HK1212919B - Wt1 vaccine - Google Patents

Description

WT1 vaccine

CROSS-REFERENCE TO RELATED APPLICATIONS

Priority of U.S. provisional application No.61/737,094, filed on 12/13/2012 of the present application, is hereby incorporated by reference in its entirety.

Technical Field

The present invention relates to Wilm's tumor (WT1) immunogens and nucleic acid molecules encoding the immunogens. The invention also relates to vaccines comprising such WT1 immunogens and/or nucleic acid molecules. The invention also relates to methods of using the vaccines to elicit an immune response and to prevent and/or treat subjects having tumors that express WT 1.

Background

Cancer remains a leading cause of death in the united states and worldwide. The cancer vaccine market is rapidly growing. Lymphoma vaccines account for approximately 0.5% of the market share. An effective tumor vaccine may be used to prevent tumor growth and/or may be used as a more effective, less toxic alternative treatment to standard treatment for patients with advanced cancer. The antigen associated with cancer and thus the target for an anti-tumor vaccine is WT 1.

Wilms' tumor suppressor gene 1(WT1) was identified as the cause of renal embryonic malignancy, affecting infants around 1/10,000. It occurs in both sporadic and genetic forms. Inactivation of WT1 leads to the development of Wilms' tumor and Denys-Drash syndrome (DDS). The result is kidney disease and possible genital abnormalities. The WT1 protein has been found to interact with a number of cytokines, including the major tumor regulatory gene p53, which p53 is also a tumor suppressor transcription factor.

The WT1 gene is expressed in many tumor types and has been more widely implicated in many cancers. For example, WT1 protein was localized in 75% of mesothelioma nuclei (14,200 cases per year worldwide with the highest incidence in the united states) and 93% of ovarian serous carcinomas (190,000 ovarian cancer cases worldwide in 2010). In addition, WT1 has been implicated in pancreatic cancer, leukemia, lung cancer, breast cancer, colon cancer, glioblastoma, head and neck cancer, as well as benign mesothelioma and cervical and ovarian cancer, among others. WT1 is a target for gene therapy or immunotherapy as a method for cancer treatment.

WT1 encodes a transcription factor containing 4 zinc finger motifs on the C-terminus and a proline/glutamine rich DNA binding domain on the N-terminus. It plays an indispensable role in the normal development of the urogenital system. However, it is less necessary in adults, suggesting that it is a target for immunotherapy. The multiple transcript variants resulting from alternative splicing on the two coding exons are well characterized. It would be considered advantageous to maximize CTL coverage with the entire reading frame.

Due to the conservation of the WT1 antigen, most of the efforts to generate strong immunity against this gene target were unsuccessful. Vaccines have been previously studied using DAN vaccine technology, poxvirus vaccine technology, adenoviral vaccine technology, peptide vaccine technology and protein-based vaccine technology. The vaccines studied use the true gene structure, i.e. the natural "normal" gene. Only low or non-functional T cell immunity was achieved in these studies.

There are several major problems with the development of a more potent WT1 immunogen. Due to the similarity of the WT1 antigen to host WT1, a strong suppressor T cell response was generated, blocking immune induction. Furthermore, the gene itself is significantly processed at the RNA level in order to generate multiple cleaved transcripts with unknown and possibly competing values. In addition, expression of WT1 delivered was low, resulting in poor immunity.

Vaccines for the treatment and prevention of cancer are of great interest. Existing vaccines targeting WT1 are limited by poor antigen expression in vivo. Therefore, there remains a need in the art to develop safe and effective vaccines suitable for WT 1-expressing tumors, thereby providing treatment for such cancers and increasing the survival rate of the cancers.

Disclosure of Invention

SUMMARY

The present invention relates to isolated nucleic acid molecules 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 having at least 98% identity to SEQ ID NO.2, a nucleic acid sequence encoding a fragment comprising at least 90% of the length of a protein having at least 98% identity 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 having at least 98% identity to SEQ ID NO.4, and a nucleic acid sequence encoding a fragment comprising at least 90% of the length of a protein having at least 98% identity to SEQ ID NO. 4.

The invention also relates to an isolated nucleic acid molecule comprising one or more nucleic acid sequences selected from the group consisting of: 1, a fragment comprising at least 90% of the length of SEQ ID No.1, a nucleic acid sequence having at least 98% identity to SEQ ID No.1, a fragment comprising at least 90% of the length of a nucleic acid sequence having at least 98% identity 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 having at least 98% identity to SEQ ID No.3, and a fragment comprising at least 90% of the length of a nucleic acid sequence having at least 98% identity to SEQ ID No. 3.

The nucleic acid molecules described above may be incorporated into plasmid or viral vectors. The invention also relates to compositions comprising one or more of the above-described nucleic acid molecules. The invention also relates to vaccines comprising one or more of the above-described nucleic acid molecules.

The present invention also relates to methods of treating an individual having a WT 1-expressing tumor comprising administering an amount of the above vaccine effective to slow the growth of, reduce or eliminate a WT 1-expressing tumor.

The present invention also relates to a method of preventing a WT 1-expressing tumor in an individual, comprising administering an amount of the above vaccine effective to inhibit the formation or growth of a WT 1-expressing tumor.

The invention also 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 having at least 98% identity to SEQ ID NO 2, a fragment comprising at least 90% of the length of an amino acid sequence having at least 98% identity 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 having at least 98% identity to SEQ ID NO 4, and a fragment comprising at least 90% of the length of an amino acid sequence having at least 98% identity to SEQ ID NO 4.

The invention also relates to vaccines comprising the nucleic acid molecules. 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 can 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. 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 the amino 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 amino acid sequence set forth in SEQ ID NO. 4.

The nucleic acid molecule may comprise an expression vector. The vaccine may further comprise a pharmaceutically acceptable excipient. The vaccine may further comprise an adjuvant.

The invention also relates to vaccines comprising the nucleic acid molecules. 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 amino 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 amino acid sequence set forth in SEQ ID NO. 4. 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 the amino 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 amino acid sequence set forth in SEQ ID NO. 4.

The nucleic acid molecule may comprise an expression vector. The vaccine may further comprise a pharmaceutically acceptable excipient. The vaccine may further comprise an adjuvant.

The invention also relates to nucleic acid molecules comprising the nucleic acid sequence shown in SEQ ID NO. 1.

The invention also relates to nucleic acid molecules comprising the nucleic acid sequence shown in SEQ ID NO. 3.

The invention also relates to peptides comprising the amino acid sequence shown in SEQ ID NO 2.

The invention also relates to peptides comprising the amino acid sequence shown in SEQ ID NO. 4.

The invention also relates to vaccines comprising an antigen, wherein the antigen can be 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 shown in SEQ ID NO.2 or SEQ ID NO. 4. The antigen may comprise the amino acid sequence shown in SEQ ID NO. 2. The antigen may comprise the amino acid sequence shown in SEQ ID NO. 4.

The invention also relates to vaccines comprising the peptides. The peptide may comprise an amino acid sequence having at least about 90% identity over the entire length of the amino acid sequence set forth in SEQ ID NO. 2. The peptide may comprise an amino acid sequence having at least about 90% identity over the entire length of the amino acid sequence set forth in SEQ ID NO. 4. The peptide may comprise the amino acid sequence shown in SEQ ID NO. 2. The peptide may comprise the amino acid sequence shown in SEQ ID NO 4.

Drawings

FIG. 1 shows a plot of the immune group versus 10 each⁶Pattern of spot-forming units (SFU) of individual splenocytes.

FIG. 2 shows a plot of the immune group versus 10 each⁶Map of SFU of individual splenocytes.

Figure 3 shows an immunoblot.

FIG. 4 shows a schematic of ConWT1-L and ConWT 1-S.

FIG. 5 shows an alignment of the respective amino acid sequences of ConWT1-L and ConWT 1.

FIG. 6 shows in (A) the nucleotide sequence encoding ConWT 1-L; and (B) the amino acid sequence of ConWT 1-L.

FIG. 7 shows in (A) the nucleotide sequence encoding ConWT 1-S; and (B) the amino acid sequence of ConWT 1-S.

FIG. 8 shows staining of transfected cells.

Figure 9 shows an immunoblot.

FIG. 10 shows a schematic illustrating an immunization protocol.

FIG. 11 shows a plot of the immune group versus 10 each⁶Map of SFU of individual splenocytes.

FIG. 12 shows a plot of the immune group versus 10 each⁶Map of SFU of individual splenocytes.

FIG. 13 shows staining of transfected cells.

Figure 14 shows an immunoblot.

Detailed Description

The present invention relates to vaccines comprising WT1 antigen. WT1 is expressed in many tumors. Thus, the vaccine provides treatment of cancer or cancer-based WT 1-expressing tumors.

The WT1 antigen may be a consensus WT1 antigen derived from sequences of WT1 from different species, and thus, the consensus WT1 antigen is unique. The consensus WT1 antigen is also unique in that the zinc fingers are modified or removed together. Modifications may include substitutions of cysteine and histidine residues that coordinate the zinc structure.

Surprisingly, when the consensus WT1 antigen had a modified zinc finger or no zinc finger, a significant immune response was elicited in response to WT 1. The immune responses elicited include both humoral and cellular immune responses, wherein the cellular immune response is elicited about a 400-fold enhancement relative to or compared to the cellular immune response elicited by a vaccine comprising native WT1 or WT1 optimized for expression.

1. Definition of

Unless defined otherwise, 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 document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

As used herein, the terms "comprises/comprising," "includes," "having," "has," "can," "containing," and variations thereof, are intended to be open-ended transition phrases, terms, or words that do not exclude the possibility of additional acts or structure. The singular forms "a", "an" and "the" include plural references unless the context clearly dictates otherwise. The present disclosure also encompasses other embodiments that "comprise" the embodiments or components set forth herein, "consist of" and "consist essentially of" the embodiments or components, whether or not explicitly shown.

As used herein, "adjuvant" may mean 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 below.

"antibody" may mean an antibody of the class IgG, IgM, IgA, IgD or IgE or fragments, fragments thereof or derivatives thereof, including Fab, F (ab')2, Fd and single chain antibodies, diabodies, bispecific antibodies, bifunctional antibodies and derivatives thereof. The antibody may be an antibody isolated from a serum sample of a mammal, a polyclonal antibody, an affinity purified antibody, or a mixture thereof, which antibody exhibits sufficient binding specificity to a desired epitope or sequence derived therefrom.

By "antigen" is meant: a protein having a mutated WT1 amino acid sequence comprising SEQ ID NO. 2; fragments thereof having the length shown herein, variants, i.e. proteins having a sequence with identity to SEQ ID No.2 as shown herein, fragments of variants having the length shown herein, SEQ ID No. 4; fragments thereof having the lengths shown herein, variants, i.e., proteins having a sequence with identity to SEQ ID No.4 as shown herein, fragments of variants having the lengths shown herein, and combinations thereof. The antigen may optionally comprise signal peptides such as those from other proteins.

