WT1 vaccine

Abstract

To provide: Wilm's tumor (WT1) immunogens and nucleic acid molecules which encode the immunogens; vaccines including such WT1 immunogens and / or nucleic acid molecules; and methods of using the vaccines for inducing immune responses and for preventing and / or treating subjects having tumors that express WT1.SOLUTION: Disclosed herein are nucleic acid molecules comprising one or more nucleic acid sequences that encode a mutated WT1 antigen. Vectors, compositions and vaccines comprising one or more nucleic acid sequences that encode a mutated WT1 antigen are disclosed. Methods of treating an individual who has a WT1-expressing tumor and methods of preventing a WT1-expressing tumor are disclosed. A mutated WT1 antigen is disclosed.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Provisional Patent Application No. 61 / 737,094, filed December 13, 2012, the entire teachings of which are incorporated herein by reference.

[0002] The present invention relates to Wilms' tumor (WT1) immunogens and nucleic acid molecules encoding the immunogens. The present invention also relates to vaccines comprising such WT1 immunogens and / or nucleic acid molecules. The present invention further relates to methods of using the vaccines to induce immune responses and for the prevention and / or treatment of subjects with tumors expressing WT1. [Background technology]

[0003] Cancer remains the leading cause of death in the United States and worldwide. The cancer vaccine market is expanding rapidly. Lymphoma vaccines account for approximately 0.5% of the market. An effective tumor vaccine may be useful in preventing tumor growth and / or may be useful as an effective and less toxic alternative to standard treatments for patients suffering from advanced cancer. An antigen associated with cancer and therefore a target for antitumor vaccines is WT1.

[0004] Wilms tumor suppressor gene 1 (WT1) has been identified as a cause of embryonal malignant tumors of the kidney (nephroblastoma), which affects approximately 1 in 10,000 infants. The gene occurs in both sporadic and inherited forms. Inactivation of WT1 leads to the development of Wilms tumor and Denys-Drash syndrome (DDS), which can result in reproductive abnormalities as well as nephropathy. The WT1 protein has been found to interact with many cellular factors, including the master tumor regulator gene p53, which is also a tumor suppressor transcription factor.

[0005] WT1 is expressed in many tumor types and is more widely involved in many cancers.For example, WT1 protein is localized in the cell nucleus of 75% mesothelioma (14,200 cases worldwide each year, with the highest incidence in the United States) and 93% serous ovarian cancer (190,000 ovarian cancer cases worldwide in 2010).In addition, WT1 is involved in pancreatic cancer, leukemia, lung cancer, breast cancer, colon cancer, glioblastoma, head cancer, neck cancer, as well as benign mesothelioma, cervical cancer, and ovarian cancer, among others.WT1 is the target of gene therapy or immunotherapy as an approach to cancer treatment.

[0006] WT1 encodes a transcription factor containing four zinc finger motifs at the C-terminus and a proline / glutamine-rich DNA-binding domain at the N-terminus. WT1 plays an important role in normal development of the urogenital system. However, it is less essential in adults, making it a suggested target for immunotherapy. Multiple transcript variants resulting from alternative splicing of the two coding exons have been well characterized. Using the entire reading frame to maximize CTL coverage would be an advantage.

[0007] Due to the conservation of the WT1 antigen, most attempts to generate strong immunity against this genetic target have been unsuccessful. Vaccine studies have been conducted using DNA, pox virus, adenovirus, peptide, and protein-based vaccine technologies. The vaccines studied used true genetic constructs, i.e., the natural "normal" gene. These studies achieved only low levels or non-functional T cell immunity.

[0008] There are several major problems in developing a more effective WT1 immunogen: the WT1 antigen resembles host WT1, which generates a strong inhibitory T cell response, which inhibits immune induction; the gene itself is significantly processed at the RNA level, which generates a large number of cleaved transcripts with unknown and potentially competing values; and the delivered WT1 expression is low, which leads to immune deficiency.

[0009] Vaccines for the treatment and prevention of cancer are of great interest.Existing vaccines that target WT1 are limited by low in vivo expression of antigen.Therefore, there is a need in the art to develop safe and effective vaccines that can be applied to tumors that express WT1, and provide treatment for such cancer and promote survival rate. Summary of the Invention

[0010] The present invention relates to an isolated nucleic acid molecule comprising one or more nucleic acid sequences selected from the group consisting of 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 98% identical to SEQ ID NO:2, a nucleic acid sequence encoding a fragment comprising at least 90% of the length of a protein that is at least 98% identical to SEQ ID NO:2, a nucleic acid sequence encoding SEQ ID NO:4, a nucleic acid sequence encoding a fragment comprising at least 90% of the length of SEQ ID NO:4, a nucleic acid sequence encoding a protein that is at least 98% identical to SEQ ID NO:4, and a nucleic acid sequence encoding a fragment comprising at least 90% of the length of a protein that is at least 98% identical to SEQ ID NO:4.

[0011] The present invention also relates to isolated nucleic acid molecules comprising one or more nucleic acid sequences selected from the group consisting of SEQ ID NO:1, a fragment comprising at least 90% of the length of SEQ ID NO:1, a nucleic acid sequence that is at least 98% identical to SEQ ID NO:1, a fragment comprising at least 90% of the length of a nucleic acid sequence that is at least 98% identical to SEQ ID NO:1, SEQ ID NO:3, a fragment comprising at least 90% of the length of SEQ ID NO:3, a nucleic acid sequence that is at least 98% identical to SEQ ID NO:3, and a fragment comprising at least 90% of the length of a nucleic acid sequence that is at least 98% identical to SEQ ID NO:3.

[0012] The nucleic acid molecules can be incorporated into a plasmid or a viral vector. Furthermore, the present invention relates to a composition comprising one or more of the nucleic acid molecules. The present invention also relates to a vaccine comprising one or more of the nucleic acid molecules.

[0013] The present invention further relates to a method for treating an individual having a WT1-expressing tumor, the method comprising administering the above-described vaccine in an amount effective to slow the growth of, shrink or eliminate the WT1-expressing tumor.

[0014] The present invention also relates to a method for preventing a WT1-expressing tumor in an individual, the method comprising administering the above-described vaccine in an amount effective to inhibit the formation or growth of a WT1-expressing tumor.

[0015] Furthermore, the present invention relates to a protein comprising an amino acid sequence selected from the group consisting of 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 98% identical to SEQ ID NO:2, a fragment comprising at least 90% of the length of an amino acid sequence that is at least 98% identical to SEQ ID NO:2, SEQ ID NO:4, a fragment comprising at least 90% of the length of SEQ ID NO:4, an amino acid sequence that is at least 98% identical to SEQ ID NO:4, and a fragment comprising at least 90% of the length of an amino acid sequence that is at least 98% identical to SEQ ID NO:4.

[0016] The present invention also relates to a vaccine comprising the nucleic acid molecule. The nucleic acid molecule may comprise a nucleic acid sequence having at least about 90% identity over the entire length of the nucleic acid sequence set forth in SEQ ID NO:1. The nucleic acid molecule may comprise a nucleic acid sequence having at least about 90% identity over the entire length of said nucleic acid sequence set forth in SEQ ID NO:3. The vaccine may further comprise a peptide. The peptide may comprise an amino acid sequence having at least about 90% identity over the entire length of said nucleic acid sequence set forth in SEQ ID NO:2. The peptide may comprise an amino acid sequence having at least about 90% identity over the entire length of said nucleic acid sequence set forth in SEQ ID NO:4.

[0017] The nucleic acid molecule may comprise an expression vector. The vaccine may further comprise a pharma- ceutically acceptable excipient. The vaccine may further comprise an adjuvant.

[0018] The present invention also relates to a vaccine comprising the nucleic acid molecule. The nucleic acid molecule can encode a peptide comprising an amino acid sequence having at least about 90% identity over the entire length of the nucleic acid sequence set forth in SEQ ID NO:2. The nucleic acid molecule can encode a peptide comprising an amino acid sequence having at least about 90% identity over the entire length of the nucleic acid sequence set forth in SEQ ID NO:4. The vaccine can further comprise a peptide. The peptide can comprise an amino acid sequence having at least about 90% identity over the entire length of the nucleic acid sequence set forth in SEQ ID NO:2. The peptide can comprise an amino acid sequence having at least about 90% identity over the entire length of the nucleic acid sequence set forth in SEQ ID NO:4.

[0019] The nucleic acid molecule may comprise an expression vector. The vaccine may further comprise a pharma- ceutically acceptable excipient. The vaccine may further comprise an adjuvant.

[0020] Furthermore, the present invention relates to a nucleic acid molecule comprising the nucleic acid sequence set forth in SEQ ID NO:1.

[0021] The present invention also relates to a nucleic acid molecule comprising the nucleic acid sequence set forth in SEQ ID NO:3.

[0022] Furthermore, the present invention relates to a peptide comprising the amino acid sequence set forth in SEQ ID NO:2.

[0023] The present invention also relates to a peptide comprising the amino acid sequence set forth in SEQ ID NO:4.

[0024] The present invention further relates to a vaccine comprising an antigen, the antigen being encoded by SEQ ID NO: 1 or SEQ ID NO: 3. The antigen may be encoded by SEQ ID NO: 1. The antigen may be encoded by SEQ ID NO: 3. The antigen may comprise the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4. The antigen may comprise the amino acid sequence set forth in SEQ ID NO: 2. The antigen may comprise the amino acid sequence set forth in SEQ ID NO: 4.

[0025] The present invention also relates to a vaccine comprising the peptide. The peptide may comprise an amino acid sequence having at least about 90% identity over the entire length of the nucleic acid sequence set forth in SEQ ID NO:2. The peptide may comprise an amino acid sequence having at least about 90% identity over the entire length of the nucleic acid sequence set forth in SEQ ID NO:4. The peptide may comprise an amino acid sequence set forth in SEQ ID NO:2. The peptide may comprise an amino acid sequence set forth in SEQ ID NO:4. [Brief description of the drawings]

[0026] [Figure 1] A graph is shown plotting immunization group versus spot forming units (SFU) per 106 splenocytes. [Diagram 2] A graph is shown plotting immunization group versus SFU per 106 splenocytes. [Diagram 3] Immunoblots are shown. [Figure 4] Schematic diagrams of ConWT1-L and ConWT1-S are shown. [Diagram 5] An alignment of the amino acid sequences of ConWT1-L and ConWT1 is shown. [Figure 6] (A) shows the nucleotide sequence encoding ConWT1-L, and (B) shows the amino acid sequence of ConWT1-L. [Figure 7] (A) shows the nucleotide sequence encoding ConWT1-S, and (B) shows the amino acid sequence of ConWT1-S. [Figure 8] Staining of transfected cells is shown. [Figure 9] Immunoblots are shown. [Figure 10] The immunization schedule is shown diagrammatically. [Figure 11] A graph is shown plotting immunization group versus SFU per 106 splenocytes. [Figure 12] A graph is shown plotting immunization group versus SFU per 106 splenocytes. [Figure 13] Staining of transfected cells is shown. [Figure 14]Immunoblots are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] The present invention relates to a vaccine comprising a WT1 antigen. WT1 is expressed in many tumors. Therefore, the vaccine provides treatment for cancer or cancerous tumors expressing WT1.

[0028] The WT1 antigen is unique because it can be a consensus WT1 antigen derived from WT1 sequences from different species. The consensus WT1 antigen is also unique in that all of the zinc fingers have been modified or removed. Modifications can include substitution of cysteine ​​and histidine residues that coordinate the zinc structure.

[0029] Surprisingly, when the consensus WT1 antigen has either an altered zinc finger or no zinc finger, a significant immune response reactive to WT1 is induced, including both humoral and cellular immune responses, and the cellular immune response is increased by about 400-fold compared to the cellular immune response induced by a vaccine containing native WT1 or WT1 optimized for expression. 1.Definition

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present specification, including definitions, will prevail. In carrying out or testing the present invention, methods and materials similar or equivalent to those described herein can be used, but suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. It should be noted that the materials, methods, and examples disclosed herein are merely illustrative and are not intended to be limiting.

[0031] As used herein, the terms "comprise(s)", "include(s)", "having", "has", "can", "contain(s)", and variations thereof are intended to be open-ended transitional phrases, terms, or words that do not preclude or limit the possibility of additional acts or structures. The singular forms "a", "and", and "the" may also have plural meanings 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 set forth herein, whether or not expressly stated.

[0032] As used herein, "adjuvant" may refer to any molecule added to the DNA plasmid vaccines described herein to enhance the antigenicity of one or more antigens encoded by the DNA plasmids and encoding nucleic acid sequences described herein below.

