (opt rh IL-12) as an adjuvant (group 3), formulated in SSC in an injection volume of 1.0 mL. Immune injections were administered at weeks 0, 4, 8, and 12, with an optional fifth immunization. Therefore, all immunizations were administered IM with a 1 ml injection volume formulated in sterile WFI using a CELLECTRA® 2000 5P-IM EP device in alternating contralateral limbs. EP conditions were as follows: OpBlock 0070-IM, 0.5 Amp, 3 pulses, 52 msec, 0.2 sec pulse interval. Mesothelin immunogenicity was assessed at weeks 2, 6, 10, and 14.

[0214] Animal identification, origin, sex, and weight in groups 1-3 are provided in Table 11. [Table 11]

[0215] PBMC isolation Non-human primate whole blood was collected in sodium citrate cell preparation tubes (CPT CPT's BD Biosciences) containing anticoagulant and a gel barrier. The whole blood is spun immediately after collection (within 2 hours) to separate and enrich the PMBCs before shipping by overnight courier. Red blood cells and neutrophils are pelleted to the bottom of the tube and held in place by the gel barrier. Plasma and lymphocytes remain on the gel barrier. Each CPT can hold approximately 8 mL of blood and is shipped at room temperature. Upon arrival at the preclinical laboratory in San Diego, the spun CPT tubes were processed for PBMC isolation. After red blood cell lysis with ammonium chloride potassium (ACK) buffer, viable cells were counted using an Invitrogen CountessTM automated cell counter and resuspended in complete medium (RPMI1640 supplemented with 10% FBS, antibiotics, and β-mercaptoethanol). Upon completion of the assays described in this report, the remaining PBMCs were frozen and stored long term in freezing medium (10% DMSO from Sigma in 90% FBS from Seradigm) in cryovials in liquid nitrogen.

[0216] IFNγ ELISPOT To evaluate vaccine-induced antigen-specific cellular responses, monkey IFNγ ELISpot assays were performed at each time point on isolated PMBCs using a kit (MabTech IFNγ ELISpotPro, no. 3421M-2APW-10). Briefly, 96-well plates pre-coated with anti-monkey IFNγ antibody (mAb MT126L) were washed with PBS and blocked with complete culture medium (RPMI1640 supplemented with 10% FBS, antibiotics, and β-mercaptoethanol) for 2 h at room temperature. NHP PBMCs were resuspended in R10 medium (and then added in triplicate at an input cell count of 2x105 cells / well). A set of peptides was synthesized (GenScript), each containing 15 amino acid residues overlapping by 11 amino acids representing the entire synthetic consensus protein sequence. These sets of peptides were resuspended in DMSO (Sigma) and pooled into pools at a concentration of approximately 2μg / ml of each peptide, respectively. All antigen-specific pooled peptides were used at a dilution of 1:100, resulting in a final dilution of 1:200 in each well when combined with PBMCs. Four pools were generated for mesothelin.

[0217] Anti-CD3 (mAh CD-2 Mabtech) and / or PMA (Sigma) with ionomycin (Sigma) were used as positive controls. Complete R10 culture medium was used as negative control. Plates were incubated for approximately 18 hours at 37°C in an incubator with 5% CO2 atmosphere. After removal of cells and addition of ALP-conjugated anti-monkey IFNγ detection antibody (MabTech Ab 7-B6-1-ALP), plates are incubated for 2 hours at room temperature. A sandwich immunoenzymatic assay is then developed using BCIP / NBT substrate solution according to the kit manufacturer's instructions (MabTech). A deep blue precipitate forms as a spot, representing each individual IFNγ producing cell. The spots are then scanned and counted by a CTL ImmunoSpot® analyzer and software (Cellular Technology), with quality controlled by a trained operator. IFNγ responses were assessed using 1×10 SFU greater than the SFU in the medium only control. 6 Reported as spot forming units (SFU) for PMBC.