As used herein, "coding sequence" or "coding nucleic acid" can refer to a nucleic acid (RNA or DNA molecule) that comprises a nucleotide sequence that encodes an antigen as set forth herein. The coding sequence may also include initiation and termination signals operably linked to regulatory elements (including promoters and polyadenylation signals) capable of directing expression in the cells of the individual or mammal to which the nucleic acid is administered. The coding sequence may also include a sequence encoding a signal peptide.

As used herein, "complementary" or "complementary" can mean a nucleic acid, can mean Watson-Crick (e.g., A-T/U and C-G) or Hoogsteen base pairing between nucleotides or nucleotide analogs of a nucleic acid molecule.

As used herein, "consensus" or "consensus sequence" can mean a synthetic nucleic acid sequence or corresponding polypeptide sequence constructed based on analysis of alignments of particular antigens of multiple subtypes. The sequences can be used to elicit broad immunity against a particular antigen of multiple subtypes, serotypes, or strains. Synthetic antigens such as fusion proteins can be manipulated into consensus sequences (or consensus antigens).

As used herein, "constant current" defines the current received or experienced by a tissue or cells defining the tissue throughout the duration of an electrical pulse delivered to the same tissue. Electrical pulses are delivered from the electroporation devices described herein. This current maintains a constant amperage in the tissue throughout the electrical pulse, as the electroporation devices provided herein have a feedback element, preferably instantaneous feedback. The feedback element may measure the resistance (resistance) of the tissue (or cells) throughout the duration of the pulse and cause the electroporation device to change its electrical energy output (e.g., increase the voltage) so that the current in the same tissue remains constant throughout the electrical pulse (on the order of microseconds) and between pulses. In some embodiments, the feedback element comprises a controller.

As used herein, "current feedback" or "feedback" may be used interchangeably and may mean an active response of the electroporation device provided that includes measuring the current between electrodes in the tissue and correspondingly varying the energy output delivered by the EP device to maintain the current at a constant level. The constant level is preset by the user before starting the pulse sequence or electrical treatment. Feedback may be implemented by an electroporation component, such as a controller of an electroporation device, as circuitry therein is capable of continuously monitoring the current between electrodes in tissue and comparing the monitored current (or current within the tissue) to a preset current, and continuously making energy output adjustments to maintain the monitored current at a preset level. The feedback loop may be instantaneous in that it is an analog closed loop feedback.

As used herein, "dispersed current" may mean a pattern of current delivered from the various needle electrode arrays of the electroporation devices described herein, wherein the pattern minimizes or preferably eliminates the occurrence of electroporation-related heat stress on any region of the tissue to be electroporated.

As used interchangeably herein, "electroporation," "electro-permeabilization," or "electrokinetic enhancement" ("EP") can refer to the use of transmembrane electric field pulses to induce microscopic channels (pores) in a biological membrane; their presence allows biomolecules such as plasmids, oligonucleotides, siRNA, drugs, ions and water to pass from one side of the cell membrane into the other.

As used herein, a "feedback mechanism" may refer to a process by software or hardware (or firmware) that receives and compares the desired impedance of the tissue (before, during, and/or after delivery of the pulse of energy) to an existing value (preferably the current), and then adjusts the pulse of energy delivered to reach a preset value. The feedback mechanism may be performed by an analog closed loop circuit.

By "fragment" can be meant a polypeptide fragment of an antigen capable of eliciting an immune response in a mammal. In each case with or without a signal peptide and/or a methionine at position 1, a fragment of the antigen may be 100% identical to the full length, except for the loss of at least one amino acid from the N-and/or C-terminus. 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 percent of the length of a particular full-length antigen, excluding any added heterologous signal peptide. Fragments may comprise fragments of the polypeptide that have 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater identity to the antigen, and additionally comprise an N-terminal methionine or a heterologous signal peptide that is not included when calculating percent identity. Fragments may also comprise an N-terminal methionine and/or a signal peptide, such as an immunoglobulin signal peptide, for example an IgE or IgG signal peptide. The N-terminal methionine and/or signal peptide may be linked to a fragment of the antigen.

In each case with or without a sequence encoding a signal peptide and/or a methionine at position 1, a fragment of the nucleic acid sequence encoding the antigen may be 100% identical to the full length, except for the loss of at least one nucleotide from the 5 'and/or 3' end. 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 percent of the length of the full length of a particular coding sequence, excluding any added heterologous signal peptide. Fragments may comprise fragments encoding polypeptides having 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater identity to the antigen, and additionally optionally comprising sequences encoding an N-terminal methionine or a heterologous signal peptide not included when calculating percent identity. Fragments may also comprise an N-terminal methionine and/or a signal peptide, such as the coding sequence for an immunoglobulin signal peptide, e.g., an IgE or IgG signal peptide. The coding sequence encoding the N-terminal methionine and/or the signal peptide may be linked to a fragment of the coding sequence.

As used herein, "genetic construct" refers to a DNA or RNA molecule comprising a nucleotide sequence encoding a protein. The coding sequence includes initiation and termination signals operably linked to regulatory elements, including a promoter and polyadenylation signal, capable of directing expression in the cells of the individual to which the nucleic acid molecule is administered. As used herein, the term "expressible form" refers to a genetic construct that contains the necessary regulatory elements operatively linked to a coding sequence encoding a protein such that the coding sequence will be expressed when present in the cells of an individual.

As used herein in the context of two or more nucleic acid or polypeptide sequences, "identical" or "identity" can mean that the sequences have a specified percentage of residues that are identical over a specified region. The percentage can be calculated by: the two sequences are optimally aligned, the two sequences are compared over a specified region, the number of positions where the identical residue is present in the two sequences is determined to give the number of matched positions, the number of matched positions is divided by the total number of positions in the specified region, and the result is multiplied by 100 to give the percentage of sequence identity. Where two sequences are of different lengths or are aligned to produce one or more staggered ends and the specified region of comparison includes only a single sequence, the residues of the single sequence are included in the denominator, rather than the numerator, of the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. Identity is obtained manually or by using a computer sequence algorithm such as BLAST or BLAST 2.0.

"impedance" as used herein may be used when discussing the feedback mechanism, and may be converted to a current value in accordance with ohm's law, thereby enabling comparison with a preset current.

As used herein, "immune response" may mean the activation of the immune system of a host, e.g., a mammal, in response to the introduction of one or more antigens described herein (via a vaccine described herein). The immune response may exist in the form of a cellular or humoral response, or both.

As used herein, a "nucleic acid" or "oligonucleotide" or "polynucleotide" can mean at least two nucleotides covalently linked together. The description of single strands also defines the sequence of the complementary strand. Thus, nucleic acids also include the complementary strand of the single strand described. Many variants of a nucleic acid may be used for the same purpose as a given nucleic acid. Thus, nucleic acids also include substantially identical nucleic acids and complements thereof. The single strand provides a probe that can hybridize to a target sequence under stringent hybridization conditions. Thus, nucleic acids also include probes that hybridize under stringent hybridization conditions.

The nucleic acid may be single-stranded or double-stranded, or may contain portions of both double-stranded and single-stranded sequence. The nucleic acid can be DNA (genomic DNA and cDNA), RNA, or hybrids, where the nucleic acid can contain a combination of deoxyribonucleotides and ribonucleotides, as well as combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine, and isoguanine. Nucleic acids can be obtained by chemical synthesis or by recombinant methods.

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

As used herein, "peptide," "protein," or "polypeptide" may refer to a linked sequence of amino acids, and may be natural, synthetic, or modified or a combination of natural and synthetic.

As used herein, "promoter" may mean a molecule of synthetic or natural origin that is capable of conferring, activating or enhancing expression of a nucleic acid in a cell. A promoter may comprise one or more specific transcriptional regulatory sequences to further enhance expression of the sequence and/or alter spatial and/or temporal expression thereof. A promoter may also comprise distal enhancer or repressor elements that are as much as several thousand base pairs from the transcription start site. Promoters may be derived from sources including viruses, bacteria, fungi, plants, insects, and animals. The promoter may regulate expression of a gene component constitutively, or differentially, either against the cell, tissue or organ in which expression occurs, or against the developmental stage in which expression occurs, or in response to an external stimulus such as a physiological stress, pathogen, metal ion or inducer. Representative examples of promoters include the phage T7 promoter, the phage T3 promoter, the SP6 promoter, the lac operator-promoter, the tac promoter, the SV40 late promoter, the SV40 early promoter, the RSV-LTR promoter, the CMVIE promoter, the SV40 early promoter or the SV40 late promoter, and the CMV IE promoter.

"Signal peptide" and "leader sequence" are used interchangeably herein and refer to amino acid sequences that can be ligated to the amino terminus of the proteins shown herein. The signal peptide/leader sequence generally directs the localization of the protein. The signal peptide/leader sequence used herein preferably promotes secretion of the protein from the cell in which it is produced. After secretion from the cell, the signal peptide/leader sequence is typically cleaved from the remainder of the protein (often referred to as the mature protein). The signal peptide/leader sequence is attached to the N-terminus of the protein.

As used herein, "subject" can mean a mammal who 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.

As used herein, "stringent hybridization conditions" can mean conditions under which a first nucleic acid sequence (e.g., a probe) will hybridize to a second nucleic acid sequence (e.g., a target), such as in a complex mixture of nucleic acids. Stringent conditions are sequence dependent and will be different in different circumstances. The thermal melting point (T) at a defined ionic strength, pH, can be selected for a specific sequence_m) About 5-10 ℃ lower as stringent conditions. T is_mIt may be that 50% of the probes complementary to the target are in equilibrium (at T due to the excess presence of target sequence_mNext, 50% of the probes are occupied at equilibrium) temperature (at defined ionic strength, pH and nucleic acid concentration) at which they hybridize to the target sequence. Stringent conditions may be a salt concentration of less than about 1.0M sodium ion, such as a sodium ion concentration of about 0.01-1.0M at pH 7.0 to 8.3(or other salts) and temperatures of at least about 30 ℃ (for short probes (e.g., about 10-50 nucleotides)) and at least about 60 ℃ (for long probes (e.g., greater than about 50 nucleotides.) stringent conditions can also be achieved by the addition of destabilizing agents such as formamide.

As used herein, "substantially complementary" can mean that the complement of the first sequence and the second sequence have 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% identity 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, or that the two sequences hybridize under stringent hybridization conditions.

As used herein, "substantially identical" can mean that the first and second 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 over a region of 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 or amino acids or with respect to the nucleic acid if the first sequence is substantially complementary to the complement of the second sequence.

As used herein, "treating" or "treatment" can mean protecting an animal from a disease by preventing, inhibiting, suppressing, or completely eliminating the disease. Preventing disease comprises administering a vaccine of the invention to an animal prior to the onset of disease. Inhibiting a disease comprises administering a vaccine of the invention to an animal after initiation of the disease but prior to its clinical manifestation. Suppressing the disease comprises administering the vaccine of the invention to the animal after clinical manifestation of the disease.