[0033] "Antibody" may refer to antibodies of class IgG, IgM, IgA, IgD, or IgE, or fragments or derivatives thereof, including Fab, F(ab')2, Fd, as well as 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 a mixture thereof, which exhibits sufficient binding specificity for the desired epitope or a sequence derived therefrom.

[0034] "Antigen" refers to a protein having a mutant WT1 amino acid sequence, including SEQ ID NO:2; fragments thereof of lengths described herein, variants, i.e., a protein having a sequence having identity to SEQ ID NO:2 as described herein, fragments of variants of lengths described herein, SEQ ID NO:4; fragments thereof of lengths described herein, variants, i.e., a protein having a sequence having identity to SEQ ID NO:4 as described herein, fragments of variants of lengths described herein, and combinations thereof. Antigens may optionally include signal peptides, such as those derived from other proteins.

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

[0036] As used herein, "complement" or "complementary" can mean that a nucleic acid exhibits 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" may refer to a synthetic nucleic acid sequence or corresponding polypeptide sequence constructed based on the alignment analysis of multiple subtypes of a particular antigen. The sequence can be used to induce broad immunity against multiple subtypes, serotypes, or strains of a particular antigen. Synthetic antigens, such as fusion proteins, can be engineered into consensus sequences (or consensus antigens).

[0038] As used herein, "constant current" defines the current that a tissue, or cells defining the tissue, receive or experience during the duration of an electrical pulse delivered to the tissue. The electrical pulse is delivered from an electroporation device as described herein. This current remains at a constant amount in the tissue during the life of the electrical pulse because the electroporation device provided herein has a feedback element, preferably with instantaneous feedback. The feedback element measures the resistance of the tissue (or cells) during the duration of the pulse and can cause the electroporation device to change its electrical energy output (e.g., increase the voltage) so that the current in the tissue remains constant throughout the electrical pulse (approximately microseconds) and between pulses. In some embodiments, the feedback element includes a controller.

[0039] As used herein, "current feedback" or "feedback" are used interchangeably and may refer to the active response of the provided electroporation device, which includes measuring the current in the tissue between the electrodes and appropriately varying the energy output delivered by the EP device to maintain the current at a constant level. This constant level is preset by the user before starting the pulse sequence or electrical treatment. Feedback may be achieved by the electroporation components of the electroporation device, such as a controller, so that the electrical circuitry in the electroporation device continuously monitors the current in the tissue between the electrodes, compares the monitored current (or the current in the tissue) with a preset current, and continuously adjusts the energy output to maintain the monitored current at a preset level. The feedback loop may be instantaneous, since it is an analog closed-loop feedback.

[0040] As used herein, "distributed current" may refer to patterns of current delivered from the various needle electrode arrays of the electroporation devices described herein that minimize or preferably eliminate the occurrence of electroporation-related thermal stress to any tissue region being electroporated.

[0041] As used interchangeably herein, "electroporation," "electropermeabilization," and "electrokinetic enhancement" ("EP") can refer to the use of transmembrane electric field pulses to generate microscopic pathways (pores) in biological membranes. The presence of these microscopic pathways allows the passage of biological molecules, such as plasmids, oligonucleotides, siRNA, drugs, ions, and water, from one side of the cell membrane to the other.

[0042] As used herein, a "feedback mechanism" may refer to a process, executed by either software or hardware (or firmware), that receives the desired tissue impedance (before, during, and / or after delivery of an energy pulse), compares it to a current value, preferably the current, and adjusts the delivered energy pulse to achieve a pre-set value. The feedback mechanism may be implemented by an analog closed loop circuit.

[0043] "Fragment" may refer to a polypeptide fragment capable of eliciting an immune response in a mammal. A fragment of an antigen may be 100% identical to the full length except for the absence of at least one amino acid from the N- and / or C-terminus, with or without a signal peptide and / or methionine at position 1. A fragment may comprise 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more of the length of a particular full length antigen, excluding any added heterologous signal peptide. Fragments may include fragments of polypeptides that are 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identical to the antigen and further include an N-terminal methionine or a heterologous signal peptide that is not included in calculating the percent identity. Fragments may further include an N-terminal methionine and / or a signal peptide, such as an immunoglobulin signal peptide, e.g., an IgE or IgG signal peptide. The N-terminal methionine and / or signal peptide may be attached to a fragment of the antigen.

[0044] A fragment of a nucleic acid sequence encoding an antigen may be 100% identical to the full length except for lacking at least one nucleotide from the 5' and / or 3' end, either with or without a sequence encoding a signal peptide and / or methionine at position 1. A fragment may comprise 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more of the length of the particular full length coding sequence excluding any added heterologous signal peptide. Fragments may include fragments encoding a polypeptide that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the antibody, and optionally further comprising a sequence encoding an N-terminal methionine or a heterologous signal peptide that is not included in calculating percent identity. Fragments may further comprise a coding sequence for an N-terminal methionine and / or a signal peptide, such as an immunoglobulin signal peptide, e.g., an IgE or IgG signal peptide. The coding sequence encoding the N-terminal methionine and / or signal peptide may be attached to the fragment of the coding sequence.

[0045] As used herein, "genetic construct" refers to a DNA or RNA molecule that contains a nucleotide sequence that codes for a protein. The coding sequence includes a start and end signal that is operably linked to a regulatory element that includes a promoter and a polyadenylation signal that can induce expression in the cells of an individual to which the nucleic acid molecule is administered. As used herein, the term "expressible form" refers to a genetic construct that includes 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 an individual.

[0046] "Identical" or "identity" as used herein in the context of two or more nucleic acid or polypeptide sequences can mean that the sequences have a specified percentage of identical residues over a specified region. To calculate this percentage, the two sequences are optimally aligned, the two sequences are compared over a specified region, and the number of positions where identical residues occur in both sequences is determined to obtain the number of matching positions, the number of matching positions is divided by the total number of positions in the specified region, and the result is multiplied by 100 to obtain the percentage of sequence identity. If the two sequences are of different length or the alignment results in one or more sticky ends, and only a single sequence is included in the specified comparison region, 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 calculations can be performed manually or using computer sequence algorithms such as BLAST, BLAST 2.0, etc.

[0047] As used herein, "impedance" is used when discussing feedback mechanisms and can be converted to a current value according to Ohm's law, so that it can be compared to a preset current.

[0048] As used herein, "immune response" can mean activation of a host's immune system, e.g., a mammal's immune system, in response to the introduction of an antigen as described herein via one or more of the vaccines described herein. This immune response can be in the form of cellular or humoral, or both.

[0049] As used herein, "nucleic acid" or "oligonucleotide" or "polynucleotide" can mean at least two nucleotides covalently linked to each other. By describing a single strand, the sequence of the complementary strand is also defined. Thus, nucleic acid also encompasses the complementary strand of the described single strand. Many variants of a nucleic acid can be used for the same purpose as a given nucleic acid. Thus, nucleic acid also encompasses substantially identical nucleic acids and their complements. A single strand provides a probe that can hybridize with a target sequence under stringent hybridization conditions. Thus, nucleic acid also encompasses probes that hybridize under stringent hybridization conditions.

[0050] Nucleic acids may be single-stranded or double-stranded, or may contain portions of both double-stranded and single-stranded sequences. Nucleic acids may be DNA, genomic DNA, cDNA, RNA, or hybrids, and may contain combinations of deoxyribonucleotides and ribonucleotides, and may contain combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine. Nucleic acids may be obtained by chemical synthesis or recombinant methods.

[0051] As used herein, "operably linked" may mean that a gene is expressed under the control of a promoter that is spatially connected to the gene. The promoter may be located 5' (upstream) or 3' (downstream) of the gene under its control. The distance between the promoter and the gene may be approximately the same as the distance between the promoter and the gene it controls in the gene from which it is derived. As is known in the art, changes in this distance can be accommodated without loss of promoter function.

[0052] As used herein, "peptide," "protein," or "polypeptide" can refer to a bound sequence of amino acids, which may be natural, synthetic, or a combination of natural and synthetic modifications.

[0053] As used herein, a "promoter" may refer to a synthetic or naturally occurring molecule capable of conferring, activating, or enhancing expression of a nucleic acid in a cell. A promoter may contain one or more specific transcriptional regulatory sequences to further enhance expression and / or modify its spatial and / or temporal expression. A promoter may also contain distal enhancer or repression elements, which may be located as far away as several thousand base pairs from the transcription start site. Promoters may be derived from sources including viruses, bacteria, fungi, plants, insects, and animals. A promoter may regulate the expression of genetic components constitutively or differentially with respect to the cell, tissue, or organ in which expression occurs, or with respect to the developmental stage in which expression occurs, or may regulate the expression of genetic components in response to external stimuli, such as physiological stress, pathogens, metal ions, inducers, etc. Representative examples of promoters include a bacteriophage T7 promoter, a bacteriophage T3 promoter, an SP6 promoter, a lac operator promoter, a tac promoter, an SV40 late promoter, an SV40 early promoter, an RSV-LTR promoter, a CMV IE promoter, an SV40 early promoter, an SV40 late promoter, and a CMV IE promoter.

[0054] "Signal peptide" and "leader sequence" are used interchangeably herein and refer to an amino acid sequence that can be attached to the amino terminus of a protein described herein. Generally, a signal peptide / leader sequence directs the localization of a protein. As used herein, a signal peptide / leader sequence preferably facilitates secretion of the protein from the cell in which it is produced. The signal peptide / leader sequence is often cleaved from the remainder of the protein (often referred to as the mature protein) upon secretion from the cell. The signal peptide / leader sequence is attached to the N-terminus of the protein.

[0055] As used herein, "subject" refers to a mammal that desires or needs to be immunized with a vaccine described herein. The mammal may be a human, chimpanzee, dog, cat, horse, cow, mouse, or rat.

[0056] As used herein, "stringent hybridization conditions" can refer to conditions under which a first nucleic acid sequence (e.g., a probe) hybridizes to a second nucleic acid sequence (e.g., a target), for example in a complex mixture of nucleic acids. Stringent conditions are sequence-dependent and therefore will vary from situation to situation. Stringent conditions are those that meet the melting temperature (T m ) is selected to be about 5 to 10°C lower than T m is 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 sequences are present in excess, so T m (In this case, 50% of the probes are occupied at equilibrium). Stringent conditions can be salt concentrations of less than about 1.0 M sodium ion, such as about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3, and temperatures of at least about 30° C. for short probes (e.g., 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 destabilizing agents 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 the following: 50% formamide, 5x SSC, and 1% SDS, incubation at 42° C.; or 5x SSC, 1% SDS, incubation at 65° C., washing in 0.2x SSC, 0.1% SDS at 65° C.

[0057] As used herein, "substantially complementary" refers to a region in which a first sequence is identical to the complement of a second sequence over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more nucleotides or amino acids. It can 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, or that the two sequences hybridize under stringent hybridization conditions.

[0058] As used herein, "substantially identical" refers to a sequence that is 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, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100 or more nucleotides. "Complementary" can mean that a first sequence and a second sequence are at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical over a region of amino acids or, with respect to nucleic acids, where a first sequence is substantially complementary to the complement of a second sequence.

[0059] As used herein, "treatment" or "treating" can mean protecting an animal from a disease by preventing, inhibiting, suppressing, or completely eliminating the disease. Preventing a disease involves administering a vaccine of the invention to an animal prior to the onset of the disease. Suppressing a disease involves administering a vaccine of the invention to an animal after induction of the disease and prior to clinical appearance of the disease. Suppressing a disease involves administering a vaccine of the invention to an animal after clinical appearance of the disease.

[0060] As used herein, a "variant" with respect to a nucleic acid may mean: (i) a portion or fragment of a reference nucleotide sequence; (ii) a complement of a reference nucleotide sequence or a portion thereof; (iii) a nucleic acid that is substantially identical to a reference nucleic acid or its complement; or (iv) a nucleic acid that hybridizes under stringent conditions to a reference nucleic acid, its complement, or a substantially identical sequence thereof.

[0061] A "variant" as used in reference to a peptide or polypeptide is one that differs in amino acid sequence by insertion, deletion, or conservative substitution of amino acids, but retains at least one biological activity. A variant can also refer to a protein having an amino acid sequence that is substantially identical to a reference protein and retains at least one biological activity. Conservative substitutions of amino acids, i.e., replacing one amino acid with another of similar properties (e.g., hydrophilicity, degree, and distribution of charged regions), are recognized in the art as usually involving minor changes. Such 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 a consideration of its hydrophobicity and charge. It is known in the art that amino acids with similar hydropathic indices can be substituted and still retain protein function. In one embodiment, amino acids with hydropathic indices of ±2 are substituted. The hydrophilicity of amino acids can also be used to identify substitutions that result in proteins that retain biological function. By considering the hydrophilicity of amino acids in the context of a peptide, the maximum local average hydrophilicity of the peptide can be calculated, a useful measure that has been reported to correlate well with antigenicity and immunogenicity. U.S. Patent No. 4,554,101, which is incorporated herein by reference in its entirety. As is understood in the art, substitutions of amino acids with similar hydrophilicity values ​​result in peptides that retain biological activity, e.g., immunogenicity. Substitutions can be made with amino acids whose hydrophilicity values ​​are 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 these findings, amino acid substitutions that are compatible with biological function are understood to depend on the relative similarity of amino acids, particularly the relative similarity of the side chains of the amino acids, as manifested by hydrophobicity, hydrophilicity, charge, size, and other properties.