[0218] immunology results Mesothelin-specific IFNγ responses are shown in Figures 11A-11C for groups 1-3 immunized with mesothelin alone (Figure 12A), mesothelin with a low dose of IL-12 (0.04 mg) (Figure 11B), or mesothelin with a high dose of IL-12 (0.2 mg) (Figure 11C). These results show responses at each time point 2 weeks after dosing. Overall, all groups and individual animals had an increased response by the end of the study 2 weeks after the 4th dose compared to the pre-bleed baseline. Figures 11A-11C show a trend for IL-12 providing an adjuvant effect in IFNγ responses to synthetic consensus mesothelin up to PD3.

[0219] 12A-12C show the averaged mesothelin-specific IFNγ responses, with immunization with mesothelin alone (FIG. 12A); Mesothelin containing a low dose of IL-12 (0.04 mg) (FIG. 12B) or a high dose of IL-12 (0.2 mg) (FIG. 12C) are shown in FIGs. 12A-12C to show that IL-12 increased the magnitude, but not the breadth, of the IFNγ response to synthetic consensus mesothelin.

[0220] Physiological parameters There were no differences in any of the physiological parameters measured following immunization for groups 1, 2, and 3, as shown in Tables 12, 13, and 14, respectively. No significant differences were noted for RBC, HCT, neutrophils, lymphocytes, monocytes, eosinophils (data not shown). These values ​​are within the expected range for animals of this species, sex, and age undergoing similar experimental procedures. Any variation from the defined normal ranges is of a sporadic nature, represents only one sex, and is not related to dose level or timing. [Table 12] [Table 13] [Table 14]

[0221] For all groups, there was no significant change in body weight over the course of the study, with the normal range being 4 to 12, as shown in Table 15 below. [Table 15]

[0222] Overall, these results indicate that synthetic consensus mesothelin administered alone is capable of inducing an immune response in 100% of NHPs. The addition of an IL-12 adjuvant did not significantly improve this.

[0223] It is understood that the foregoing detailed description and the accompanying examples are merely illustrative and are not to be construed as limitations on the scope of the invention, which is defined solely by the appended claims and their equivalents.

[0224] Various changes and modifications to the embodiments of the present disclosure will be apparent to those skilled in the art. Such changes and modifications to the embodiments of the present disclosure, including but not limited to those related to the chemical structures, substituents, derivatives, intermediates, syntheses, compositions, formulations, or methods of use of the present invention, can be made without departing from the spirit and scope thereof. [Table 16] [Table 17]

Claims

1. A promoter; (a) a nucleic acid sequence encoding amino acids 19-607 of SEQ ID NO:2; and (b) a nucleic acid sequence encoding a protein that is at least 96% identical to amino acids 19-607 of SEQ ID NO:2, the protein comprising, relative to SEQ ID NO:2, an alanine at amino acid position 303, a threonine at amino acid position 583, and a valine at amino acid position 585. A vector comprising a nucleic acid molecule comprising one or more nucleic acid sequences selected from the group consisting of:

2. A promoter; (a) nucleotides 55 to 1821 of SEQ ID NO:1, and (b) a fragment that is at least 96% identical to nucleotides 55 to 1821 of SEQ ID NO:1, which, relative to SEQ ID NO:2, encodes an alanine at amino acid position 303, a threonine at amino acid position 583, and a valine at amino acid position 585. A vector comprising a nucleic acid molecule comprising one or more nucleic acid sequences selected from the group consisting of:

3. A promoter; (a) a nucleic acid sequence encoding SEQ ID NO:2, and (b) a nucleic acid sequence encoding a protein that is at least 96% identical to SEQ ID NO:2, the protein comprising, relative to SEQ ID NO:2, an alanine at amino acid position 303, a threonine at amino acid position 583, and a valine at amino acid position 585. A vector comprising a nucleic acid molecule comprising one or more nucleic acid sequences selected from the group consisting of:

4. A promoter; (a) SEQ ID NO:1, and (b) a fragment that is at least 96% identical to SEQ ID NO:1, which, relative to SEQ ID NO:2, encodes an alanine at amino acid position 303, a threonine at amino acid position 583, and a valine at amino acid position 585. A vector comprising a nucleic acid molecule comprising one or more nucleic acid sequences selected from the group consisting of:

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

1.

6. The vector according to any one of claims 1 to 5, wherein the promoter is upstream of the nucleic acid molecule.

7. The vector of claim 6, wherein the nucleic acid molecule is operably linked to a regulatory element comprising a poly-adenylation signal.