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

With respect to a peptide or polypeptide, a "variant" refers to a peptide or polypeptide that differs in amino acid sequence by insertion, deletion, or conservative substitution of amino acids, but retains at least one biological activity. A variant may also mean a protein having an amino acid sequence that is substantially identical to a reference protein having an amino acid sequence that retains at least one biological activity. Conservative substitutions of amino acids, i.e., substitutions of amino acids with different amino acids having similar properties (e.g., hydrophobicity, extent and distribution of charged regions), are considered in the art to generally include minor variations. As understood in the art, these minor changes may be identified in part by considering the hydropathic index (hydropathic index) of amino acids. Kyte et al, J.mol.biol.157:105-132 (1982). The hydropathic index of an amino acid is based on 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 aspect, amino acids having a hydropathic index of ± 2 are substituted. The hydrophilicity of amino acids can also be used to show substitutions in proteins that can result in retention of biological function. Taking into account the hydrophilicity of amino acids in the context of a peptide allows the calculation of the maximum local average hydrophilicity of the peptide, a useful metric that has been reported to be closely related to antigenicity and immunogenicity. U.S. Pat. No.4,554,101, incorporated herein by reference in its entirety. As understood in the art, substitution of amino acids with similar hydrophilicity values can result in peptides that retain biological activity, e.g., immunogenicity. Substitutions may be made with amino acids having hydrophilicity values within + -2 of each other. The hydrophobicity index and hydrophilicity value of an amino acid are affected by the particular side chain of that amino acid. Consistent with this observation, amino acid substitutions that are compatible with biological function are believed to be dependent on the amino acids, and in particular the relative similarity of the side chains of those amino acids, as shown by hydrophobicity, hydrophilicity, charge, size, and other properties.

Variants can be nucleic acid sequences that are substantially identical over the entire gene sequence or fragments thereof. The nucleic acid sequence may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical over the full-length gene sequence or a fragment thereof. A variant may be an amino acid sequence that is substantially identical over the entire amino acid sequence or a fragment thereof. An 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.

As used herein, "vector" may mean a nucleic acid sequence comprising an origin of replication. The vector may be a plasmid, a phage, a bacterial artificial chromosome, or a yeast artificial chromosome. The vector may be a DNA or RNA vector. The vector may be an autonomously replicating extrachromosomal vector or a vector which integrates into the host genome.

For recitation of numerical ranges herein, each intervening number therebetween is explicitly included with equal precision. For example, for the range of 6-9, the values 7 and 8 are included in addition to 6 and 9, and for the range of 6.0-7.0, the values 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 and 7.0 are explicitly included.

2. Vaccine

Provided herein are vaccines comprising antigens, fragments thereof, variants thereof, or combinations thereof. The vaccine may be capable of generating an immune response in a mammal against the antigen. The vaccine may comprise a plasmid or plasmids as described in more detail below. The vaccine may induce a therapeutic or prophylactic immune response.

The vaccine may be used against cancers such as cancers or tumors that express wilms tumor suppressor gene 1(WT 1). The vaccine may be used for the prevention and/or treatment of WT 1-expressing tumors in a subject in need thereof. The vaccine may induce cellular and/or antibody responses against WT1 and WT1 expressing tumors.

The vaccine may induce or elicit an immune response in a subject to whom the vaccine is administered. An immune response in a subject administered the vaccine can be induced 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, the immune response of the subject administered the vaccine can be induced at least about 300-fold, 325-fold, 350-fold, 375-fold, 400-fold, 425-fold, 450-fold, 475-fold, or 500-fold.

The vaccine may induce a humoral and/or cellular immune response in a subject administered the vaccine. The induced humoral immune response may include antibodies that immunoreact with the antigen. The induced immune response may include T cells that produce interferon-gamma (IFN- γ) and that immunoreact with the antigen. A cellular immune response in a subject administered the vaccine can be induced 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, the cellular immune response of the subject administered the vaccine can be induced at least about 300-fold, 325-fold, 350-fold, 375-fold, 400-fold, 425-fold, 450-fold, 475-fold, or 500-fold.

The vaccines 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. In the case of delivery of multiple antigens, the vaccine may comprise multiple compositions or a single composition. Delivery may include a single plasmid that may be used to encode multiple different antigens or different portions of the same antigen. In other embodiments, delivery may include different plasmids encoding different antigens or different portions of the same antigen.

The vaccine may be a nucleic acid vaccine, a peptide vaccine or a combination vaccine of nucleic acids and peptides. The nucleic acid vaccine may comprise a nucleic acid molecule. The nucleic acid vaccine may comprise multiple copies of a single nucleic acid molecule such as a single plasmid, multiple copies of two or more different nucleic acid molecules such as two or more different plasmids.

The nucleic acid vaccine can comprise nucleic acid molecules, such as plasmids, which can collectively contain coding sequences for a single antigen, heterologous coding sequences for two or more antigens. A nucleic acid vaccine comprising heterologous coding sequences for two antigens can be on a single nucleic acid molecule, such as a single plasmid, or the nucleic acid vaccine can comprise two different nucleic acid molecules, such as two different plasmids, wherein one nucleic acid molecule comprises heterologous coding sequences for one antigen and the other nucleic acid molecule comprises heterologous coding sequences for a different antigen. The nucleic acid vaccine may comprise two different nucleic acid molecules, such as two different plasmids, wherein one nucleic acid molecule comprises a heterologous coding sequence for a first part of the antigen and the other nucleic acid molecule comprises a heterologous coding sequence for a second part of the antigen.

Similarly, a nucleic acid vaccine comprising heterologous coding sequences for 3 antigens may comprise a single nucleic acid molecule such as a single plasmid, 2 different nucleic acid molecules, or 3 different nucleic acid molecules. Likewise, a nucleic acid vaccine comprising heterologous coding sequences for 4 antigens may comprise a single nucleic acid molecule such as a single plasmid, 2 different nucleic acid molecules, 3 different nucleic acid molecules, or 4 different nucleic acid molecules.

The 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 further comprise additional sequences encoding a linker, leader and/or tag sequence linked to the antigen by peptide bonds.

In some embodiments, the nucleic acid vaccine can further comprise a coding sequence for a molecular adjuvant, which in some cases can be IL-12, IL-15, IL-28, IL-31, IL-33, and/or RANTES, and in some cases is a checkpoint inhibitor, including anti-cytotoxic T-lymphocyte antigen 4(CTLA-4), anti-programmed death receptor-1 (PD-1), and anti-lymphocyte-activation gene (LAG-3). The coding sequence for IL-12, IL-15, IL-28, IL-31, IL-33, and/or RANTES may be included on one or more nucleic acid molecules that include a 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 contained on a separate nucleic acid molecule, such as a separate plasmid.

The peptide vaccine may include an antigenic peptide, an antigenic protein, variants thereof, fragments thereof, or combinations thereof. The combination DNA and peptide vaccines may comprise one or more of the nucleic acid sequences described above encoding an antigen and an antigenic peptide or antigenic protein.

The vaccines of the present invention may have desirable properties for an effective vaccine, such as being safe so that the vaccine itself does not cause disease or death; has protective effect against diseases; inducing neutralizing antibodies; inducing protective T cells; as well as providing ease of administration, little side effects, biostability, and low cost per dose.

a. Antigens

As described herein, the vaccine may comprise an antigen. The antigen may be wilms tumor suppressor gene 1(WT1), a fragment thereof, a variant thereof, or a combination thereof. WT1 is a transcription factor containing a proline/glutamine rich DNA binding domain at the N-terminus and 4 zinc finger motifs at the C-terminus. WT1 plays a role in the normal development of the urogenital system and interacts with a number of factors, for example, p53 (a known tumor suppressor) and the serine protease HtrA2 (which cleaves WT1 at multiple sites following treatment with cytotoxic drugs).

Mutations in WT1 may lead to the formation of tumors or cancers such as wilms' tumors or WT1 expressing tumors. Wilms' tumors typically form in one or both kidneys before metastasizing to other tissues such as, but not limited to, liver tissue, urinary tract tissue, lymphatic tissue, and lung tissue. Thus, wilms' tumor can be considered a metastatic tumor. Wilms' tumors typically occur in young children (e.g., younger than 5 years of age) and occur in sporadic and genetic forms.

Thus, the vaccine may be used to treat a subject having a wilms tumor. The vaccine may also be used to treat a subject having a cancer or tumor that expresses WT1 to prevent such tumor from developing in the subject. The WT1 antigen may differ from the native "normal" WT1 gene, thereby providing treatment or prevention against tumors expressing the WT1 antigen. Thus, provided herein are WT1 antigen sequences that differ from the native WT1 gene (i.e., mutated WT1 gene or sequence).

The transcript of the native WT1 gene can be processed into various mRNAs, and the resulting proteins are not completely equivalent to elicit an immune response. The mutated WT1 gene described herein avoids selective processing, thereby producing a full length transcript and resulting in a stronger induction of effector T and B cell responses. The WTI sequence of the first mutation was designated CON WT1 with a modified zinc finger or ConWT 1-L. SEQ ID NO 1 is a 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 mutated WT1 sequence was designated CON WT1 without zinc fingers or ConWT 1-S. SEQ ID NO 3 is a nucleic acid sequence encoding the WTI antigen CON WT1 without zinc fingers. SEQ ID NO 4 is the amino acid sequence of WT1 antigen CON WT1 without modified zinc fingers.

Isolated nucleic acid molecules comprising the above-described heterologous sequences are provided. Isolated nucleic acid molecules consisting of the above-described heterologous sequences are provided. The isolated nucleic acid molecule comprising the above-described heterologous sequences can be incorporated into vectors such as plasmids, viral vectors, and other forms of nucleic acid molecules as described below. Provided herein are nucleic acid sequences encoding the WT1 antigen. The coding sequence encoding the WT1 antigen has the sequence as described above.

Protein molecules comprising the above-described heterologous amino acid sequences are provided. Protein molecules comprised of the above-described heterologous amino acid sequences are provided. Proteins and polypeptides having the sequences described above are provided herein. The proteins and polypeptides may be referred to as WT1 antigen and WT1 immunogen. The WT1 antigen was able to elicit an immune response against tumors expressing WT1 antigen.

In one aspect of the invention, it is desirable that the consensus antigen provides increased transcription and translation, including one or more of: a low GC content leader sequence that increases transcription; mRNA stability and codon optimization and elimination of cis-acting sequence motifs (i.e., internal TAT a boxes) as much as possible.

In some aspects 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: integrating all available full-length sequences; a computationally generated sequence using the most commonly occurring amino acids at each position; and enhancing cross-reactivity between strains.

The WT1 antigen may be a consensus antigen (or immunogen) sequence derived from two or more species. The WT1 antigen may comprise consensus sequences and/or modifications for increased expression. Modifications may include codon optimization, RNA optimization, addition of kozak sequences (e.g., GCC ACC) to increase translation initiation, and/or addition of immunoglobulin leader sequences to increase immunogenicity of the WT1 antigen. The WT1 antigen may comprise a signal peptide such as an immunoglobulin signal peptide, for example but not limited to immunoglobulin e (ige) or immunoglobulin g (igg) signal peptide. In some embodiments, the WT1 consensus antigen may comprise a Hemagglutinin (HA) tag. The WT1 consensus antigen may be designed to elicit stronger and broader cellular and/or humoral immune responses than the corresponding codon optimized WT1 antigen.