[0062] A variant may be a nucleic acid sequence that is substantially identical over the entire 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 entire length of the gene sequence or a fragment thereof. A variant may be an amino acid sequence that is substantially identical over the entire 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 entire length of the amino acid sequence or a fragment thereof.

[0063] As used herein, "vector" may refer to a nucleic acid sequence that includes a replication origin. A vector may be a plasmid, a bacteriophage, a bacterial artificial chromosome, or a yeast artificial chromosome. A vector may be a DNA vector or an RNA vector. A vector may be either an autonomously replicating extrachromosomal vector or a vector that integrates into a host genome.

[0064] For the recitation of numerical ranges herein, each intervening number is expressly contemplated to the same degree of precision. For example, in the range 6 to 9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and in the range 6.0 to 7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are expressly contemplated.

[0065] 2. Vaccines Provided herein is a vaccine comprising an antigen, a fragment thereof, a variant thereof, or a combination thereof. The vaccine can generate an immune response against the antigen in a mammal. The vaccine can comprise a plasmid or multiple plasmids, as described in more detail below. The vaccine can induce a therapeutic or prophylactic immune response.

[0066] The vaccine can be used to protect against cancer, for example, cancer or tumor that expresses Wilms tumor suppressor gene 1 (WT1).The vaccine can be used to prevent and / or treat tumor that expresses WT1 in a subject in need thereof.The vaccine can induce cellular and / or antibody responses against WT1 and tumor that expresses WT1.

[0067] The vaccine can induce or induce an immune response in the subject administered with the vaccine.The immune response in the subject administered with the vaccine can be induced at least about 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 150 times, 200 times, 225 times, 250 times, 275 times, 300 times, 325 times, 350 times, 375 times, 400 times, 425 times, 450 times, 475 times, 500 times, 525 times, 550 times, 575 times, 600 times, 650 times, 700 times, 750 times, 800 times, 850 times, 900 times, 950 times, 1000 times, 1500 times, 2000 times, 2500 times, 3000 times, 3500 times, or 4000 times. In some embodiments, an immune response in a subject receiving the vaccine can be induced by at least about 300-fold, 325-fold, 350-fold, 375-fold, 400-fold, 425-fold, 450-fold, 475-fold, or 500-fold.

[0068] The vaccine can induce a humoral and / or cellular immune response in a subject administered the vaccine. The induced humoral immune response can include antibodies immunoreactive with the antigen. The induced cellular immune response can include T cells that produce interferon-gamma (IFN-γ) and are immunoreactive with the antigen. A cellular immune response in a subject receiving the vaccine can be induced by at least about 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 150-fold, 200-fold, 225-fold, 250-fold, 275-fold, 300-fold, 325-fold, 350-fold, 375-fold, 400-fold, 425-fold, 450-fold, 475-fold, 500-fold, 525-fold, 550-fold, 575-fold, 600-fold, 650-fold, 700-fold, 750-fold, 800-fold, 850-fold, 900-fold, 950-fold, 1000-fold, 1500-fold, 2000-fold, 2500-fold, 3000-fold, 3500-fold, or 4000-fold. In some embodiments, a cellular immune response in a subject receiving the vaccine can be induced by at least about 300-fold, 325-fold, 350-fold, 375-fold, 400-fold, 425-fold, 450-fold, 475-fold, or 500-fold.

[0069] The vaccine can be used to deliver one or more antigens selected from the group consisting of antigens, fragments of such antigens, variants of such antigens, and fragments of variants.When delivering multiple antigens, the vaccine can include multiple compositions or a single composition.Delivery can include a single plasmid that is used to code multiple different antigens or different parts of the same antigen.In other embodiments, delivery can include different plasmids that code different antigens or different parts of the same antigen.

[0070] The vaccine can be a nucleic acid vaccine, a peptide vaccine, or a combination of nucleic acid and peptide vaccines. The nucleic acid vaccine can include a nucleic acid molecule. The nucleic acid vaccine can include multiple copies of a single nucleic acid molecule, such as a single plasmid, or multiple copies of two or more different nucleic acid molecules, such as two or more different plasmids.

[0071] A nucleic acid vaccine may comprise a nucleic acid molecule, such as a plasmid, that contains the coding sequence of a single antigen, the heterologous coding sequence of two or more antigens together. A nucleic acid vaccine that contains the heterologous coding sequence of two antigens may be on a single nucleic acid molecule, such as a single plasmid, or the nucleic acid vaccine may comprise two different nucleic acid molecules, such as two different plasmids, where one nucleic acid molecule contains the heterologous coding sequence of one antigen and the other nucleic acid molecule contains the heterologous coding sequence of a different antigen. A nucleic acid vaccine may comprise two different nucleic acid molecules, such as two different plasmids, where one nucleic acid molecule contains the heterologous coding sequence of a first portion of an antigen and the other nucleic acid molecule contains the heterologous coding sequence of a second portion of an antigen.

[0072] Similarly, a nucleic acid vaccine that contains heterologous coding sequences for three antigens may contain a single nucleic acid molecule, such as a single plasmid, two different nucleic acid molecules, or three different nucleic acid molecules.Similarly, a nucleic acid vaccine that contains heterologous coding sequences for four antigens may contain a single nucleic acid molecule, such as a single plasmid, two different nucleic acid molecules, three different nucleic acid molecules, or four different nucleic acid molecules.

[0073] A nucleic acid vaccine may comprise one or more nucleotide sequences encoding an antigen. The nucleic acid sequence may be DNA, RNA, cDNA, variants thereof, fragments thereof, or combinations thereof. The nucleic acid sequence may also comprise additional sequences encoding linkers, leaders, and / or tag sequences that are linked to the antigen by a peptide bond.

[0074] In some embodiments, the nucleic acid vaccine may further comprise a coding sequence for 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 such as 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, IL-31, IL-33, and / or RANTES may be included on 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 included on a separate nucleic acid molecule, such as a separate plasmid.

[0075] A peptide vaccine may include an antigenic peptide, an antigenic protein, a variant thereof, a fragment thereof, or a combination thereof. A combination of DNA and peptide vaccines may include one or more of the above nucleic acid sequences encoding an antigen, an antigenic peptide, or an antigenic protein.

[0076] The vaccines of the invention possess the necessary characteristics of an effective vaccine, including being safe, so that the vaccine itself does not cause illness or death, protects against disease, induces neutralizing antibodies; induces protective T cells, is easy to administer, has few side effects, is biologically stable, and has a low cost per dose.

[0077] a. Antigen As mentioned above, the vaccine may include an antigen. The antigen may be Wilms' tumor suppressor gene 1 (WT1), its fragment, its variant, or a combination thereof. WT1 is a transcription factor that contains a proline / glutamine-rich DNA-binding domain at the N-terminus and four zinc finger motifs at the C-terminus. WT1 is involved in the normal development of the urogenital system and interacts with many factors, such as p53, known as a tumor suppressor, and the serine protease HtrA2, which cleaves WT1 at multiple sites after treatment with cytotoxic drugs.

[0078] Mutation of WT1 can lead to tumorigenesis or cancer formation, for example, Wilms' tumor or tumor expressing WT1.Wilms' tumor often forms in one or both kidneys before metastasizing to other tissues, for example, but not limited to, liver tissue, urinary system tissue, lymphatic tissue, and lung tissue.Therefore, Wilms' tumor can be considered as a metastatic tumor.Wilms' tumor usually occurs in young children (for example, under 5 years old) in both sporadic and hereditary forms.

[0079] Therefore, the vaccine can be used to treat subjects suffering from Wilms' tumor. The vaccine can also be used to treat subjects with cancer or tumors that express WT1, and prevent the development of such tumors in the subject. WT1 antigen is different from the natural "normal" WT1 gene, and can provide treatment or prevention for WT1 antigen-expressing tumors. Thus, WT1 antigen sequences (i.e., mutant WT1 genes or mutant WT1 sequences) that are different from the natural WT1 gene are provided herein.

[0080] The transcript of the native WT1 gene is processed into various mRNAs, and the resulting proteins are not all equally valuable in inducing immune responses. The mutant WT1 gene described herein avoids selective processing, produces one full-length transcript, and induces stronger effector T and B cell responses. The first mutant WT1 sequence is referred to as CON WT1 with modified zinc fingers, i.e., ConWT1-L. SEQ ID NO: 1 is the nucleic acid sequence encoding WT1 antigen CON WT1 with modified zinc fingers. SEQ ID NO: 2 is the amino acid sequence of WT1 antigen CON WT1 with modified zinc fingers. The second mutant WT1 sequence is referred to as CON WT1 without zinc fingers, i.e., ConWT1-S. SEQ ID NO: 3 is the nucleic acid sequence encoding WT1 antigen CON WT1 without zinc fingers. SEQ ID NO: 4 is the amino acid sequence of WT1 antigen CON WT1 without modified zinc fingers.

[0081] An isolated nucleic acid molecule is provided that comprises the above-mentioned heterologous sequence. An isolated nucleic acid molecule is provided that consists of the above-mentioned heterologous sequence. The isolated nucleic acid molecule that comprises the above-mentioned heterologous sequence may be incorporated into a vector such as a plasmid, a viral vector, and other forms of nucleic acid molecule as described below. A nucleic acid sequence that codes for a WT1 antigen is provided herein. The coding sequence that codes for a WT1 antigen has the above-mentioned sequence.

[0082] The protein molecule is provided that comprises the heterologous amino acid sequence described above. The protein molecule is provided that consists of the heterologous amino acid sequence described above. The protein and polypeptide having the sequence described above are provided herein. The protein and polypeptide can be referred to as WT1 antigen and WT1 immunogen. The WT1 antigen can induce immune response against tumors that express the WT1 antigen.

[0083] In one embodiment of the invention, the consensus antigen desirably provides improved transcription and translation, including one or more of the following: increased transcription through a low GC content leader sequence; mRNA stability and codon optimization, and removal of cis-acting sequence motifs (i.e., internal TATA boxes) whenever possible.

[0084] In some embodiments of the invention, it is desirable to generate consensus antigens that generate a broad immune response across multiple strains, including one or more of the following: incorporating all available full-length sequences; computer-generating sequences that utilize the most commonly occurring amino acids at each position; and increasing cross-reactivity between strains.

[0085] The WT1 antigen may be a consensus antigen (or immunogen) sequence from two or more species. The WT1 antigen may include a consensus sequence and / or modifications for improved 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 WT1 antigen. The WT1 antigen may include a signal peptide, such as, but not limited to, an immunoglobulin signal peptide, for example, an immunoglobulin E (IgE) signal peptide or an immunoglobulin G (IgG) signal peptide. In some embodiments, the WT1 consensus antigen may include a hemagglutinin (HA) tag. The WT1 consensus antigen may be designed to elicit a stronger and broader cellular and / or humoral immune response than the corresponding codon-optimized WT1 antigen.

[0086] The WT1 consensus antigen comprises one or more mutations in one or more zinc fingers, which can induce stronger and broader cellular and / or humoral immune responses than the corresponding codon-optimized WT1 antigen. The one or more mutations can be substitutions of one or more amino acids that coordinate the zinc ion in one or more zinc fingers. The one or more amino acids that coordinate the zinc ion can be a CCHH motif. Thus, in some embodiments, the one or more mutations can be substitutions of one, two, three, or all four amino acids in the CCHH motif.

[0087] In other embodiments, the one or more mutations are such that residues 312, 317, 342, and 347 of SEQ ID NO:2 are any residue other than cysteine ​​(C) and residues 330, 334, 360, and 364 of SEQ ID NO:2 are any residue other than histidine (H). In particular, the one or more mutations are such that residues 312, 317, 330, 334, 342, 347, 360, and 364 of SEQ ID NO:2 are glycine (G).

[0088] In other embodiments, one or more zinc fingers can be removed from the WT1 consensus antigen. One, two, three, or all four zinc fingers can be removed from the WT1 consensus antigen.