8. 8. The vector of claim 7, wherein the promoter is selected from the group consisting of a human cytomegalovirus (hCMV) promoter, a simian virus 40 (SV40) promoter, a mouse mammary tumor virus (MMTV) promoter, a human immunodeficiency virus (HIV) promoter, a Moloney virus promoter, an avian leukosis virus (ALV) promoter, an Epstein-Barr virus (EBV) promoter, a Rous sarcoma virus (RSV) promoter, a human actin gene promoter, a human myosin gene promoter, a human hemoglobin gene promoter, a human muscle creatine gene promoter, and a human mesothelin gene promoter.

9. The vector of claim 7 , wherein the promoter is a modified human cytomegalovirus (hCMV) promoter.

10. The vector of claim 7, wherein the poly-adenylation signal is the bovine growth hormone poly-adenylation signal (bGH polyA).

11. The vector according to any one of claims 1 to 10, wherein the vector is a plasmid or a viral vector.

12. A composition comprising one or more vectors according to any one of claims 1 to 11.

13. The composition of claim 12, further comprising a pharma- ceutically acceptable carrier.

14. (a) amino acids 19 to 607 of SEQ ID NO:2, and (b) an amino acid sequence that is at least 95% identical to amino acids 19 to 607 of SEQ ID NO:2, which contains, relative to SEQ ID NO:2, an alanine at amino acid position 303, a threonine at amino acid position 583, and a valine at amino acid position 585. The vector according to any one of claims 1 to 11, which encodes a protein comprising an amino acid sequence selected from the group consisting of:

15. (a) SEQ ID NO:2, and (b) an amino acid sequence that is at least 96% identical to SEQ ID NO:2, the amino acid sequence including, relative to SEQ ID NO:2, an alanine at amino acid position 303, a threonine at amino acid position 583, and a valine at amino acid position 585. The vector according to any one of claims 1 to 11, which encodes a protein comprising an amino acid sequence selected from the group consisting of:

16. The vector according to any one of claims 1 to 11, which encodes a protein comprising the amino acid sequence set forth in SEQ ID NO:

2.

17. A vaccine comprising the vector of any one of claims 1 to 11, wherein the nucleic acid molecule encodes an antigen comprising the amino acid sequence set forth in SEQ ID NO:

2.

18. The vaccine of claim 17, wherein the nucleic acid molecule comprises the sequence of SEQ ID NO:

1.

19. The nucleic acid molecule encodes a peptide, the peptide comprising:

12. A vaccine comprising the vector of any one of claims 1 to 11, comprising an amino acid sequence having at least 96% identity over the entire length of the amino acid sequence set forth in SEQ ID NO:2, and having an alanine at amino acid position 303, a threonine at amino acid position 583, and a valine at amino acid position 585, relative to SEQ ID NO:

2.

20. 12. A vaccine comprising the vector of any one of claims 1 to 11, wherein the nucleic acid molecule has at least 96% identity over the entire length of the nucleic acid sequence set forth in SEQ ID NO:1 and comprises a nucleic acid sequence encoding an alanine at amino acid position 303, a threonine at amino acid position 583, and a valine at amino acid position 585, relative to SEQ ID NO:

2.

21. 21. The vaccine of claim 17 or 20, further comprising a pharma- ceutically acceptable excipient.

22. 21. The vaccine of claim 17 or 20, further comprising an adjuvant.

23. 23. The vaccine of claim 22, wherein the adjuvant is IL-12, IL-15, IL-28, or RANTES.

24. A vaccine according to any one of claims 17 to 20 for use in the treatment of cancers expressing mesothelin.

25. A vaccine according to any one of claims 21 to 23 for use in the treatment of cancers expressing mesothelin.

26. A vaccine according to any one of claims 17 to 25 for use in vaccination against mesothelin expressing cancers.

27. A vector according to any one of claims 1 to 11 for use as a medicament.

28. A vector according to any one of claims 1 to 11 for use as a medicament in the treatment of cancer.

29. A vector according to any one of claims 1 to 11 for use in the preparation of a medicament.

30. A vector according to any one of claims 1 to 11 for use in the preparation of a medicament for the treatment of cancer.

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