The WT1 consensus antigen may comprise one or more mutations in one or more zinc fingers, thereby eliciting stronger and broader cellular and/or humoral immune responses than the corresponding codon optimized WT1 antigen. The one or more mutations may be a substitution of one or more amino acids that coordinate a zinc ion in one or more zinc fingers. One or more amino acids coordinating the zinc ion may be a CCHH motif. Thus, in some embodiments, the one or more mutations can replace 1,2, 3, or all 4 amino acids of the CCHH motif.

In other embodiments, the one or more mutations is a mutation wherein 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). Specifically, the one or more mutations is a mutation of residues 312, 317, 330, 334, 342, 347, 360 and 364 of SEQ ID NO 2 to glycine (G).

In other embodiments, one or more zinc fingers may be removed from WT1 consensus antigen. 1,2, 3 or all 4 zinc fingers may be removed from the WT1 consensus antigen.

The WT1 consensus antigen can be the nucleic acid SEQ ID NO:1 encoding SEQ ID NO:2 (FIGS. 6A and 6B). In some embodiments, a WT1 consensus antigen may 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 amino acid sequence set forth in SEQ ID No. 2.

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 amino acid sequence set forth in SEQ ID NO:2, provided 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 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 amino acid sequence set forth in SEQ ID NO:2, provided that residues 312, 317, 330, 334, 342, 347, 360, and 364 of SEQ ID NO:2 are glycine (G).

The WT1 consensus antigen may be the amino acid sequence SEQ ID NO 2. In some embodiments, a WT1 consensus antigen may 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 amino 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 amino acid sequence set forth in SEQ ID NO:2, provided 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 sequence may 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 amino acid sequence set forth in SEQ ID NO:2, provided that residues 312, 317, 330, 334, 342, 347, 360, and 364 of SEQ ID NO:2 are glycine (G).

The WT1 consensus antigen can be the nucleic acid SEQ ID NO:3, encoding SEQ ID NO:4 (FIGS. 7A and 7B). In some embodiments, a WT1 consensus antigen may 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 amino acid sequence set forth in SEQ ID No. 4.

The WT1 consensus antigen may be the amino acid sequence SEQ ID NO 4. In some embodiments, a WT1 consensus antigen may 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 amino acid sequence set forth in SEQ ID No. 4.

Immunogenic fragments of SEQ ID NO 2 and SEQ ID NO 4 are provided. An 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 and/or SEQ ID NO 4. In some embodiments, an immunogenic fragment can 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, provided that these residues are any residues other than cysteine (C) if residues 312, 317, 342, and 347 of SEQ ID No.2 are present in the immunogenic fragment, and provided that these residues are any residues other than histidine (H) if residues 330, 334, 360, and 364 of SEQ ID No.2 are present in the immunogenic fragment. In other embodiments, the immunogenic fragment can 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, provided that if residues 312, 317, 330, 334, 342, 347, 360, and 364 of SEQ ID No.2 are present in the immunogenic fragment, these residues are glycine (G).

In some embodiments, the immunogenic fragment comprises a leader sequence, e.g., an immunoglobulin leader sequence, such as an immunoglobulin e (ige) leader sequence. In some embodiments, the immunogenic fragment does not contain a leader sequence.

Immunogenic fragments of proteins having amino acid sequences with identity to the immunogenic fragments of SEQ ID NO 2 and 4 can be provided. Such 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 a protein having 95% or greater identity to SEQ ID No.2 and/or SEQ ID No. 4. Some embodiments relate to immunogenic fragments that have 96% or greater identity to the immunogenic fragments of WT1 protein sequences herein. Some embodiments relate to immunogenic fragments that have 97% or greater identity to the immunogenic fragments of WT1 protein sequences herein. Some embodiments relate to immunogenic fragments that have 98% or greater identity to the immunogenic fragments of WT1 protein sequences herein. Some embodiments relate to immunogenic fragments that have 99% or greater identity to the immunogenic fragments of WT1 protein sequences herein. In some embodiments, the immunogenic fragment comprises a leader sequence, e.g., an immunoglobulin leader sequence such as an IgE leader sequence. In some embodiments, the immunogenic fragment does not contain a leader sequence.

Some embodiments relate to immunogenic fragments of SEQ ID NO 1 and SEQ ID NO 3. 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.1 and/or SEQ ID No. 3. In some embodiments, the immunogenic fragment comprises a sequence encoding a leader sequence, e.g., a leader sequence of an immunoglobulin, such as the IgE leader sequence. In some embodiments, the immunogenic fragment does not contain a coding sequence that encodes a leader sequence. Immunogenic fragments of nucleic acids having nucleotide sequences with identity to the immunogenic fragments of SEQ ID NO.1 and SEQ ID NO.3 can be provided. Such 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 nucleic acids having 95% or more identity to SEQ ID No.1 and/or SEQ ID No. 3. Some embodiments relate to immunogenic fragments that have 96% or greater identity to the immunogenic fragments of WT1 nucleic acid sequences herein. Some embodiments relate to immunogenic fragments that have 97% or greater identity to the immunogenic fragments of WT1 nucleic acid sequences herein. Some embodiments relate to immunogenic fragments that have 98% or greater identity to the immunogenic fragments of WT1 nucleic acid sequences herein. Some embodiments relate to immunogenic fragments that have 99% or greater identity to the immunogenic fragments of WT1 nucleic acid sequences herein. In some embodiments, the immunogenic fragment comprises a sequence encoding a leader sequence, e.g., an immunoglobulin leader sequence such as the IgE leader sequence. In some embodiments, the immunogenic fragment does not contain a coding sequence that encodes a leader sequence.

b. Carrier

The vaccine may comprise one or more vectors comprising a heterologous nucleic acid encoding the antigen. The one or more vectors may be capable of expressing the antigen in an amount effective to elicit an immune response in a mammal. The vector may comprise a heterologous nucleic acid encoding the antigen. The vector may have a nucleic acid sequence comprising 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 an autonomously replicating extrachromosomal vector or a vector which integrates into the host genome.

The vector or vectors may be expression constructs, which are typically plasmids used to introduce a particular gene into a target cell. Once the expression vector is inside the cell, the protein encoded by the gene is produced by the cellular transcription and translation machinery ribosomal complex. The plasmids are typically engineered to contain regulatory sequences that act as enhancers and promoter regions and result in efficient transcription of the genes carried on the expression vector. The vectors of the present invention express a large amount of stable messenger RNA, thereby expressing proteins.

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

(1) Expression vector

The vector may be a circular plasmid or a linear nucleic acid. The circular plasmid and linear nucleic acid are capable of directing expression of a particular nucleotide sequence in an appropriate subject cell. The vector may have a promoter operably linked to a nucleotide sequence encoding an antigen operably linked to a termination signal. The vector may also contain sequences required for proper translation of the nucleotide sequence. The vector comprising the nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous to at least one other component thereof. Expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or an inducible promoter that initiates transcription only when the host cell is exposed to some specific external stimulus. In the case of multicellular organisms, the promoter may also be specific to a particular tissue or organ or developmental stage.

(2) Plasmids

The vector may be a plasmid. The plasmids can be used to transfect cells with nucleic acids encoding antigens, and the transformed host cells can be cultured and maintained under conditions in which expression of the antigens occurs.

The plasmid may comprise nucleic acid sequences encoding one or more of the various antigens disclosed hereinabove, including sequences encoding synthetic, consensus antigens 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 (which consists of a combination of consensus proteins and/or fragments of variants of consensus proteins).

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

In some embodiments, the plasmid may further comprise a coding sequence encoding CCR20 alone or as part of one such plasmid. Similarly, the plasmid may also contain IL-12, IL-15 and/or IL-28 coding sequence.

The plasmid may further comprise a start codon that may be upstream of the coding sequence, and a stop codon that may be downstream of the coding sequence. The start and stop codons can be in frame with the coding sequence.

The plasmid may further comprise 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), Mouse Mammary Tumor Virus (MMTV) promoter, Human Immunodeficiency Virus (HIV) promoter such as the Bovine Immunodeficiency Virus (BIV) Long Terminal Repeat (LTR) promoter, Moloney virus (Moloney virus) promoter, Avian Leukemia Virus (ALV) promoter, Cytomegalovirus (CMV) promoter such as the CMV immediate early promoter, Epstein-Barr virus (EBV) promoter or Rous Sarcoma Virus (RSV) promoter. The promoter may also be a promoter from a human gene such as human actin, human myosin, human hemoglobin, human muscle creatine or human metallothionein. The promoter may also be a tissue-specific promoter, such as a muscle or skin-specific promoter (natural or synthetic). Examples of such promoters are described in U.S. patent application publication No. us20040175727, the contents of which are incorporated herein in their entirety.

The plasmid may also comprise a polyadenylation signal, which may be located 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 the pCEP4 plasmid (Invitrogen, San Diego, CA).

The plasmid may further comprise an upstream enhancer in the coding sequence. The enhancer may be human actin, human myosin, human hemoglobin, human muscle creatine or a viral enhancer such as from CMV, FMDV, RSV or EBV. Polynucleotide functional enhancers are described in U.S. Pat. Nos. 5,593,972, 5,962,428, and W094/016737, the contents of each of which are fully incorporated by reference.

The plasmid may also comprise 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 may contain an epstein-barr virus origin of replication and the nuclear antigen EBNA-1 coding region, which can produce high copy episomal replication without integration. The backbone of the plasmid may be pA V0242. The plasmid may be a replication-defective adenovirus type 5 (Ad5) plasmid.

The plasmid may also contain regulatory sequences which may be well suited for gene expression in cells to which the plasmid is administered. The coding sequence may comprise codons that allow for more efficient transcription of the coding sequence in a host cell.

The coding sequence may also comprise an Ig leader sequence. The leader sequence may be 5 "of the coding sequence. The consensus antigen encoded by this sequence may comprise the N-terminal Ig leader sequence followed by the consensus antigenic protein. The N-terminal Ig leader sequence may be IgE or IgG.

The plasmid may be pSE420(Invitrogen, San Diego, Calif.) which can be used to produce proteins in e. The plasmid may also be p YES2(Invitrogen, san Diego, Calif.) which can be used to produce proteins in a Saccharomyces cerevisiae strain of yeast. The plasmid may also be a MAXBAC^TMPlasmids of the complete baculovirus expression system (Invitrogen, San Diego, Calif), which can be used for the production of proteins in insect cells. The plasmid may also be pcDNA I or pcDNA3(Invitrogen, San Diego, Calif.) which can be used to produce proteins in mammalian cells such as Chinese Hamster Ovary (CHO) cells.

(3) Circular and linear vectors

The vector may be a circular plasmid, which may transform a target cell by integration into the genome of the cell or may exist extrachromosomally (e.g., an autonomously replicating plasmid with an origin of replication).