[0089] The WT1 consensus antigen can be nucleic acid SEQ ID NO:1 encoding SEQ ID NO:2 (FIGS. 6A and 6B). In some embodiments, the WT1 consensus antigen can be a nucleic acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity over the entire length of the nucleic acid sequence set forth in SEQ ID NO:1. In other embodiments, the WT1 consensus antigen may be a nucleic acid sequence encoding an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity over the entire length of the nucleic acid sequence set forth in SEQ ID NO:2.

[0090] In still another embodiment, the WT1 consensus antigen may be a nucleic acid sequence encoding an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity over the entire length of the nucleic acid sequence set forth in SEQ ID NO: 2, with the proviso that residues 312, 317, 342, and 347 of SEQ ID NO: 2 are any residue other than cysteine ​​(C), and residues 330, 334, 360, and 364 of SEQ ID NO: 2 are any residue other than histidine (H). In another embodiment, the WT1 consensus antigen can be a nucleic acid sequence encoding an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity over the entire length of the nucleic acid sequence set forth in SEQ ID NO:2, with the proviso that residues 312, 317, 330, 334, 342, 347, 360, and 364 of SEQ ID NO:2 are glycine (G).

[0091] The WT1 consensus antigen can be the amino acid sequence of SEQ ID NO: 2. In some embodiments, the WT1 consensus antigen can be an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity over the entire length of the nucleic acid sequence set forth in SEQ ID NO:2. The WT1 consensus antigen can be an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity over the entire length of the nucleic acid sequence set forth in SEQ ID NO:2, except that residues 312, 317, 342, and 347 of SEQ ID NO:2 are any residue other than cysteine ​​(C), and residues 330, 334, 360, and 364 of SEQ ID NO:2 are any residue other than histidine (H). In some embodiments, the WT1 consensus antigen can be an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity over the entire length of the nucleic acid sequence set forth in SEQ ID NO: 2, with the proviso that residues 312, 317, 330, 334, 342, 347, 360, and 364 of SEQ ID NO: 2 are glycine (G).

[0092] The WT1 consensus antigen can be nucleic acid SEQ ID NO:3 encoding SEQ ID NO:4 (FIGS. 7A and 7B). In some embodiments, the WT1 consensus antigen can be a nucleic acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity over the entire length of the nucleic acid sequence set forth in SEQ ID NO:3. In other embodiments, the WT1 consensus antigen may be a nucleic acid sequence encoding an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity over the entire length of the nucleic acid sequence set forth in SEQ ID NO:4.

[0093] The WT1 consensus antigen can be the amino acid sequence of SEQ ID NO: 4. In some embodiments, the WT1 consensus antigen can be an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity over the entire length of the nucleic acid sequence set forth in SEQ ID NO: 4.

[0094] Immunogenic fragments of SEQ ID NO:2 and SEQ ID NO:4 are obtained. Immunogenic fragments may comprise at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of SEQ ID NO:2 and / or SEQ ID NO:4. In some embodiments, immunogenic fragments may comprise at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of SEQ ID NO:2. with the proviso that residues 312, 317, 342, and 347 of SEQ ID NO:2, when present in the immunogenic fragment, are any residue other than cysteine ​​(C), and residues 330, 334, 360, and 364 of SEQ ID NO:2, when present in the immunogenic fragment, are any residue other than histidine (H). In other embodiments, the immunogenic fragment may comprise at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of SEQ ID NO:2 with the proviso that residues 312, 317, 330, 334, 342, 347, 360, and 364 of SEQ ID NO:2, when present in the immunogenic fragment, are glycine (G).

[0095] In some embodiments, an immunogenic fragment includes a leader sequence, e.g., an immunoglobulin leader sequence, such as an immunoglobulin E (IgE) leader sequence. In some embodiments, an immunogenic fragment does not include a leader sequence.

[0096] Immunogenic fragments of proteins having amino acid sequences identical to immunogenic fragments of SEQ ID NO:2 and 4 are obtained. Such fragments may comprise at least 60%, at least 65%, at least 70%, at least 75%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of a protein having 95% or more identity to SEQ ID NO:2 and / or SEQ ID NO:4. Some embodiments relate to immunogenic fragments having 96% or more identity to immunogenic fragments of WT1 protein sequences described herein. Some embodiments relate to immunogenic fragments having 97% or more identity to immunogenic fragments of WT1 protein sequences described herein. Some embodiments relate to immunogenic fragments having 98% or more identity to immunogenic fragments of WT1 protein sequences described herein. Some embodiments relate to immunogenic fragments having 99% or more identity to immunogenic fragments of WT1 protein sequences described herein. In some embodiments, an immunogenic fragment includes a leader sequence, e.g., an immunoglobulin leader sequence, such as an IgE leader sequence. In some embodiments, an immunogenic fragment does not include a leader sequence.

[0097] Some embodiments relate to immunogenic fragments of SEQ ID NO:1 and SEQ ID NO:3. Immunogenic fragments may comprise at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of SEQ ID NO:1 and / or SEQ ID NO:3. In some embodiments, immunogenic fragments comprise a sequence encoding a leader sequence, e.g., an immunoglobulin leader sequence, such as an IgE leader sequence. In some embodiments, immunogenic fragments do not comprise a coding sequence encoding a leader sequence. Immunogenic fragments of nucleic acids having nucleotide sequences having identity to the immunogenic fragments of SEQ ID NO:1 and SEQ ID NO:3 are obtained. Such fragments may comprise at least 60%, at least 65%, at least 70%, at least 75%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of a nucleic acid having 95% or greater identity to SEQ ID NO:1 and / or SEQ ID NO:3. Some embodiments relate to immunogenic fragments having 96% or greater identity to immunogenic fragments of WT1 nucleic acid sequences described herein. Some embodiments relate to immunogenic fragments having 97% or greater identity to immunogenic fragments of WT1 nucleic acid sequences described herein. Some embodiments relate to immunogenic fragments having 98% or greater identity to immunogenic fragments of WT1 nucleic acid sequences described herein. Some embodiments relate to immunogenic fragments having 99% or greater identity to immunogenic fragments of WT1 nucleic acid sequences described herein. In some embodiments, the immunogenic fragment comprises a sequence encoding a leader sequence, e.g., an immunoglobulin leader sequence, such as an IgE leader sequence. In some embodiments, the immunogenic fragment does not include the coding sequence that encodes a leader sequence.

[0098] b. Vector The vaccine may include one or more vectors that contain a heterologous nucleic acid that encodes an antigen. The one or more vectors can express an effective amount of the antigen to induce an immune response in a mammal. The vector may include a heterologous nucleic acid that encodes an 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 a host genome.

[0099] One or more vectors can be expression constructs, which are generally 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. The plasmid contains regulatory sequences that act as enhancer and promoter regions, and is often engineered to provide efficient transcription of the gene carried on the expression vector. The vector of the present invention expresses large amounts of stable messenger RNA, and therefore large amounts of stable protein.

[0100] 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, a transcription termination sequence and insertion of a PTIS (portable translation initiation sequence).

[0101] (1) Expression vector The vector can be a circular plasmid or a linear nucleic acid. Circular plasmids and linear nucleic acids can result in the expression of a particular nucleotide sequence in a suitable target cell. The vector can have a promoter operably linked to the antigen-encoding nucleotide sequence, which can be operably linked to a termination signal. The vector can also include sequences required for proper translation of the nucleotide sequence. The vector containing the nucleotide sequence of interest can be chimeric, meaning that 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 can be under the control of a constitutive or inducible promoter, which initiates transcription only when the host cell is exposed to some specific external stimulus. In the case of a multicellular organism, the promoter can be specific to a particular tissue or organ or stage of development.

[0102] (2) Plasmid 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.

[0103] The plasmid may contain a nucleic acid sequence encoding one or more of the various antigens described above, including a coding sequence encoding a synthetic antigen, a consensus antigen capable of eliciting an immune response against the antigen, a fragment of such a protein, a variant of such a protein, a fragment of a variant, or a fusion protein consisting of a combination of a consensus protein and / or a fragment of a consensus protein and / or a variant of a consensus protein and / or a fragment of a variant of a consensus protein.

[0104] A single plasmid may contain the coding sequence for a single antigen, the coding sequence for two antigens, the coding sequence for three antigens, or the coding sequence for four antigens.

[0105] In some embodiments, the plasmids may further comprise coding sequences encoding only CCR20 or as part of these plasmids. Similarly, the plasmids may further comprise coding sequences for IL-12, IL-15, and / or IL-28.

[0106] 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.

[0107] The plasmid may include a promoter operably linked to the coding sequence. The promoter operably linked to the coding sequence may be a promoter from Simian Virus 40 (SV40), a mouse mammary tumor virus (MMTV) promoter, a human immunodeficiency virus (HIV) promoter, such as 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, an Epstein-Barr virus (EBV) promoter, or a Rous sarcoma virus (RSV) promoter. The promoter may be a promoter from a human gene, such as human actin, human myosin, human hemoglobin, human muscle creatine, or human metallothionein. The promoter may be a tissue-specific promoter, such as a muscle or skin specific promoter, and may be natural or synthetic. Examples of such promoters are described in U.S. Patent Publication US20040175727, the contents of which are incorporated herein in their entirety.

[0108] The plasmid may contain 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, or a human β-globin polyadenylation signal. The SV40 polyadenylation signal may be a polyadenylation signal from pCEP4 plasmid (Invitrogen, San Diego, Calif.).

[0109] The plasmid may 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 CMV, FMDV, RSV, or EBV.Polynucleotide function enhancers are described in U.S. Patent Nos. 5,593,972, 5,962,428, and WO94 / 016737, the contents of each of which are incorporated by reference in their entirety.

[0110] The plasmid may also contain a mammalian origin of replication to maintain the plasmid extrachromosomally and to produce multiple copies of the plasmid in the cell. The plasmid may be pVAXI, pCEP4, or pREP4 from Invitrogen (San Diego, CA), which contain the Epstein-Barr virus origin of replication and the coding region for the nuclear antigen EBNA-1, resulting in high copy episomal replication without integration. The plasmid backbone may be pAV0242. The plasmid may be a replication-deficient adenovirus type 5 (Ad5) plasmid.

[0111] The plasmid may also contain regulatory sequences, which may be sufficient for gene expression in a cell to which the plasmid is administered. The coding sequence may contain codons that allow for more efficient transcription of the coding sequence in the host cell.

[0112] The coding sequence may include an 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 following the consensus antigen protein. The N-terminal Ig leader may be IgE or IgG.

[0113] The plasmid may be pSE420 (Invitrogen, San Diego, CA), which may be used for protein production in Escherichia coli (E. Coli). The plasmid may be pYES2 (Invitrogen, San Diego, CA), which may be used for protein production in Saccharomyces cerevisiae strains of yeast. The plasmid may be of the MAXBAC™ Complete Baculovirus Expression System (Invitrogen, San Diego, CA), which may be used for protein production in insect cells. The plasmid may be pcDNA I or pcDNA3 (Invitrogen, San Diego, CA), which may be used for protein production in mammalian cells, such as Chinese Hamster Ovary (CHO) cells.

[0114] (3) Circular and linear vectors The vector may be a circular plasmid, which may transform the target cell into the cellular genome by integration, or it may exist extrachromosomally (eg, an autonomously replicating plasmid having an origin of replication).

[0115] The vector may be pVAX, pcDNA3.0, provax, or any other expression vector capable of expressing DNA encoding an antigen and allowing the cell to translate the sequence into an antigen that is recognized by the immune system.

[0116] Also provided herein is a linear nucleic acid vaccine or linear expression cassette ("LEC") that can be efficiently delivered to a subject via electroporation and express one or more desired antigens. The LEC may be any linear DNA lacking any phosphate backbone. The DNA may encode one or more antigens. The LEC may include a promoter, an intron, a stop codon, and / or a polyadenylation signal. Expression of the antigen may be controlled by a promoter. The LEC may not include any antibiotic resistance genes and / or a phosphate backbone. The LEC may not include other nucleic acid sequences not related to the desired antigen gene expression.

[0117] The LEC may be derived from any plasmid that can be linearized. The plasmid may be capable of expressing an antigen. The plasmid may be pNP (Puerto Rico / 34) or pM2 (New Caledonia / 99). The plasmid may be WLV009, pVAX, pcDNA3.0, provax, or any other expression vector capable of expressing DNA encoding an antigen and allowing the cell to translate the sequence into an antigen that is recognized by the immune system.

[0118] The LEC may be pcrM2. The LEC may be pcrNP. The pcrNP and pcrMR may be derived from pNP (Puerto Rico / 34) and pM2 (New Caledonia / 99), respectively.

[0119] (4) Promoter, intron, stop codon, and polyadenylation signal The vector may have a promoter. The promoter may be any promoter capable of driving gene expression and regulating the expression of the isolated nucleic acid. Such promoter is a cis-acting sequence element required for transcription via DNA-dependent RNA polymerase, and transcribes the antigen sequence described herein. The promoter used to direct the expression of the heterologous nucleic acid is selected depending on the specific application. The promoter may be located at approximately the same distance from the transcription start site of the vector as it is from the transcription start site in its natural setting. However, variation in this distance may be accommodated without loss of promoter function.

[0120] The promoter can be operably linked to the antigen-encoding nucleic acid sequence and to signals required for efficient polyadenylation of the transcript, ribosome binding sites, and translation termination.

[0121] The promoter may be a CMV promoter, an SV40 early promoter, an SV40 late promoter, a metallothionein promoter, a mouse mammary tumor virus promoter, a Rous sarcoma virus promoter, a polyhedrin promoter, or other promoters which show efficient expression in eukaryotic cells.

[0122] The vector may contain an enhancer and an intron with functional splice donor and functional splice acceptor sites. The vector may contain a transcription termination region downstream of the structural gene to provide for efficient termination. The termination region may be obtained from the same gene as the promoter sequence or may be obtained from a different gene.

[0123] (5) Method for preparing vector Provided herein is a method for preparing a vector comprising the DNA vaccine disclosed herein, which, after a final step of subcloning into a mammalian expression plasmid, can be used to inoculate cell cultures in large-scale fermentation tanks using methods known in the art.

[0124] The vectors used in the EP device can be formulated or manufactured using a combination of known equipment and techniques, as described in detail below, but preferably they are manufactured using the optimized plasmid manufacturing techniques described in co-pending U.S. Provisional Patent Application No. 60 / 939,792, filed May 23, 2007, which is subject to grant. In some instances, the DNA plasmids used in these studies can be formulated at concentrations of 10 mg / mL or greater. The manufacturing techniques include and incorporate a variety of equipment and protocols generally known to those of skill in the art, including those described in U.S. Patent Application No. 60 / 939,792, as well as those described in U.S. Patent Application No. 7,238,522, which is subject to grant, issued July 3, 2007. The previously referenced applications and patents, U.S. Patent Application No. 60 / 939,792 and U.S. Patent No. 7,238,522, are each incorporated herein in their entirety.

[0125] c. Vaccine excipients and other components The vaccine may further comprise a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient may be a functional molecule as a vehicle, carrier, or diluent. The pharmaceutically acceptable excipient may be a transfection enhancer, which may comprise a surfactant, such as immune stimulating complexes (ISCOMS), Freund's incomplete adjuvant, LPS analogs including monophosphoryl lipid A, muramyl peptides, quinone analogs, vesicles such as squalene and squalene, hyaluronic acid, lipids, liposomes, calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other known transfection enhancers.

[0126] The transfection facilitating agent is a polyanion, polycation, or lipid, including poly-L-glutamate (LGS). The transfection facilitating agent is poly-L-glutamate, which may be present in the vaccine at a concentration of less than 6 mg / ml. The transfection facilitating agent may include surfactants, such as immune stimulating complexes (ISCOMS), Freund's incomplete adjuvant, LPS analogs, including monophosphoryl lipid A, muramyl peptides, quinone analogs, and vesicles, such as squalene and squalene, and may be administered in conjunction with the genetic construct using hyaluronic acid. The DNA plasmid vaccine may include a transfection facilitating agent, such as lipids, liposomes, including other liposomes known in the art, such as lecithin liposomes or DNA liposome mixtures (see, for example, WO09324640), calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other known transfection facilitating agents. The transfection facilitating agent is a polyanion, polycation, or lipid, including poly-L-glutamate (LGS). 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.

[0127] The pharma- ceutically acceptable excipient may be one or more adjuvants, which may be other genes expressed from a separate plasmid or delivered as proteins in combination with the plasmids described above 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), TNFα, 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, MHC, CD80, CD86, IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-18, MCP-1, MIP-1a, MIP-1, IL-8, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, pl50.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, mutant 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 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 deleted and optionally including a different signal peptide, such as a signal peptide from IgE, or a coding sequence encoding a different signal peptide, such as a signal peptide 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, TNFβ, GM-CSF, epidermal growth factor (EGF), IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-18, or a combination thereof.

[0128] In some embodiments, the adjuvant may be one or more proteins and / or nucleic acid molecules encoding a protein selected from the group consisting of CCL-20, IL-12, IL-15, IL-28, CTACK, TECK, MEC, or RANTES. Examples of IL-12 constructs and sequences are disclosed in PCT Application No. PCT / US1997 / 019502 and corresponding U.S. Patent Application No. 08 / 956,865, and U.S. Provisional Patent 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 Patent Application No. 10 / 560,650, and PCT Application No. PCT / US07 / 00886 and corresponding US Patent Application No. 12 / 160,766, and PCT Application No. PCT / US10 / 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 Patent Application No. 12 / 936,192, each of which is incorporated herein by reference. Examples of RANTES, other constructs, and sequences are disclosed in PCT Application No. PCT / US1999 / 004332 and corresponding US Patent 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, other constructs, and sequences are disclosed in PCT Application No. PCT / US1999 / 004332 and corresponding US Patent 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 Patent Application No. 11 / 719,646, each of which is incorporated herein by reference.Examples of OX40 and other immune regulators are disclosed in U.S. Patent Application No. 10 / 560,653, which is incorporated herein by reference. Examples of DR5 and other immune regulators are disclosed in U.S. Patent Application No. 09 / 622452, which is incorporated herein by reference.

[0129] Other genes that may be useful as adjuvants include MCP-1, MIP-la, MIP-1p, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, pl50.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, mutant 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, D R5, 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.

[0130] The vaccine may further comprise a genetic vaccine facilitator as described in US patent application Ser. No. 021,579, filed Apr. 1, 1994, which is incorporated by reference in its entirety.

[0131] The vaccine may contain the antigen and plasmid in an amount of about 1 nanogram to about 100 milligrams; about 1 microgram to about 10 milligrams; preferably about 0.1 microgram to about 10 milligrams; more preferably about 1 milligram to about 2 milligrams. In some preferred embodiments, the vaccine according to the present invention contains about 5 nanograms to about 1000 micrograms of DNA. In some preferred embodiments, the vaccine may contain about 10 nanograms to about 800 micrograms of DNA. In some preferred embodiments, the vaccine may contain about 0.1 to about 500 micrograms of DNA. In some preferred embodiments, the vaccine may contain about 1 to about 350 micrograms of DNA. In some preferred embodiments, the vaccine may contain about 25 to about 250 micrograms, about 100 to about 200 micrograms, about 1 nanogram to 100 milligrams, about 1 microgram to about 10 milligrams, about 0.1 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.

[0132] The vaccine may be formulated according to the mode of administration 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 vasoconstrictor. The isotonic solution may include phosphate buffered saline. The vaccine may further include a stabilizer, including gelatin and albumin. Stabilizers such as LGS or polycations or polyanions allow the formulation to be stable for long periods at room or ambient temperature.

[0133] 3. Vaccine delivery method Provided herein are methods of delivering vaccines to provide genetic constructs and antigens that contain epitopes that are particularly effective against target immunogens that can induce an immune response. Methods of delivering vaccines or administering vaccines to induce therapeutic and prophylactic immune responses can be provided. The vaccination process can generate an immune response to the antigen in a mammal. The vaccine can deliver the vaccine to an individual to modulate the activity of the mammal's immune system and enhance the immune response. The delivery of the vaccine can be the transfection of the antigen as a nucleic acid molecule delivered to the cell surface that is expressed within the cell and then recognized by the immune system to induce a cellular response, a humoral response, or a cellular and humoral response. The delivery of the vaccine can be used to induce or elicit an immune response in a mammal against the antigen by administering the mammal with a vaccine as discussed above.

[0134] Once the vaccine and plasmids are delivered to mammalian cells, the transfected cells express and secrete antigens for each of the injected plasmids from the vaccine. These antigens are recognized as foreign by the immune system, and antibodies are generated against them. These antibodies are maintained by the immune system and are able to respond effectively to the antigens.

[0135] The vaccine can be administered to a mammal to elicit an immune response in the mammal, which may be a human, a primate, a non-human primate, a cow, a cattle, a sheep, a goat, an antelope, a bison, a buffalo, a bison, a bovine, a deer, a hedgehog, an elephant, a llama, an alpaca, a mouse, a rat, and a chicken.

[0136] Combination treatment Other proteins and / or proteins that may be present include CCL20, α-interferon, γ-interferon, platelet-derived growth factor (PDGF), TNFα, 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, including IL-15 with a deleted signal sequence and optionally including a different signal peptide, such as the signal peptide of IgE, MHC, CD80, CD86, IL-28, IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-18, MCP-1, MIP-1α, MIP-1β, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD 34, 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, mutant 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, TRIC K2, DR6, caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, inactive NIK, SAP The vaccine can be administered in combination with genes encoding 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, and functional fragments or combinations thereof.In some embodiments, the vaccine is administered in combination with one or more of the following nucleic acid molecules and / or proteins: a nucleic acid molecule selected from the group consisting of a nucleic acid molecule comprising a coding sequence encoding one or more of CCL20, IL-12, IL-15, IL-28, CTACK, TECK, MEC, and RANTES, or a functional fragment thereof, and a protein selected from the group consisting of CCL20, an IL-12 protein, an IL-15 protein, an IL-28 protein, a CTACK protein, a TECK protein, a MEC protein, a RANTES protein, or a functional fragment thereof.

[0137] The vaccine may be administered by different routes, including orally, parenterally, sublingually, transdermally, rectally, transmucosally, topically, by inhalation, buccal administration, intrathoracically, intravenously, intraarterially, intraperitoneally, subcutaneously, intramuscularly, intranasally, intrathecally, and intraarticularly, or a combination thereof. For veterinary use, the vaccine may be administered in an appropriately acceptable formulation, according to normal veterinary practice. A veterinarian can easily determine the administration regimen and route of administration that is most suitable for a particular animal. The vaccine may be administered by traditional syringe, needleless injection device, "microparticle bombardment gene gun", or other physical methods, such as electroporation (EP), "hydrodynamic methods", or ultrasound.

[0138] Vaccine plasmids can be delivered to mammals by several well-known techniques, including in vivo electroporation, liposome-mediated delivery, nanoparticle facilitation, DNA injection (also referred to as DNA vaccination) with and without recombinant vectors such as recombinant adenoviruses, recombinant adenovirus-associated viruses, and recombinant vaccinia viruses. DNA injection can be used to deliver antigens or immunogens in conjunction with in vivo electroporation.

[0139] B. Electroporation Vaccines can be prepared according to standard techniques well known to those skilled in the pharmaceutical arts. Such compositions can be administered in doses and by techniques well known to those skilled in the medical arts, taking into account factors such as the age, sex, weight, and condition of the particular subject, as well as the route of administration. The subject can be a mammal, such as a human, horse, cow, pig, sheep, cat, dog, rat, or mouse.

[0140] Vaccines can be administered prophylactically or therapeutically. In prophylactic administration, vaccines are administered in an amount sufficient to induce an immune response. In therapeutic applications, vaccines are administered to subjects in need thereof in an amount sufficient to induce a therapeutic effect. An amount sufficient to accomplish this is defined as a "therapeutically effective dose". The effective amount for this use will depend, for example, on the specific composition of the vaccine therapy administered, the mode of administration, the stage and severity of the disease, the patient's general health, and the judgment of the prescribing physician.

[0141] Vaccines are administered by methods well known in the art, such as those described in Donnelly et al. (Ann. Rev. Immunol. 15:617-648 (1997); Felgner et al. (U.S. Patent No. 5,580,859 issued December 3, 1996); Felgner et al. (U.S. Patent No. 5,703,055 issued December 30, 1997); and Carson et al. (U.S. Patent No. 5,679,647 issued October 21, 1997), the contents of which are incorporated herein by reference in their entireties. The DNA of the vaccine can be complexed, for example, with particles or beads and administered to an individual using a vaccine gun. One of skill in the art will know that the choice of a pharma- ceutically acceptable carrier, including a physiologically acceptable compound, will depend, for example, on the route of administration of the expression vector.

[0142] Vaccines may be delivered by a variety of routes. Typical delivery routes include parenteral administration, e.g., intradermal, intramuscular, or subcutaneous delivery. Other routes include oral administration, intranasal, and intravaginal routes. In particular with respect to DNA vaccines, vaccines can be delivered to the interstitial space of tissues of an individual (Felgner et al., U.S. Pat. Nos. 5,580,859 and 5,703,055, the contents of which are incorporated herein by reference in their entirety). Vaccines may be administered into muscle, or through intradermal or subcutaneous injection, or through transdermal administration by iontophoresis. Epidermal administration of vaccines can also be used. Epidermal administration can include mechanically or chemically irritating the outermost layer of the epidermis to stimulate an immune response to the irritant (Carson et al., U.S. Pat. No. 5,679,647, the contents of which are incorporated herein by reference in their entirety).