The vector may be pVAX, pcdna3.0 or provax, or any other expression vector capable of expressing DNA encoding an antigen and enabling the cell to translate the sequence into an antigen recognized by the immune system.

Also provided herein are linear nucleic acid vaccines or linear expression cassettes ("LECs") that can be efficiently delivered to a subject via electroporation and express one or more desired antigens. The LECs may be any linear DNA without any phosphate backbone present. The DNA may encode one or more antigens. The LECs may contain promoters, introns, stop codons and/or polyadenylation signals. Expression of the antigen may be under the control of a promoter. The LECs may not contain any antibiotic resistance genes and/or phosphate backbones. The LECs may be free of other nucleic acid sequences unrelated to the desired gene expression of the antigen.

The LECs may be derived from any plasmid capable of being 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 or provax, or any other expression vector capable of expressing the DNA encoding the antigen and enabling the cell to translate the sequence into an antigen recognized by the immune system.

The LEC may be pcrM 2. The LEC may be pcrNP. pcrNP and pcrMR were derived from pNP (Puerto Rico/34) and pM2(New Caledonia/99), respectively.

(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 expression of the isolated nucleic acid. Such promoters are cis-acting sequence elements required for transcription by DNA-dependent RNA polymerases that transcribe the antigen sequences described herein. The choice of promoter used to direct expression of the heterologous nucleic acid depends on the particular application. The promoter may be placed in the vector at about the same distance from the transcription start site as it is in its natural background. However, this change in distance is acceptable without any loss of promoter function.

The promoter may be operably linked to nucleic acid sequences encoding signals, ribosome binding sites and translation termination required for efficient polyadenylation of the antigen and transcript.

The promoter may be a CMV promoter, an SV40 early promoter, an SV40 late promoter, a metallothionein promoter, a murine mammary tumor virus promoter, a rous sarcoma virus promoter, a polyhedrin promoter, or another promoter that has been shown to be effective for expression in eukaryotic cells.

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

(5) Method for preparing carrier

Provided herein are methods for preparing vectors comprising the DNA vaccines discussed herein. After cloning into mammalian expression plasmids by a final subcloning step, the vectors can be used to inoculate cell cultures in large scale fermentors using methods known in the art.

Vectors for use with EP devices described in more detail below can be formulated or manufactured using a combination of known devices and techniques, but preferably they are manufactured using optimized plasmid manufacturing techniques described in approved, co-pending U.S. provisional application serial No.60/939,792, filed on 23/5/2007. In some examples, the DNA plasmid used in these studies may be formulated at a concentration of greater than or equal to 10 mg/mL. The manufacturing techniques include or encompass various devices and protocols that are well known to those of ordinary skill in the art, in addition to those described in U.S. serial No.60/939792 (including approved patents issued on 7/3/2007, those described in U.S. patent No.7,238,522). The above-mentioned applications and patents (U.S. serial No.60/939,792 and U.S. patent No.7,238,522, respectively) are hereby incorporated in their entirety.

c. Excipients and other components of vaccines

The vaccine may further comprise a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient may be a functional molecule such as a vehicle, carrier or diluent. The pharmaceutically acceptable excipient may be a transfection facilitating agent, which may include surfactants such as Immune Stimulating Complexes (ISCOMS), Freunds incomplete adjuvant (Freunds 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 facilitating agents.

The transfection facilitating agent is a polyanion, a polycation, including poly-L-glutamate (LGS) or a lipid. The transfection facilitating agent is poly-L-glutamate and the poly-L-glutamate may be present in the vaccine at a concentration of less than 6 mg/ml. The transfection facilitating agent may also include surfactants such as Immune Stimulating Complexes (ISCOMS), freunds incomplete adjuvant, LPS analogs, including monophosphoryl lipid a, muramyl peptides, quinone analogs, and vesicles such as squalene and squalene, and hyaluronic acid may also be administered in combination with the genetic construct. The DNA plasmid vaccine may also include a transfection facilitating agent such as lipids, liposomes, including lecithin liposomes or other liposomes known in the art, as DNA-liposome mixtures (see, e.g., W09324640), calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other known transfection facilitating agents. The transfection facilitating agent is a polyanion, polycation, including poly-L-glutamate (LGS) or lipid. The concentration of transfection reagent in the vaccine is less than 4mg/ml, less than 2mg/ml, less than 1mg/ml, less than 0.750mg/ml, less than 0.500mg/ml, less than 0.250mg/ml, less than 0.100mg/ml, less than 0.050mg/ml or less than 0.010 mg/ml.

The pharmaceutically acceptable excipient may be one or more adjuvants. The adjuvant may be another gene expressed in an alternative plasmid or delivered as a protein in a vaccine in combination with the above plasmid. The one or more adjuvants may be selected from the group consisting of: CCL20, interferon-alpha (IFN-alpha), interferon-beta (IFN-beta), interferon-gamma, Platelet Derived Growth Factor (PDGF), TNF alpha, TNF beta, GM-CSF, Epidermal Growth Factor (EGF), cutaneous T cell mobilization chemokine (CTACK), thymic epithelial cell expression chemokine (TECK), mucosa-associated epithelial chemokine (MEC), IL-12, IL-15, IL-28, MHC, CD80, CD86, IL-L, IL-2, IL-4, IL-5, IL-6, IL-10, IL-18, MCP-1, MIP-1a, MIP-1, IL-8, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, and LFC-1, VLA-1, Mac-1, pl50.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, mutant forms of IL-18, CD40, CD40L, vascular growth factor, fibroblast growth factor, IL-7, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Flt, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DRS, KILLER, TRAIL-R2, TRICK2, DR6, caspase, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAILF 6, IkB, NIK, SAP 37, Barec-1, TRAR 369685, TRAIL-R369638, TRAIL-R369685, TRAR, TRACK, RANK ligand, Ox40, Ox40 ligand, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAPI, TAP2, IL-15 (coding sequence with a signal sequence or coding sequence encoding a deleted signal sequence and optionally comprising 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), as well as functional fragments thereof or combinations thereof. The adjuvant may be IL-12, IL-15, IL-28, CTACK, TECK, Platelet Derived Growth Factor (PDGF), TNF α, TNF β, 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.

In some embodiments, an 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 US application sequence No.08/956,865 and US provisional application sequence No 61/569600 filed 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 U.S. application Ser. No.10/560,650, and PCT application No. PCT/US07/00886 and corresponding U.S. application Ser. No.12/160,766 and PCT application No. PCT/USI0/048827, each of which is incorporated herein by reference. Examples of iL-28 constructs and sequences are disclosed in PCT application No. PCT/US09/039648 and the corresponding US application sequence No.12/936,192, each of which is incorporated herein by reference. Examples of RANTES and other constructs and sequences are disclosed in PCT application No. PCT/US1999/004332 and corresponding US application sequence No.09/622452, each of which is incorporated herein by reference. Further examples of RANTES constructs and sequences are disclosed in PCT application No. PCT/US11/024098, which is incorporated herein by reference. Examples of RANTES and other constructs and sequences are disclosed in PCT application No. PCT/US1999/004332 and corresponding US application sequence No.09/622452, each of which is incorporated herein by reference. Further 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 U.S. application sequence No.11/719,646, each of which is incorporated herein by reference. Examples of OX40 and other immunomodulators are disclosed in U.S. application Ser. No.10/560,653, which is incorporated herein by reference. Examples of DR5 and other immunomodulators are disclosed in U.S. application serial No.09/622452, which is incorporated herein by reference.

Other genes that may be used as adjuvants include those encoding: 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 forms of IL-18, CD40, CD40L, vascular growth factors, fibroblast growth factors, IL-7, IL-22, nerve growth factors, vascular endothelial growth factors, Fas, TNF receptors, Flt, Apo-1, P55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KIER 2, TRICK2, caspase, Fodr 6, caspase, FoMP, c-jun, Sp-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, inactive NIK, SAP K, SAP-1, JNK, interferon responsive genes, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK ligand, Ox40, Ox40 ligand, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1, TAP2 and functional fragments thereof.

The vaccine may also comprise a gene vaccine facilitator as described in U.S. sequence No.021,579 (which is incorporated by reference in its entirety) filed on month 4 and 1 of 1994.

The vaccine may comprise the antigen and the plasmid in an amount of about 1 ng to 100mg, about 1 ng to about 10mg, or preferably about 0.1 ng to about 10mg, or more preferably about 1mg to about 2 mg. In some preferred embodiments, the vaccine according to the invention comprises about 5 nanograms to about 1000 micrograms of DNA. In some preferred embodiments, the vaccine may contain from about 10 nanograms to about 800 micrograms of DNA. In some preferred embodiments, the vaccine may contain 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 from about 25 to about 250 micrograms, from about 100 to about 200 micrograms, from about 1 nanogram to 100 milligrams, from about 1 microgram to about 10 milligrams, from about 0.1 micrograms to about 10 milligrams, from about 1 milligram to about 2 milligrams, from about 5 nanograms to about 1000 micrograms, from about 10 nanograms to about 800 micrograms, from about 0.1 to about 500 micrograms, from about 1 to about 350 micrograms, from about 25 to about 250 micrograms, from about 100 to about 200 micrograms of antigen or plasmid thereof.

The vaccine may be formulated according to the mode of administration to be used. Injectable vaccine pharmaceutical compositions can be sterile, pyrogen free and particle free. Isotonic formulations or solutions may be used. Additives for isotonicity may include sodium chloride, dextrose, mannitol, sorbitol, and lactose. The vaccine may comprise a vasoconstrictor. The isotonic solution may include phosphate buffered saline. The vaccine may also contain stabilizers including gelatin and albumin. The stabilizing agent may render the formulation stable at room or ambient temperature for an extended period of time, including LGS or polycations or polyanions.

3. Vaccine delivery method

Provided herein are methods for delivering vaccines to provide genetic constructs and antigens comprising epitopes that make them particularly effective against target immunogens against which an immune response can be elicited. Methods of delivering vaccines or vaccinations may be provided to elicit therapeutic and prophylactic immune responses. The vaccination method may generate an immune response in the mammal against the antigen. The vaccine can be delivered to an individual to modulate the activity of the immune system and enhance the immune response of a mammal. Delivery of a vaccine may be transfection of an antigen as a nucleic acid molecule expressed in and delivered to the surface of a cell, the immune system recognizing the antigen on the cell surface and eliciting a cellular, humoral, or both cellular and humoral response. Delivery of the vaccine can be used to induce or elicit an immune response in a mammal against the antigen by administering the vaccine as described above to the mammal.

When the vaccine and plasmid are delivered into cells of a mammal, the transfected cells will express and secrete the antigen of each plasmid injected from the vaccine. The antigen will be recognized by the immune system as foreign and antibodies will be raised against them. These antibodies will be maintained by the immune system and allow an effective response against the antigen.