[0143] Vaccines can also be prepared for administration via the intranasal route. Formulations in which the carrier is solid and suitable for nasal administration include coarse powders having a particle size, for example, in the range of about 10 microns to about 500 microns, and are administered as if snorted, i.e., by rapid inhalation through the nasal passages from a powder container held close to the nose. Formulations may also be nasal sprays, nasal drops, or aerosolized by nebulizer. Formulations may include aqueous or oily solutions of the vaccine.

[0144] The vaccine may be a liquid preparation, such as a suspension, syrup, or elixir, or may be in a formulation for parenteral, subcutaneous, intradermal, intramuscular, or intravenous administration (e.g., by injection), such as a sterile suspension or emulsion.

[0145] Vaccines can be incorporated into liposomes, microspheres, or other polymer matrices (Felgner et al., U.S. Pat. No. 5,703,055; Gregoriadis, Liposome Technology, Vols. I-III (2nd ed., 1993), the contents of which are incorporated herein by reference in their entireties). Liposomes are composed of phospholipids or other lipids and can be non-toxic, physiologically acceptable, metabolizable carriers that are relatively simple to make and administer.

[0146] Administration of the vaccine by electroporation of the vaccine plasmid can be accomplished using an electroporation device that can be configured to deliver an energy pulse to the desired mammalian tissue that is effective to form reversible pores in the cell membrane, preferably the energy pulse is a constant current close to a preset current input by the user. The electroporation device can include an electroporation component and an electrode assembly or a handle assembly. The electroporation component can include or incorporate one or more of the various components 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 can be accomplished using an in vivo electroporation device that facilitates transfection of cells with plasmids, such as the CELLECTRA® EP system (VGX Pharmaceuticals, Blue Bell, PA) or the Elgen electroporator (Genetronics, San Diego, CA).

[0147] The electroporation component may function as one element of the electroporation device, and the other element is a separate element (or components) that communicates with the electroporation component. The electroporation component may function as more than one element of the electroporation device, and it may also communicate with other elements of the electroporation device that are separate from the electroporation component. An element of the electroporation device that exists as part of one electromechanical or mechanical device may not be limited as the element may function as one device or as separate elements that communicate with each other. The electroporation component may be capable of delivering an energy pulse that generates a constant current in the desired tissue, and includes a feedback mechanism. The electrode assembly may include an electrode array having multiple electrodes in a spatial arrangement, and the electrode assembly receives an energy pulse from the electroporation component and delivers it to the desired tissue through the electrodes. At least one of the multiple electrodes is neutral during delivery of the energy pulse and measures the impedance in the desired tissue and communicates the impedance to the electroporation component. A feedback mechanism may receive the measured impedance and can adjust the energy pulses delivered by the electroporation component to maintain a constant current.

[0148] The electrodes may deliver energy pulses in a distributed pattern. The electrodes may deliver energy pulses in a distributed pattern via electrode control under a programmed sequence, the programmed sequence being input by a user into the electroporation component. The programmed sequence may include multiple pulses delivered in sequence, with each pulse of the multiple pulses being delivered by at least two active electrodes, including one neutral electrode that measures impedance, and a subsequent pulse of the multiple pulses being delivered by a different one of the at least two active electrodes, including one neutral electrode that measures impedance.

[0149] The feedback mechanism may be implemented by either hardware or software. The feedback mechanism may be implemented by an analog closed circuit. The feedback occurs every 50 μs, 20 μs, 10 μs or 1 μs, but is preferably real-time feedback or instantaneous (i.e., substantially instantaneous as determined by available techniques for determining response time). The neutral electrode may measure the impedance in the desired tissue, which is communicated to the feedback mechanism, which responds to the impedance to adjust the energy pulse to maintain a constant current at a value similar to the preset current. The feedback mechanism may maintain the constant current continuously and instantaneously during the delivery of the energy pulse.

[0150] Examples of electroporation devices and electroporation methods that may facilitate delivery 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 methods that may be used to facilitate delivery of DNA vaccines include those provided in 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 Patent Application No. 60 / 852,149, filed October 17, 2006, and U.S. Provisional Patent Application No. 60 / 978,982, filed October 10, 2007, all of which are incorporated herein by reference in their entireties.

[0151] US Patent No. 7,245,963 by Draghia-Akli et al. describes a modular electrode system and its use for facilitating the introduction of biomolecules into cells of selected tissues in a body or plant. The modular electrode system may include a plurality of needle electrodes; a hypodermic needle; an electrical connector providing a conductive connection from a programmable constant current pulse controller to the plurality of needle electrodes; and a power source. An operator can grasp the plurality of needle electrodes mounted on a support structure and firmly insert them into selected tissues in a body or plant. The biomolecules are then delivered to the selected tissue via the hypodermic needle. The programmable constant current pulse controller is activated to apply a constant current electrical pulse to the plurality of needle electrodes. The applied constant current electrical pulse promotes the introduction of biomolecules into cells between the plurality of electrodes. The entire contents of US Patent No. 7,245,963 are incorporated herein by reference.

[0152] US Patent Publication No. 2005 / 0052630 filed by Smith et al. describes an electroporation device that can be used to effectively promote the introduction of biomolecules into cells of selected tissues in the body or plant. The electroporation device includes an electrokinetic device ("EKD device") whose operation is specified by software or firmware. The EKD device generates a series of programmable constant current pulse patterns between a row of electrodes and pulse parameter input based on user control, allowing current waveform data to be stored and retrieved. The electroporation device also includes a replaceable electrode disk with an array of needle electrodes, a central injection channel for injection needles, and a removable guide disk. The entire contents of US Patent Publication No. 2005 / 0052630 are incorporated herein by reference.

[0153] The electrode arrays and methods described in U.S. Patent No. 7,245,963 and U.S. Patent Publication No. 2005 / 0052630 can be adapted to penetrate deep into tissues such as muscles, as well as other tissues or organs. Depending on the configuration of the electrode array, the injection needle (delivering the selected biomolecule) is also 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.

[0154] In addition, as anticipated in some embodiments incorporating electroporation devices and their use, there are electroporation devices described in the following patents: 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, 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 including subject matter provided in U.S. Patent No. 6,697,669 issued February 24, 2004, which relates to delivery 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 DNA injection, are contemplated herein. The foregoing patents are incorporated by reference in their entirety.

[0155] Vaccine can be administered via electroporation by the method described in U.S. Patent No. 7,664,545, the contents of which are incorporated herein by reference. Electroporation can be by the method and / or device described in U.S. Patent Nos. 6,302,874; 5,676,646; 6,241,701; 6,233,482; 6,216,034; 6,208,893; 6,192,270; 6,181,964; 6,150,148; 6,120,493; 6,096,020; 6,068,650; and 5,702,359, the contents of which are incorporated herein by reference in their entirety. Electroporation can also be carried out via a minimally invasive device.

[0156] A minimally invasive electroporation device ("MID") may be a device that injects the vaccine and associated fluids described above into body tissue. The device may include a hollow needle, a DNA cassette, and a fluid delivery means, the device being adapted to activate the fluid delivery means in use to simultaneously (e.g., automatically) inject the DNA into the body tissue while the needle is being inserted. This has the advantage that the ability to gradually inject the DNA and associated fluids while the needle is being inserted results in a more uniform distribution of the fluids in the body tissue. Also, the distribution of the injected DNA over a larger area may reduce the pain experienced during injection.

[0157] The MID can inject the vaccine into tissue without the use of a needle. The MID can inject the vaccine as a small stream or jet with such force that the vaccine penetrates the tissue surface and enters the underlying tissue and / or muscle. The force behind the small stream or jet can be provided by the instantaneous expansion of a compressed gas such as carbon dioxide through a micro-orifice. Examples of minimally invasive electroporation devices and methods of their use are described in U.S. Published Patent Application No. 2008 / 0234655; U.S. Patent No. 6,520,950; U.S. Patent No. 7,171,264; U.S. Patent No. 6,208,893; U.S. Patent No. 6,009,347; U.S. Patent No. 6,120,493; U.S. Patent No. 7,245,963; U.S. Patent No. 7,328,064; and U.S. Patent No. 6,763,264, the contents of each of which are incorporated herein by reference.

[0158] MID can include a syringe that generates high-speed jet of liquid that penetrates tissue painlessly.Such needleless syringes are commercially available.Examples of needleless syringes that can be utilized herein include those described in U.S. Patent No. 3,805,783; No. 4,447,223; No. 5,505,697; and No. 4,342,310, the contents of each of which are incorporated herein by reference.

[0159] The desired vaccine in a form suitable for direct or indirect electrotransport can be introduced (e.g., injected) into the tissue to be treated using a needleless syringe.This is usually done by contacting the syringe with the tissue surface and jetting the agent with sufficient force to make the vaccine penetrate the tissue.For example, if the tissue to be treated is mucous membrane, skin, or muscle, the agent is fired toward the mucous membrane or skin surface with sufficient force to make the agent penetrate through the stratum corneum into the skin layer or into the underlying tissue and muscle, respectively.

[0160] Needleless injectors are suitable for delivering vaccines to all types of tissues, especially skin and mucous membranes. In some embodiments, needleless injectors can be used to advance liquids containing vaccines to surfaces and to the skin or mucous membranes of subjects. Representative examples of different types of tissues that can be treated using the methods of the present invention include pancreas, larynx, nasopharynx, hypopharynx, oropharynx, lips, throat, lung, heart, kidney, muscle, breast, colon, prostate, thymus, testis, skin, mucosal tissue, ovary, blood vessels, or any combination thereof.

[0161] MIDs can have needle electrodes that electroporate tissue. For example, generating pulses between pairs of electrodes in a multiple electrode array set in a rectangular or square pattern provides better results than generating pulses between a pair of electrodes. US Patent No. 5,702,359, entitled "Needle Electrodes for Mediated Delivery of Drugs and Genes," discloses a needle array that can generate pulses in pairs of needles during therapeutic treatment. In that application, the needles are arranged in a circular array, but have connectors and switching devices that can generate pulses between opposing pairs of needle electrodes. A pair of needle electrodes can be used to deliver recombinant expression vectors to cells. Such devices and systems are described in US Patent No. 6,763,264, the contents of which are incorporated herein by reference. Alternatively, a single-needle device that can inject DNA and perform electroporation through a single needle similar to a regular hypodermic needle could be used to apply a lower voltage pulse than can be delivered by currently used devices, reducing the electrical sensation experienced by patients.

[0162] The MID may include one or more electrode arrays. The array may include two or more needles of the same or different diameters. The needles may be equally or unequally spaced. The needles may be 0.005 inches to 0.03 inches; 0.01 inches to 0.025 inches; or 0.015 inches to 0.020 inches. The needles may be 0.0175 inches in diameter. The needles may be spaced 0.5 mm, 1.0 mm, 1.5 mm, 2.0 mm, 2.5 mm, 3.0 mm, 3.5 mm, 4.0 mm, or more apart.

[0163] The MID may consist of a pulse generator and two or more needle vaccine syringes that deliver the vaccine and provide electroporation pulses in a single step. The pulse generator allows for flexible programming of pulse and injection parameters via a flash card operated by a personal computer, as well as comprehensive recording and storage of electroporation and patient data. The pulse generator can deliver short, variable voltage pulses. For example, the pulse generator can deliver three 100 millisecond (ms) long 15 volt pulses. An example of such an MID is the Elgen1000 system manufactured by Inovio Biomedical Corporation, described in U.S. Pat. No. 7,328,064, the contents of which are incorporated herein by reference.

[0164] The MID may be a CELLECTRA (Innovio Pharmaceuticals, Inc., Blue Bell, PA) device and system, which is a modular electrode system for facilitating the introduction of macromolecules, such as DNA, into cells of selected tissues of an organism or plant. The modular electrode system may include a plurality of needle electrodes; a hypodermic needle; an electrical connector providing a conductive connection from a programmable constant current pulse controller to the plurality of needle electrodes; and a power source. An operator may grasp the plurality of needle electrodes mounted on a support structure and firmly insert it into the selected tissue of an organism or plant. The macromolecules are then delivered into the selected tissue via the hypodermic needle. The programmable constant current pulse controller is activated to apply a constant current electrical pulse to the plurality of needle electrodes. The applied constant current electrical pulse promotes the introduction of the macromolecules into the cells between the plurality of electrodes. Cell death due to overheating of the cells is minimized by limiting power consumption in the tissue with the constant current pulse. The Cellectra device and system are described in U.S. Pat. No. 7,245,963, the contents of which are incorporated herein by reference.