The vaccine can be administered to a mammal to elicit an immune response in the mammal. The mammal can be a human, primate, non-human primate, cow, sheep, goat, antelope, bison, buffalo, bison, bovidae, deer, hedgehog, elephant, camel, alpaca, mouse, rat, and chicken.

a. Combination therapy

The vaccine may be administered in combination with other proteins and/or genes encoding: CCL20, a-interferon, y-interferon, Platelet Derived Growth Factor (PDGF), TNF α, TNF β, GM-CSF, Epidermal Growth Factor (EGF), cutaneous T-cell delivery chemokine (CTACK), thymic epithelial cell expressing chemokine (TECK), mucosa associated epithelial chemokine (MEC), IL-12, IL-15 including IL-15 with a deleted signal sequence and optionally comprising different signal peptides such as IgE signal peptide, MHC, CD80, CD86, IL-28, IL-L, IL-2, IL-4, IL-5, IL-6, IL-10, IL-18, MCP-1, MIP-1 α, MIP-1 β, IL-8, TES, 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 forms of IL-18, CD40, CD40L, vascular growth factor, fibroblast growth factor, IL-7, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Flt, Apo-1, p55, WSL-1, DR 7, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, rec, KILLER, TRAIL-R2, CK TRI 2, DR6, caspase, Fos, c-jun, Sp-1, Ap-2, p38, p65 Reak, 46D 88, IRIL, NIkF 6, TRAIkB, DRS, BAK-25, DRS-inducible genes, DRS, DRC-I, JNK, SAP-17, DRC-7, SAP-IRF-7, TRAIL-3, DRC-17, DRS, DRC-7, SAP-7, TRAIL-3, ASP-3, ASR-1, DRS-1, DR, TRAIL-R4, RANK ligand, Ox40, Ox40 ligand, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAPI, TAP2 and functional fragments thereof 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 functional fragments thereof, and a protein selected from the group consisting of CCL20, IL-12 protein, IL-15 protein, IL-28 protein, CTACK protein, TECK protein, MEC protein, or RANTES protein, or functional fragments thereof.

The vaccine may be administered by different routes including oral, parenteral, sublingual, transdermal, rectal, transmucosal, topical, via inhalation, buccal, intrapleural, intravenous, intraarterial, intraperitoneal, subcutaneous, intramuscular, intrathecal and intraarticular or a combination thereof. For veterinary use, the vaccine may be administered as a suitably acceptable formulation in accordance with normal veterinary practice. Veterinarians can readily determine the dosage regimen and route of administration that is most appropriate for a particular animal. The vaccine may be administered by conventional syringes, needleless injection devices, "particle bombardment gene guns", or other physical methods such as electroporation ("EP"), "hydrodynamic methods", or ultrasound.

The plasmids of the vaccines can be delivered to mammals using several well-known techniques, including DNA injection (also known as DNA vaccination) with and without in vivo electroporation, liposome-mediated, nanoparticle-facilitated recombinant vectors such as recombinant adenoviruses, recombinant adeno-associated viruses, and recombinant vaccinia viruses. The antigen or immunogen can be delivered via DNA injection and in conjunction with in vivo electroporation.

b. Electroporation

The vaccine may be formulated according to standard techniques well known to those skilled in the pharmaceutical art. Such compositions may be administered in doses using 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 and the route of administration. The subject may be a mammal, such as a human, horse, cow, pig, sheep, cat, dog, rat, or mouse.

The vaccine can be administered prophylactically or therapeutically. In prophylactic administration, the vaccine may be administered in an amount sufficient to elicit an immune response. In therapeutic applications, the vaccine is administered to a subject in need thereof in an amount sufficient to elicit a therapeutic effect. An amount sufficient to achieve this effect is defined as a "therapeutically effective dose". The amount effective for this use will depend, for example, on the particular composition of the vaccine regimen being administered, the mode of administration, the stage and severity of the disease, the general health of the patient, and the judgment of the prescribing physician.

Such vaccines can be obtained, for example, by Donnelly et al (Ann. Rev. Immunol.15:617-648 (1997)); felgner et al (U.S. Pat. No.5,580,859, issued on 3.12.1996); felgner (U.S. Pat. No.5,703,055, granted 12 months and 30 days 1997); and Carson et al (U.S. patent No.5,679,647, issued on 21/10/1997) (the contents of all documents and U.S. patents are incorporated herein by reference in their entirety). The DNA of the vaccine can be compounded into particles or beads that can be administered to an individual, for example, using a vaccine gun. One skilled in the art will appreciate that the choice of a pharmaceutically acceptable carrier, including a physiologically acceptable compound, will depend, for example, on the route of administration of the expression vector.

The vaccine can be administered via a variety of routes. Typical routes of delivery include parenteral administration, e.g., intradermal, intramuscular or subcutaneous delivery. Other routes include oral administration, intranasal, and intravaginal routes. Specifically for the DNA of the vaccine, the vaccine can be delivered to the interstitial spaces of the tissue of the individual (Felgner et al, U.S. Pat. nos. 5,580,859 and 5,703,055, the contents of all of which are incorporated herein by reference in their entirety). The vaccine may also be administered to muscle, either via intradermal or subcutaneous injection, or transdermally (such as by iontophoresis). Epidermal administration of the vaccine may also be used. Epidermal administration may include mechanical or chemical stimulation of the outermost layer of the epidermis to stimulate an immune response against the stimulus (Carson et al, U.S. patent No.5,679,647, the contents of which are incorporated herein by reference in their entirety).

The vaccine may also be formulated for administration via the nasal passage. Formulations suitable for nasal administration, wherein the carrier is a solid, may comprise a coarse powder having a particle size, for example, in the range of from about 10 to about 500 microparticles, to be administered in a manner wherein inhalation from the nose, i.e., by rapid inhalation through the nasal passage from a container of the powder held close to the nose, is employed. The formulation may be administered as a nasal spray, nasal drops or aerosol with a nebulizer. The formulation may comprise an aqueous or oily solution of the vaccine.

The vaccine may be a liquid formulation such as a suspension, syrup or elixir. The vaccine may also be a formulation for parenteral, subcutaneous, intradermal, intramuscular, or intravenous administration (e.g., injectable administration), such as a sterile suspension or emulsion.

The vaccine may be incorporated into liposomes, microspheres, or other polymeric matrices (Felgner et al, U.S. Pat. No.5,703,055; Gregoriadis, Liposome Technology, Vol.I to III (2 nd edition, 1993), the contents of which are incorporated herein by reference in their entirety). Liposomes can be composed of phospholipids or other lipids, and can be nontoxic, physiologically acceptable, and metabolizable carriers that are relatively simple to produce and administer.

Administration of the vaccine via electroporation of the plasmids of the invention can be achieved using an electroporation device configured to deliver to the desired tissue of the mammal a pulse of energy effective to cause reversible pore formation in the cell membrane, and preferably the pulse of energy is a constant current similar to the current input preset by the user. An electroporation device may include an electroporation component and an electrode component or an operational component. The electroporation component may include and contain one or more of the various elements of the electroporation device, including: a controller, a current waveform generator, an impedance tester, a waveform recorder, an input element, a status reporting element, a communication port, a memory component, a power supply, and a power switch. Can be used, for example, with in vivo electroporation devices such as

Electroporation was achieved by using CELLECTRA EP systems (VGX Pharmaceuticals, Blue Bell, Pa.) or Elgen electroporation devices (Genetronics, San Diego, Calif.) to facilitate plasmid-to-cell transfection.

The electroporation component may function as one element of the electroporation device and the other elements are separate elements (or components) in communication with the electroporation component. The electroporation component may function as more than one element of an electroporation device, which may be in communication with additional other elements of the electroporation apparatus separate from the electroporation component. The elements of the electroporation device that are present as part of the electromechanical or mechanical device may be unlimited in that the elements may function as one device or as separate elements in communication with each other. The electroporation component may be capable of delivering an energy pulse that produces a constant current in a desired tissue and include a feedback mechanism. The electrode assembly may include an electrode array having a plurality of electrodes in a spatial arrangement, wherein the electrode assembly receives energy pulses from the electroporation component and delivers them through the electrodes to a desired tissue. At least one of the plurality of electrodes is neutral during delivery of the energy pulse and measures an impedance of the desired tissue and communicates the impedance to the electroporation component. A feedback mechanism may receive the measured impedance and may adjust the energy pulse delivered by the electroporation component to maintain a constant current.

The plurality of electrodes may deliver the energy pulses in a distributed pattern. The plurality of electrodes may deliver the energy pulses in a distributed pattern by controlling the electrodes in a programmed sequence, and the programmed sequence is input into the electroporation component by a user. The programmed sequence may comprise a plurality of pulses delivered in sequence, wherein each pulse of the plurality of pulses is delivered by at least two active electrodes (with one neutral electrode measuring impedance), and wherein a subsequent pulse of the plurality of pulses is delivered by a different one of the at least two active electrodes (with one neutral electrode measuring impedance).

The feedback mechanism may be performed by hardware or software. The feedback mechanism may be performed by an analog closed loop circuit. Feedback is generated once every 50, 20, 10 or 1 mus, but is preferably real-time or instantaneous (i.e., substantially simultaneous, as determined by available techniques for determining response time). The neutral electrode may measure an impedance in a desired tissue and communicate the impedance to a feedback mechanism, and the feedback mechanism responds to the impedance and adjusts the energy pulse to maintain the constant current at a value similar to a preset current. The feedback mechanism can continuously and instantaneously maintain a constant current during the delivery of the energy pulse.

Examples of electroporation devices and methods that can facilitate delivery of the DNA vaccines of the present invention include those described in U.S. patent No.7,245,963 to Draghia-Akli et al, U.S. patent publication 2005/0052630 to Smith et al, the contents of which are incorporated herein by reference in their entirety. Other electroporation devices and electroporation methods that may be used to facilitate delivery of DNA vaccines include those provided in co-pending and commonly owned U.S. patent application serial No.11/874072 filed on 17.10.2007, in accordance with the benefits of 35USC 119(e) claim U.S. provisional application serial No.60/852,149 filed on 17.10.2006 and 60/978,982 filed on 10.10.2007 (all of which are hereby incorporated herein in their entirety).

U.S. Pat. No.7,245,963 to Draghia-Akli et al describes standard electrode systems and their use for facilitating the introduction of biomolecules into cells of selected tissues of the body or plant. A standard electrode system may include a variety of needle electrodes; a hypodermic needle; an electrical connector providing a conductive connection from the programmable constant current pulse controller to the plurality of pin electrodes; and a power source. An operator may grasp a plurality of needle electrodes mounted on a support structure and insert them securely into selected tissue of a body or plant. The biomolecules are then delivered to the selected tissue via a hypodermic needle. The programmable constant current pulse controller is activated and constant current electrode pulses are applied to the plurality of needle electrodes. The applied constant current electrical pulse facilitates the introduction of the biomolecule into the cell between the plurality of electrodes. U.S. Pat. No.7,245,963 is incorporated herein by reference in its entirety.

U.S. patent publication 2005/0052630, filed by Smith et al, describes an electroporation device that can be used to effectively facilitate the introduction of biomolecules into cells of a selected tissue of a body or plant. The electroporation devices include electrokinetic devices ("EKD devices") whose operation is specified by software or firmware. The EKD device generates a series of programmable constant current pulse waveforms between electrodes in the array based on user control and input of pulse parameters and allows current waveform data to be stored and retrieved. The electroporation device also includes a replaceable electrode disk having an array of needle electrodes, a central injection channel for the injection needles, and a removable guide disk. U.S. patent publication 2005/0052630 is incorporated herein by reference in its entirety.

The electrode arrays and methods described in U.S. Pat. No.7,245,963 and U.S. patent publication 2005/0052630 may be adapted for deep penetration into not only tissues such as muscles, but also other tissues or organs. Due to the configuration of the electrode array, an injection needle (to deliver selected biomolecules) can also be inserted completely into the target organ and the injection agent administered perpendicular to the target tissue in the area pre-delineated with the electrodes. The electrodes described in U.S. Pat. No.7,245,963 and U.S. patent publication 2005/005263 are preferably 20mm long and 21 gauge.

Furthermore, in some embodiments including electroporation devices and uses thereof, the presence of electroporation devices is contemplated, which are described in the following patents: those electroporation devices described in U.S. Pat. No.5,273,525, 28/1993, U.S. Pat. No.6,110,161, 8/29/2000, 6,261,281, 17/7/2001, 6,958,060, 10/25/2005, and U.S. Pat. No.6,939,862, 6/9/2005. Further, patents designed for methods of injecting DNA encompassing the subject matter provided in us patent 6,697,669 issued 2/24 of 2004 (which involves the delivery of DNA using any of a variety of devices) and us patent 7,328,064 issued 2/5 of 2008 are contemplated herein. The above patents are incorporated by reference in their entirety.

The vaccine may be administered via electroporation, such as by the methods described in U.S. patent No.7,664,545 (the contents of which are incorporated herein by reference). The electroporation can be performed using the methods and/or apparatus described in U.S. Pat. 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. The electroporation can be performed via a minimally invasive device.

A minimally invasive electroporation device ("MID") may be a device for injecting the above-described vaccines and related fluids into body tissue. The device may comprise a hollow needle, a DNA cassette and a fluid delivery device, wherein the device is adapted to actuate the fluid delivery device in use to simultaneously (e.g. automatically) inject DNA into body tissue during insertion of the needle into the body tissue. This has the advantageous aspect that the ability to gradually inject DNA and associated fluids while the needle is inserted results in a more even distribution of fluids throughout the body tissue. The pain experienced during injection can be reduced by the distribution of injected DNA over a larger area.

MIDs can inject vaccines into tissue without the use of needles. MID may inject the vaccine in the form of a small stream or jet, with the force that causes the vaccine to penetrate the surface of the tissue and into the underlying tissue and/or muscle. The force behind the small stream or jet can be provided by expanding a compressed gas (such as carbon dioxide) through the micropores in a fraction of a second. Examples of minimally invasive electroporation devices and methods of using them are described in published U.S. patent application No.20080234655, 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.

The MID may comprise a syringe that produces a high-velocity liquid jet that penetrates tissue without pain. Such needleless injectors are commercially available. Examples of needleless injectors that may be used herein include those described in U.S. patent nos. 3,805,783, 4,447,223, 5,505,697, and 4,342,310 (the contents of each of which are incorporated herein by reference).

A desired vaccine suitable for direct or indirect electrotransport can be introduced (e.g., injected) into the tissue to be treated using a needleless syringe (typically by contacting the tissue surface with the syringe to drive delivery of a jet of agent with sufficient force to penetrate the vaccine into the tissue). For example, if the tissue to be treated is mucosal, skin, or muscle, the agent is injected toward the mucosal or skin surface with sufficient force to cause the agent to penetrate through the cortical layer and into the dermal layer, or into the underlying tissue and muscle, respectively.

Needleless injectors are particularly suitable for delivering vaccines to all types of tissue, in particular skin and mucosa. In some embodiments, a needle-free injector may be used to propel a liquid containing a vaccine to a surface and into the skin or mucosa of a subject. Representative examples of the types of tissues that can be treated using the methods of the invention include pancreas, larynx, nasopharynx, hypopharynx, oropharynx, lip, throat, lung, heart, kidney, muscle, breast, colon, prostate, thymus, testis, skin, mucosal tissue, ovary, blood vessels, or any combination thereof.

The MID may have needle electrodes that electroporate tissue. By pulsing between pairs of electrodes in a multi-electrode array (e.g., created in a rectangular or square pattern), improved results are provided over those of pulsing between a pair of electrodes. An array of needles in which pairs of needles can be pulsed during therapeutic treatment is disclosed, for example, in U.S. patent No.5,702,359 entitled "Needle Electrodes for medial Delivery of Drugs and Genes". In this application (which is incorporated by reference as if fully set forth herein), the needles are placed in a circular array, but with connectors and a switching device that enables pulses to be generated between opposing pairs of needle electrodes. A pair of needle electrodes for delivering the recombinant expression vector to the cell may be used. Such devices and systems are described in U.S. patent No.6,763,264 (the contents of which are incorporated herein by reference). Alternatively, a single needle device may be used that allows for the injection of DNA and electroporation using a single needle similar to a conventional injection needle, and the application of pulses having a lower voltage than pulses delivered by currently used devices, thereby reducing the tactile inductance experienced by the patient.

MIDs may contain one or more electrode arrays. The array may include two or more needles having the same diameter or different diameters. The needles may be evenly or unevenly spaced. The needle can be 0.005 to 0.03 inches, 0.01 to 0.025 inches, or 0.015 to 0.020 inches. The needle may be 0.0175 inches in diameter. The needles may be spaced apart by 0.5mm, 1.0mm, 1.5mm, 2.0mm, 2.5mm, 3.0mm, 3.5mm, 4.0mm or more.

The MID may consist of a pulse generator and two or more needle-type vaccine syringes that deliver vaccine and electroporation pulses in a single step. The pulse generator may allow flexible programming of pulse and injection parameters via a flash card operated personal computer, as well as comprehensive recording and storage of electroporation and patient data. The pulse generator can deliver multiple volt pulses in a very short time. For example, the pulse generator may deliver 3 15 volt pulses delivered in 100ms duration. An example of such an MID is the Elgen 1000 system provided by inovoi Biomedical Corporation, which is described in U.S. patent No.7,328,064 (the contents of which are incorporated herein by reference).

The MID may be CELLECTRA (inovoi Pharmaceuticals, Blue Bell PA) devices and systems, which are standard electrode systems that aid in the introduction of macromolecules such as DNA into cells in vivo or in selected tissues of plants. A standard electrode system may include a plurality of needle electrodes; subcutaneous needles; an electrical connector providing conductive connection from the programmable constant current pulse controller to the plurality of pin electrodes; and a power source. An operator may grasp the plurality of needle electrodes secured to the carrier structure and securely insert them into selected tissue of the body or plant. The macromolecule is then delivered into the selected tissue via a hypodermic needle. The programmable constant current pulse controller is activated and a constant current electrical pulse is applied to the plurality of needle electrodes. The applied constant current electrical pulse helps to introduce the biomolecules into the cells between the plurality of electrodes. Cell death due to overheating of the cells is minimized by limiting the power consumption in the tissue by constant current pulses. The Cellecta device and system are described in U.S. Pat. No.7,245,963 (the contents of which are incorporated herein by reference).

The MID may be the Elgen 1000 system (Inovio Pharmaceuticals). The Elgen 1000 system may include a device that provides a hollow needle; and a fluid delivery device, wherein the apparatus is adapted to actuate the fluid delivery device in use to simultaneously (e.g. automatically) inject fluid (vaccine described herein) into body tissue during insertion of the needle into the body tissue. An advantageous aspect is the ability to gradually inject fluid while inserting the needle results in a more even distribution of fluid throughout the body tissue. It is also believed that the pain experienced during injection is reduced by the distribution of the volume of fluid injected over a larger area.

Furthermore, automatic injection of fluid helps to automatically monitor and record the actual dose of injected fluid. This data may be stored by the control unit for archiving, if desired.

It will be appreciated that the injection rate may be linear or non-linear and that the injection may be performed after the needles have been inserted through the skin of the subject to be treated and as they are further inserted into the body tissue.

Suitable tissues into which the device of the invention may be used to inject fluid include tumour tissue, skin or liver tissue, but also muscle tissue.

The device also comprises a needle insertion means for guided 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 needle insertion and fluid injection can be controlled so that the rate of insertion can match the desired injection rate. It also makes the device easier for the user to operate. If desired, means for automatically inserting the needle into the body tissue may be provided.

The user may select the time at which to begin injecting fluid. Ideally, however, the injection is initiated when the needle tip has reached muscle tissue, and the device may comprise means for sensing that the needle has been inserted to a depth sufficient to initiate injection of the fluid. This means that the needle can be reminded to automatically start injecting fluid when it has reached the desired depth, which is usually the depth at which muscle tissue starts. The depth at which the muscle starts may for example be taken as a preset needle insertion depth, such as a value of 4mm that is considered sufficient for the needle to penetrate the skin layer.

The sensing device may comprise an ultrasound probe. The sensing means may comprise means for sensing a change in impedance or resistance. In this case, the device may not register the depth of the needle in the body tissue as such, but is adapted to sense a change in impedance or resistance as the needle moves from a different type of body tissue into the muscle. Any of these alternative devices provides a relatively accurate and simple means of operating that an induction injection can commence. If desired, the depth of needle insertion may also be recorded and used to control the injection of fluid so that the volume of fluid to be injected is determined while the depth of needle insertion is recorded.

The device may also include: a base for supporting the needle; and a housing for receiving the base therein, wherein the base is movable relative to the housing to retract the needle within the housing when the base is in a first rearward position relative to the housing and to extend the needle out of the housing when the base is in a second forward position within the housing. This is advantageous for the user, since the housing may be aligned on the skin of the patient, after which the needle may be inserted into the skin of the patient by moving the housing relative to the base.

As mentioned above, it is desirable to achieve rate-controlled fluid injection so that when the needle is inserted into the skin, the fluid is evenly distributed over the length of the needle. The fluid delivery device may comprise a piston drive adapted to inject fluid at a controlled rate. The piston drive can be driven, for example, by a servomotor. However, the piston drive may be driven by the base moving in an axial direction relative to the housing. It will be appreciated that alternative means for fluid delivery may be provided. Thus, for example, a closed container that can be squeezed for fluid delivery at a controlled or uncontrolled rate may be provided in place of a syringe and plunger system.

The above described device may be used for any type of injection. It is envisaged that it is particularly useful in the field of electroporation and therefore it may also comprise 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 region as the injected fluid. Historically, there have been problems with electroporation in that it has been extremely difficult to accurately align the electrodes with the previously injected fluid, so users have intended to inject a larger volume of fluid over a larger area than is required, and to apply an electric field over the higher area in an attempt to ensure overlap between the injected substance and the electric field. By using the present invention, the volume of fluid injected and the size of the applied electric field can be reduced while achieving a good fit between the electric field and the fluid.