[0165] The MID can be the Elgen1000 system (Innovio Pharmaceuticals). The Elgen1000 system may include a device with a hollow needle and a fluid delivery means, which is adapted to activate the fluid delivery means in use to simultaneously (e.g., automatically) inject fluid, which is a vaccine as described herein, into the body tissue during the insertion of the needle into the body tissue. This has the advantage that the ability to gradually inject fluid while the needle is being inserted results in a more uniform distribution of the fluid in the body tissue. It is also believed that the distribution of the injected fluid over a larger area reduces the pain experienced during injection.

[0166] Automatic injection of fluids facilitates automatic monitoring and registration of the actual dose of fluid injected. This data can be stored by the control unit for documentation purposes, if desired.

[0167] It will be understood that the injection rate may be linear or non-linear, and that the injection is carried out after the needles are inserted through the skin of the subject and as they are further inserted into the body tissue.

[0168] Tissues suitable for injecting fluid with the device of the present invention include tumor tissue, skin, or liver tissue, but may also be muscle tissue.

[0169] The device further comprises needle insertion means for guiding the insertion of the needle into the body tissue. The rate of fluid injection is controlled by the rate of needle insertion. This has the advantage that both needle insertion and fluid injection can be controlled so that the rate of insertion can be matched to the rate of injection as desired. It also makes the device easier for the user to operate. If desired, means can be provided for automatically inserting the needle into the body tissue.

[0170] The user can choose when the injection of fluid should commence. Ideally, however, injection commences when the tip of the needle reaches the muscle tissue, and the device includes means for sensing when the needle has been inserted deep enough for injection of fluid to commence. This means that injection of fluid can commence automatically when the needle has reached the desired depth (which will normally be the depth at which muscle tissue commences). The depth at which muscle tissue commences could also be taken to be a pre-set needle insertion depth, such as a value of 4mm (this value is deemed sufficient for the needle to penetrate the skin layer).

[0171] The sensing means may include an ultrasonic probe. The sensing means may include means for sensing changes in impedance or resistance, in which case the means may not itself record the depth of the needle within the body tissue, but rather sense changes in impedance or resistance as the needle moves through various types of body tissue and into the muscle. Either of these alternatives provides a relatively accurate and easy to operate means for sensing the start of injection. If desired, the needle insertion depth can further be recorded, which can be used to control the injection of the fluid, thereby determining the amount of fluid to be injected as the needle insertion depth is recorded.

[0172] The device further includes a base supporting the needle and a housing receiving the base therein, the base being movable relative to the housing such that the needle is retracted into the housing when the base is in a first rearward position relative to the housing and the needle extends from the housing when the base is in a second forward position within the housing, which is convenient for a user as the housing can be aligned over a patient's skin and the needle can then be inserted into the patient's skin by moving the housing relative to the base.

[0173] As mentioned above, it is desirable to achieve a controlled rate of fluid injection so that the fluid is evenly distributed over the length of the needle as it is inserted into the skin. The fluid delivery means may include a piston drive means adapted to inject the fluid at a controlled rate. The piston drive means may be driven, for example, by a servo motor. However, the piston drive means may be actuated by axial movement of the base relative to the housing. It will be appreciated that alternative means of fluid delivery may be provided. Thus, for example, a closed container that can be squeezed to deliver fluid at a controlled or uncontrolled rate may be provided instead of a syringe and piston system.

[0174] The above device can be used for any type of injection. However, it is believed to be particularly useful in the field of electroporation, and therefore it may further comprise a means for applying a voltage to the needle. This allows the needle to be used not only for injection, but also as an electrode during electroporation. This is particularly advantageous as it means that the electric field is applied to the same area as the fluid being injected. Traditionally, electroporation has had the problem that it is very difficult to precisely align the electrode with the previously injected fluid, and therefore, in an attempt to ensure overlap between the injected material and the electric field, users tend to inject larger amounts of fluid than necessary over a large area and apply the electric field over a large area. Using the present invention, both the amount of fluid injected and the size of the applied electric field can be reduced while still achieving a good match between the electric field and the fluid.

[0175] 4. Treatment and / or prevention Also provided herein is a method for administering the vaccine to a subject in need thereof.The disease can be cancer, for example, Wilms' tumor, metastatic cancer arising from Wilms' tumor, and cancer or tumor expressing WT1.The vaccine can be administered to a subject by the above-mentioned delivery method.The vaccine can be administered to a subject to induce or elicit an immune response in the subject by administering the vaccine to the subject.In particular, the vaccine can be administered to a subject to induce or elicit a humoral and / or cellular immune response in the subject.

[0176] In particular, the method can treat a subject having Wilms tumor, metastatic cancer arising from Wilms tumor, and / or WT1-expressing tumor or cancer, because the vaccine slows, shrinks, or removes the growth of Wilms tumor, metastatic cancer arising from Wilms tumor, and / or WT1-expressing tumor or cancer. The method can also prevent Wilms tumor, metastatic cancer arising from Wilms tumor, and / or WT1-expressing tumor or cancer in a subject, because the vaccine inhibits the formation and growth of Wilms tumor, metastatic cancer arising from Wilms tumor, and / or WT1-expressing tumor or cancer. As described above, the vaccine induces or induces a humoral and / or cellular immune response. The induced humoral and / or cellular immune response targets Wilms tumor, metastatic cancer arising from Wilms tumor, and / or WT1-expressing tumor or cancer, and can slow, reduce, or eliminate the growth of Wilms tumor, metastatic cancer arising from Wilms tumor, and / or WT1-expressing tumor or cancer in a subject administered the vaccine. The humoral and / or cellular immune response induced by the vaccine can also inhibit the formation or growth of any Wilms tumor, metastatic cancer arising from Wilms tumor, and / or WT1-expressing tumor or cancer in a subject administered the vaccine.

[0177] The dose of the vaccine may be 1 μg to 10 mg active ingredient / kg body weight / hour, or 20 μg to 10 mg active ingredient / kg body weight / hour. The vaccine may be administered every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days. The number of doses of the vaccine for effective treatment may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0178] The present invention has many aspects, which are illustrated by the following non-limiting examples. EXAMPLES

[0179] 5. Working Example The present invention will be further described below with reference to examples. These examples show preferred embodiments of the present invention, but should be understood to be provided for illustrative purposes only. From the above description and these examples, a person 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 suit various applications and conditions without departing from the spirit and scope of the present invention. Therefore, in addition to those shown and described herein, various modifications of the present invention will be apparent to a person skilled in the art from the above description. Such modifications are also intended to be included within the scope of the claims.

[0180] In the examples, a stepwise approach described herein was taken to generate WT1 immunogens. The WT1 gene was modified through several stepwise modifications. First, the WT1 RNA structure was modified to produce a single full-length transcript. Next, the RNA structure was altered at the 5' end and the codon usage was modified to enhance in vivo expression. Finally, the zinc finger domain of the gene was mutated to modify WT1 activity and delete a portion of the zinc finger binding site.

[0181] Example 1 Optimized WT-1 A stepwise approach to generate WT1 immunogens was developed. First, changes in the RNA structure were engineered to produce only a single full-length transcript, creating an immunogen with a much more controlled phenotype. The sequence also contained modified codon usage choices, and the RNA structure was altered at the 5' end to enhance in vivo expression. Thus, the WT1 immunogen was optimized for expression. Plasmids encoding the expression cassettes for this improved immunogen were generated and tested in animals for potential immunity and the ability to generate T-cell and B-cell responses. This plasmid vaccine was formulated to enhance in vivo delivery by electroporation. Specific conditions were used for in vivo delivery.

[0182] Plasmids encoding this immunogen (termed WT1-pVAX1, WT1-pVAX2, WT1-pVAX3, WT1-pVAX4, and WT1-pVAX5) were compared with the WT1 vaccines (termed WT1-pCDNA1, WT1-pCDNA2, WT1-pCDNA3, WT1-pCDNA4, and WT1-pCDNA5), which represent standard vaccines studied in the field.

[0183] A mouse study was performed in which mice vaccinated with modified WT1 vaccines (i.e., vectors WT1-pVAX1, WT1-pVAX2, WT1-pVAX3, WT1-pVAX4, and WT1-pVAX5) demonstrated greater anti-WT1 T and B cell responses than mice vaccinated with conventional WT1 vaccines containing native WT1 (i.e., WT1-pCDNA1, WT1-pCDNA2, WT1-pCDNA3, WT1-pCDNA4, and WT1-pCDNA5).

[0184] Animals (i.e., BalB / C mice) were immunized three times with identical amounts of WT-1 plasmids (either the new WT1-pVax vaccines (i.e., vectors WT1-pVAX1, WT1-pVAX2, WT1-pVAX3, WT1-pVAX4, and WT1-pVAX5) or the original WT-1 plasmid vaccines (i.e., WT1-pCDNA1, WT1-pCDNA2, WT1-pCDNA3, WT1-pCDNA4, and WT1-pCDNA5)). The results of the T cell assay (i.e., interferon-gamma (IFN-γ ELISpot assay) are shown in Figure 1 and Figure 2. It was evident that the newly designed WT-1 vaccines (i.e., vectors WT1-pVAX1, WT1-pVAX2, WT1-pVAX3, WT1-pVAX4, and WT1-pVAX5) were approximately four-fold superior in generating T cell responses than the prototype WT-1 vaccines (i.e., WT1-pCDNA1, WT1-pCDNA2, WT1-pCDNA3, WT1-pCDNA4, and WT1-pCDNA5). As shown in Figure 1 and Figure 2, only low levels or non-functional T cell immunity was observed with the conventional vaccines. This was consistent with the previously achieved data.

[0185] The ability of this novel DNA vaccine to induce antibody responses was also examined. These studies were performed by collecting sera from animals immunized with either the WT1-pCDNA vaccine or the WT1-pVAX vaccine. No seroconversion or induction of antibody responses was observed with the WT1-pCDNA vaccine (data not shown). In contrast, the WT1-pVAX immunogen induced strong Western blot reactivity with the correct specificity and molecular weight, indicating robust WT1 seroconversion and strong antibody responses (Figure 3). Collectively, these data strongly support the improvement of this vaccine immunogenicity through altered delivery and improved design of the WT1 immunogen.

[0186] These data also demonstrated that the conformational properties of the immunogen were maintained as these vaccines contained an optimized WT1 immunogen that specifically reacted with the native gene sequence in tumor cells.

[0187] The immunogen sequence was targeted by modifying the coding sequence to disrupt its native structure through two means: (1) targeting the zinc finger region and introducing mutations that alter WT1 activity, and (2) deleting sequences from critical zinc finger binding sites. These changes are summarized in Examples 2 and 3 below.

[0188] Example 2 Consensus WT1 As described above, the optimized WT1 immunogen (i.e., the WT1 gene was optimized as described in Example 1) induced humoral and cellular immune responses. To further target and modify the WT1 immunogen sequence, a consensus WT1 immunogen was generated.

[0189] Specifically, WT1 sequences from multiple species were compared with each other. As shown in Table 1 below, WT1 is highly conserved. Therefore, WT1 sequences from multiple species were used to generate a WT1 consensus sequence. The WT1 consensus sequence shared approximately 95% identity with human WT1. The resulting consensus WT1 immunogen (also referred to as ConWT1) shares 95.9% identity with human WT1 and has the amino acid sequence set forth in SEQ ID NO:5 (FIG. 5).

[0190] [Table 1]

[0191] Example 3 Zinc finger modification and removal The consensus WT1 immunogen described in Example 2 above was further modified to improve the immunogenicity of the WT1 immunogen. In particular, the consensus WT1 immunogen was modified to disrupt the zinc fingers located at the carboxy terminus (C-terminus) of the WT1 immunogen. These modifications included replacing residues that coordinate the zinc ion (i.e., CCHH motif) in the two amino-terminal (N-terminal) zinc fingers to generate a consensus WT1 immunogen with modified zinc fingers (also referred to herein as CON WT1 or ConWT1-L with modified zinc fingers) (Figures 4 and 5). The C and H residues of the CCHH motif were replaced with glycine (G). An immunoglobulin E (IgE) leader sequence was placed on the N-terminus of the ConWT1-L peptide. ConWT1-L is encoded by SEQ ID NO:1 and has the amino acid sequence set forth in SEQ ID NO:2 (Figures 6A and 6B, respectively).

[0192] Figure 5 shows an alignment of the amino acid sequences of the consensus WT1 immunogen (ConWT1) and the consensus WT1 immunogen with modified zinc fingers (ConWT1-L). The shading in Figure 5 indicates residues that differ between ConWT1 and ConWT1-L. In addition to the addition of an IgE leader sequence to ConWT1-L, residues 312, 317, 330, 334, 342, 347, 360, and 364 of ConWT1-L differed from the corresponding residues of ConWT1 (i.e., residues 295, 300, 313, 317, 325, 330, 343, and 347). These differences reflected the modification of the CCHH motif in the two N-terminal zinc fingers described above.

[0193] Additionally, the consensus WT1 immunogen was modified to remove the zinc fingers to generate a consensus WT1 immunogen lacking zinc fingers (also referred to herein as CON WT1 lacking zinc fingers or CONWT1-S) (FIG. 4). ConWT1-S is encoded by SEQ ID NO:3 and has the amino acid sequence set forth in SEQ ID NO:4 (FIGS. 7A and 7B, respectively).

[0194] Example 4 Expression analysis of constructs encoding ConWT1-L and ConWT1-S Nucleic acid sequences encoding ConWT1-L and ConWT1-S were separately placed in pVAX1 vector (Life Technologies, Carlsbad, CA). The resulting vectors were named WT1-pVAX-L and WT1-pVAX-S, respectively. WT1-pVAX-L and WT1-pVAX-S were transfected into cells together with pVAX1 to confirm the expression of ConWT1-L and ConWT1-S from these vectors. pVAX1 was used as a negative control.

[0195] After transfection, cells were stained with 4',6-diamidino-2-phenylindole (DAPI) to mark nuclei and antibodies specific for WT1. Figure 8 shows the staining results. In Figure 8, the left and center columns show DAPI and WT1 staining, and the right column shows a composite of DAPI and WT1 staining. No staining was detected with WT1 antibody in cells transfected with pVAX1. These data indicated that both ConWT1-L and ConWT1-S were expressed from their respective vectors.

[0196] The expression of ConWT1-L and ConWT1-S was further confirmed by immunoblotting of lysates from transfected cells. Specifically, the immunoblots were probed with anti-WT1 antibody. As shown in FIG. 9, ConWT1-L and ConWT1-S of the expected size were detected (see lanes labeled WT1-pVAX-L and WT1-pVAX-S, respectively). No signal was detected in lysates obtained from cells transfected with pVAX1. Thus, these data indicated that ConWT1-L and ConWT1-S were expressed in the transfected cells.

[0197] Example 5 Immune responses induced by constructs encoding ConWT1-L and ConWT1-S To determine whether the constructs encoding ConWT1-L and ConWT1-S induced an immune response, C57BL / 6 mice were immunized with 25 μg of WT1-pVAX-L or WT1-pVAX-S, respectively. C57BL / 6 mice were also immunized with 25 μg of WT1-pVAX (the optimized construct encoding WT1 described in Example 1) or 25 μg of WT1-pCDNA (the non-optimized construct encoding WT1). Naive mice were used as controls.

[0198] Specifically, the immunization schedule included administering 25 μg of each vaccine to each group of mice at weeks 0, 2, 3, and 5 ( FIG. 10 ). Blood was collected from each mouse prior to vaccination at week 0, and this week 0 blood was used as a control for antibody induction. A second blood was collected at week 5, when the mice were sacrificed. Splenocytes were isolated from sacrificed mice and used in an ELISpot assay as described below to examine T cell responses.

[0199] The cellular immune response (i.e., T cell response) against ConWT1-L and ConWT1-S was examined using an ELISpot assay, in which interferon-gamma (IFN-γ) produced by T cells was measured. As shown in Figures 11 and 12, immunization with WT1-pVAX and WT1-pCDNA constructs generated T cell responses similar to those observed in naive mice. In contrast, WT1-pVAX-L and WT1-pVAX-S constructs expressing ConWT1-L and ConWT-S, respectively, generated significantly higher T cell responses compared to the optimized and non-optimized constructs (i.e., WT1-pVAX and WT1-pCDNA, respectively). In Figures 11 and 12, error bars reflect the standard error of the mean (SEM).

[0200] In particular, the ConWT1-L and ConWT-S antigens induced T cell responses that were approximately 400-fold higher than those induced by the optimized and non-optimized constructs. Thus, these data indicate that the modification and removal of the WT1 zinc fingers significantly increased the immunogenicity of the WT1 immunogen, thereby providing a significant T cell response to the WT1 immunogen.

[0201] The humoral immune response to the ConWT1-L and ConWT1-S antigens was examined by determining whether serum from 5-week bleeds contained antigen-specific antibodies. Specifically, 293T cells were transfected with vectors WT1-pVAX-L and WT1-pVAX-S. After transfection, cells transfected with WT1-pVAX-L or WT1-pVAX-S were stained with DAPI to mark nuclei and serum from 5-week bleeds of mice immunized with WT1-pVAX-L or WT-pVAX-S. As shown in FIG. 13, ConWT1-L or ConWT1-S antigens were detected in the transfected cells from serum of mice immunized with WT1-pVAX-L or WT1-pVAX-S, respectively. Thus, these data demonstrated that immunization with constructs expressing ConWT1-L and ConWT1-S antigens resulted in the production of antibodies immunoreactive with ConWT1-L and ConWT1-S antigens.

[0202] The induction of humoral immune responses by the constructs expressing ConWT1-L and ConWT1-S was further examined by immunoblotting. Specifically, lysates were obtained from the above transfected cells and probed with sera from mice immunized with WT1-pVAX-L or WT1-pVAX-S. Figure 14 shows a representative immunoblot, and lysates from non-transfected 293T cells were used as background controls. The immunoblot in Figure 14 was probed with anti-actin antibody, demonstrating that equivalent amounts of lysates were loaded among the three lanes. These data indicated that the sera from immunized mice contained antibodies immunoreactive with ConWT1-L and ConWT1-S antigens, confirming the cell staining results described above.

[0203] In summary, constructs expressing either the ConWT1-L or ConWT1-S antigen induced significant T cell responses that produced IFN-γ and antibodies immunoreactive with the ConWT1-S and ConWT-L antigens.

[0204] It is understood that the foregoing detailed description and accompanying examples are merely illustrative and should not be taken as limiting the scope of the invention, which is defined solely by the appended claims and their equivalents.

[0205] Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications, including without limitation with respect to the chemical structures, substitutes, derivatives, intermediates, compositions, formulations and / or methods of use of the invention, can be made without departing from the spirit and scope of the invention.

Claims

1. An isolated nucleic acid molecule comprising one or more nucleic acid sequences selected from the group consisting of 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 98% identical to SEQ ID NO:2, a nucleic acid sequence encoding a fragment comprising at least 90% of the length of a protein that is at least 98% identical to SEQ ID NO:2, a nucleic acid sequence encoding SEQ ID NO:4, a nucleic acid sequence encoding a fragment comprising at least 90% of the length of SEQ ID NO:4, a nucleic acid sequence encoding a protein that is at least 98% identical to SEQ ID NO:4, and a nucleic acid sequence encoding a fragment comprising at least 90% of the length of a protein that is at least 98% identical to SEQ ID NO:

4.

2. An isolated nucleic acid molecule comprising one or more nucleic acid sequences selected from the group consisting of SEQ ID NO:1, a fragment comprising at least 90% of the length of SEQ ID NO:1, a nucleic acid sequence that is at least 98% identical to SEQ ID NO:1, a fragment comprising at least 90% of the length of a nucleic acid sequence that is at least 98% identical to SEQ ID NO:1, SEQ ID NO:3, a fragment comprising at least 90% of the length of SEQ ID NO:3, a nucleic acid sequence that is at least 98% identical to SEQ ID NO:3, and a fragment comprising at least 90% of the length of a nucleic acid sequence that is at least 98% identical to SEQ ID NO:

3.

3. The nucleic acid molecule of claim 1 or 2, wherein the molecule is incorporated into a plasmid or viral vector.

4. A composition comprising one or more nucleic acid molecules according to claim 1.

5. A composition comprising one or more nucleic acid molecules according to claim 2.

6. A vaccine comprising one or more nucleic acid molecules according to claim 1.

7. A vaccine comprising one or more nucleic acid molecules according to claim 2.

8. 1. A method for treating an individual having a WT1-expressing tumor, comprising: A method comprising administering a vaccine according to claim 6 or 7 in an amount effective to slow the growth of, shrink or eliminate a WT1-expressing tumor.

9. 1. A method for preventing a WT1-expressing tumor in an individual, comprising: A method comprising administering a vaccine according to claim 6 or 7 in an amount effective to inhibit the formation or growth of WT1-expressing tumors.

10. A protein comprising an amino acid sequence selected from the group consisting of 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 98% identical to SEQ ID NO:2, a fragment comprising at least 90% of the length of an amino acid sequence that is at least 98% identical to SEQ ID NO:2, SEQ ID NO:4, a fragment comprising at least 90% of the length of SEQ ID NO:4, an amino acid sequence that is at least 98% identical to SEQ ID NO:4, and a fragment comprising at least 90% of the length of an amino acid sequence that is at least 98% identical to SEQ ID NO:

4.

11. 1. A vaccine comprising a nucleic acid molecule, (a) the nucleic acid molecule comprises a nucleic acid sequence having at least about 90% identity over the entire length of the nucleic acid sequence set forth in SEQ ID NO:1; or (b) the nucleic acid molecule comprises a nucleic acid sequence having at least about 90% identity over the entire length of the nucleic acid sequence set forth in SEQ ID NO:

3.

12. (a) a peptide comprising an amino acid sequence having at least about 90% identity over the entire length of the nucleic acid sequence set forth in SEQ ID NO:2; or (b) a peptide comprising an amino acid sequence having at least about 90% identity over the entire length of the nucleic acid sequence set forth in SEQ ID NO:

4. The vaccine of claim 11 further comprising:

13. The vaccine of claim 11 , wherein the nucleic acid molecule comprises an expression vector.

14. 12. The vaccine of claim 11, further comprising a pharma- ceutically acceptable excipient.

15. The vaccine of claim 11 further comprising an adjuvant.

16. 1. A vaccine comprising a nucleic acid molecule, (a) the nucleic acid molecule encodes a peptide comprising an amino acid sequence having at least about 90% identity over the entire length of the nucleic acid sequence set forth in SEQ ID NO:2; or (b) the nucleic acid molecule encodes a peptide comprising an amino acid sequence having at least about 90% identity over the entire length of the nucleic acid sequence set forth in SEQ ID NO:

4.

17. (a) a peptide comprising an amino acid sequence having at least about 90% identity over the entire length of the nucleic acid sequence set forth in SEQ ID NO:2; or (b) a peptide comprising an amino acid sequence having at least about 90% identity over the entire length of the nucleic acid sequence set forth in SEQ ID NO:

4. The vaccine of claim 11 further comprising:

18. The vaccine of claim 16 , wherein the nucleic acid molecule comprises an expression vector.

19. 17. The vaccine of claim 16, further comprising a pharma- ceutically acceptable excipient.

20. 17. The vaccine of claim 16, further comprising an adjuvant.

21. A nucleic acid molecule comprising the nucleic acid sequence set forth in SEQ ID NO:

1.

22. A nucleic acid molecule comprising the nucleic acid sequence set forth in SEQ ID NO:

3.

23. A peptide comprising the amino acid sequence set forth in SEQ ID NO:

2.

24. A peptide comprising the amino acid sequence set forth in SEQ ID NO:

4.

25. 1. A vaccine comprising an antigen, A vaccine wherein the antigen is encoded by SEQ ID NO:1 or SEQ ID NO:

3.

26. 26. The vaccine of claim 25, wherein the antigen is encoded by SEQ ID NO:

1.

27. 26. The vaccine of claim 25, wherein the antigen is encoded by SEQ ID NO:

3.

28. 26. The vaccine of claim 25, wherein the antigen comprises the amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:

4.

29. 29. The vaccine of claim 28, wherein the antigen comprises the amino acid sequence set forth in SEQ ID NO:

2.

30. 29. The vaccine of claim 28, wherein the antigen comprises the amino acid sequence set forth in SEQ ID NO:

4.

31. A vaccine comprising a peptide, (a) the peptide comprises an amino acid sequence having at least about 90% identity over the entire length of the nucleic acid sequence set forth in SEQ ID NO:2; or (b) the peptide comprises an amino acid sequence having at least about 90% identity over the entire length of the nucleic acid sequence set forth in SEQ ID NO:

4.

32. 32. The vaccine of claim 31 , wherein the peptide comprises the amino acid sequence set forth in SEQ ID NO:

2.

33. 32. The vaccine of claim 31 , wherein the peptide comprises the amino acid sequence set forth in SEQ ID NO:4.

Images