4. Methods of treatment and/or prevention

Also provided herein are methods of treating, protecting against, and/or preventing a disease in a subject in need thereof by administering the vaccine to the subject in need thereof. The disease may be cancer, for example, Wilms 'tumor, metastatic cancer arising from Wilms' tumor, and WT1 expressing cancer or tumor. The vaccine may be administered to a subject as described above in the delivery method. Administration of the vaccine to a subject can induce or elicit an immune response in the subject. In particular, administration of the vaccine to a subject may induce or elicit a humoral and/or cellular immune response in the subject.

In particular, the methods can treat a subject having a wilms tumor, a metastatic cancer arising from a wilms tumor, and/or a WT1 expressing tumor or cancer because the vaccine slows the growth, reduces, and eliminates the wilms tumor, the metastatic cancer arising from a wilms tumor, and/or the WT1 expressing tumor or cancer. The method may also prevent wilms 'tumor, metastatic cancer arising from wilms' tumor, and/or WT1 expressing tumor or cancer in the 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 elicits a humoral and/or cellular immune response. The induced humoral and/or cellular immune response may target wilms 'tumors, metastatic cancers arising from wilms' tumors, and/or WT1 expressing tumors or cancers, thereby slowing the growth, reducing and eliminating any wilms 'tumors, metastatic cancers arising from wilms' tumors, and/or WT1 expressing tumors or cancers in a subject to which the vaccine is administered. The humoral and/or cellular immune response induced by the vaccine may also inhibit the formation and growth of any wilms tumor, metastatic cancer arising from wilms tumors, and/or WT1 expressing tumors or cancers in the subject to which the vaccine is administered.

The vaccine dose may be from 1 μ g to 10mg of active ingredient per kg of body weight per time, and may be from 20 μ g to 10mg of ingredient per kg of body weight per time. The vaccine may be administered once 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 vaccine for effective treatment may be 1,2, 3,4, 5,6, 7, 8,9 or 10.

The invention has a number of aspects which are illustrated by the following non-limiting examples.

5. Examples of the embodiments

The invention will be further illustrated in the following examples. It should be understood that these examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Accordingly, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

The WT1 immunogen was produced using a stepwise method as described in the examples herein. The WT1 gene was modified by several stepwise modifications. First, the WT1RNA structure was modified so that it produced a single full-length transcript. Second, codon usage was modified to alter the RNA structure at the 5' end, thereby increasing expression in vivo. Finally, the zinc finger domain of the gene is mutated to modify WT1 activity and to delete portions of the zinc finger binding site.

Example 1

Optimized WT-1

A stepwise method for the production of WT1 immunogen was developed. First changes to RNA structure are designed that result in the production of only a single full-length long transcript, thereby producing an immunogen with a more controllable phenotype. The sequences also contain modified codon usage choices and altered RNA structure at the 5' end to enhance expression in vivo. Therefore, WT1 immunogen was optimized for expression. Plasmids encoding expression cassettes for the improved immunogens are generated and tested for their immunogenic potential and ability to generate T and B cell responses in animals. The plasmid vaccine is formulated for enhanced in vivo delivery by electroporation, and specific conditions are used for in vivo delivery.

Plasmids encoding the immunogen (designated WT1-pVAX1, WT1-pVAX2, WT1-pVAX3, WT1-pVAX4, and WT1-pVAX5) were compared with WT1 vaccines (also designated WT1-pCDNA1, WT1-pCDNA2, WT1-pCDNA3, WT1-pCDNA4, and WT1-pCDNA5) which represent standard vaccines studied by the art.

A mouse study was performed. Mice were vaccinated with the modified WT1 vaccine (i.e., vectors WT1-pVAX1, WT1-pVAX2, WT1-pVAX3, WT1-pVAX4, and WT1-pVAX5) and experienced better anti-WT 1T cell and B cell responses than anti-WT 1T cell and B cell responses produced by mice vaccinated with a standard WT1 vaccine comprising native WT1 (i.e., WT1-pCDNA1, WT1-pCDNA2, WT1-pCDNA3, WT1-pCDNA4, and WT1-pCDNA 5).

Animals (i.e., BalB/C mice) were immunized 3 times with the same amount of WT-1 plasmid, i.e., novel WT1-pVax vaccine (i.e., vectors WT1-pVAX1, WT1-pVAX2, WT1-pVAX3, WT1-pVAX4, and WT1-pVAX5) or original WT-1 plasmid vaccine (i.e., WT1-pCDNA1, WT1-pCDNA2, WT1-pCDNA3, WT1-pCDNA4, and WT1-pCDNA 5). The results of the T cell assay (i.e., interferon- γ ELISpot assay) are shown in figures 1 and 2 it is clear that the newly designed WT-1 vaccine (i.e., vectors WT1-pVAX1, WT1-pVAX2, WT1-pVAX3, WT1-pVAX4, and WT1-pVAX5) is about 4 times as much as the WT-1 original vaccine (i.e., WT1-pCDNA1, WT1-pCDNA2, WT1-pCDNA3, WT1-pCDNA4, and WT1-pCDNA5) in generating T cell responses as shown in figures 1 and 2 only low levels of T cell immunity or nonfunctional T cell immunity were observed for the standard vaccine, consistent with the data that has been previously obtained.

The ability of the novel DNA vaccine to induce an antibody response was also examined. These studies were performed by collecting sera from animals immunized in WT1-pCDNA or WT1-pVAX vaccine. No seroconversion or induction of antibody responses was observed with the WT1-pCDNA vaccine (data not shown). In contrast, WT1-pVAX immunogen induced strong Western blot reactivity with correct specificity and molecular weight, showing strong WT1 seroconversion and strong antibody response (FIG. 3). These data collectively strongly support the improvement of this vaccine immunity gene by delivery variation of WT1 immunogen and improved design.

These data also show that the conformational properties of the immunogen are maintained as these vaccines containing the optimized WT1 immunogen react specifically with native gene sequences in tumor cells.

Further targeting of immunogenic sequences by modifying the coding sequence to disrupt its native structure in two ways, (1) targeting of the zinc finger region and induction of mutations that modify WT1 activity, and (2) deletion of sequences from the important Zn finger binding site. These variations are summarized below in examples 2 and 3.

Example 2

In common WT1

As described above, optimized WT1 immunogen (i.e., optimized WT1 gene as described in example 1) elicited both humoral and cellular immune responses. To further target or modify WT1 immunogen sequences, a consensus WT1 immunogen was generated.

Specifically, WT1 sequences from multiple species were compared to each other. As shown in table 1 below, WT1 is highly conserved. Thus, WT1 sequences from multiple species were used to generate WT1 consensus sequence, where WT1 consensus sequence shares about 95% identity with human WT 1. The resulting consensus WT1 immunogen (also known as ConWT1) shared 95.9% identity with human WT1 and had the amino acid sequence shown in SEQ ID NO:5 (FIG. 5).

Table 1: identity of WT1 between species.

Example 3

Zinc finger modification and removal

The consensus WT1 immunogen described in example 2 above was further modified to enhance the immunogenicity of the WT1 immunogen. Specifically, the consensus WT1 immunogen was modified to break the zinc finger located at the carboxy terminus (C-terminus) of the WT1 immunogen. These modifications included substitutions of zinc ions coordinated in two amino-terminal (N-terminal) zinc fingers to generate residues (i.e., CCHH motif) of the consensus WT1 immunogen with modified zinc fingers (also referred to herein as CON WT1 or ConWT1-L with modified zinc fingers) (fig. 4 and 5). The C and H residues of the CCHH motif are replaced with glycine (G). The immunoglobulin E (IgE) leader sequence was placed N-terminal to the ConWT1-L peptide. ConWT1-L is encoded by SEQ ID NO:1 and has the amino acid sequence shown in SEQ ID NO:2 (FIGS. 6A and 6B, respectively).

FIG. 5 shows an alignment of the amino acid sequences of consensus WT1 immunogen (ConWT1) and consensus WT1 immunogen (ConWT1-L) with modified zinc fingers. The shaded portions in FIG. 5 represent the residues that differ between ConWT1 and ConWT 1-L. In addition to the addition of the IgE leader sequence to ConWT1-L, residues 312, 317, 330, 334, 342, 347, 360 and 364 in ConWT1-L were also different from the corresponding residues in ConWT1 (i.e., residues 295, 300, 313, 317, 325, 330, 343 and 347). These differences reflect the modification of the CCHH motif in the two N-terminal zinc fingers described above.

In addition, the consensus WT1 immunogen was modified to remove the zinc fingers, resulting in a consensus WT1 immunogen without zinc fingers (also referred to herein as CON WT1 or CONWT1-S without zinc fingers) (fig. 4). ConWT1-S is encoded by SEQ ID NO:3 and has the amino acid sequence shown in SEQ ID NO:4 (FIGS. 7A and 7B, respectively).

Example 4

Expression analysis of constructs encoding ConWT1-L and ConWT1-S

The nucleic acid sequences encoding ConWT1-L and ConWT1-S were placed in pVAX1 vectors (Life technologies, Carlsbad, Calif.), respectively. The resulting vectors were designated WT1-pVAX-L and WT1-pVAX-S, respectively. WT1-pVAX-L and WT1-pVAX-S were transfected into cells along with pVAX1 to confirm the expression of ConWT1-L and ConWT1-S, respectively, from these vectors. pVAX1 was used as a negative control.

After transfection, cells were stained with 4', 6-diamidino-2-phenylindole (DAPI) to label the nucleus and antibodies specific for WT 1. Fig. 8 shows the results of this staining. In fig. 8, the left and middle columns show DAPI and WT1 staining, while the right column shows the combination of DAPI and WT1 staining. No staining was detected for WT1 antibody in cells transfected with pVAX 1. These data indicate that ConWT1-L and ConWT1-S were expressed from their respective vectors.

Expression of ConWT1-L and ConWT1-S was further confirmed by immunoblotting of lysates derived from transfected cells. Specifically, immunoblots were probed with anti-WT 1 antibody. As shown in FIG. 9, the expected sizes were detected for ConWT1-L and ConWT1-S (see lanes labeled WT1-pVAX-L and WT1-pVAX-S, respectively). No signal was detected in lysates obtained from cells transfected with pVAX 1. Thus, these data further indicate that ConWT1-L and ConWT1-S are expressed in transfected cells.

Example 5

Immune responses elicited by constructs encoding ConWT1-L and ConWT1-S

To determine whether constructs encoding ConWT1-L and ConWT1-S induced an immune response, C57BL/6 mice were immunized with 25. mu. gWT1-pVAX-L or WT1-pVAX-S, respectively. C57BL/6 mice were also immunized with 25 μ g of WT1-pVAX (which is the optimized construct encoding WT1 described in example 1) or 25 μ g of WT1-pCDNA (which is the non-optimized construct encoding WT 1). Mice from the first exposure experiment were used as controls.

Specifically, the immunization protocol included vaccination of each group of mice with 25 μ g of each vaccine at weeks 0, 2,3 and 5 (fig. 10). Each mouse was bled prior to week 0 vaccination, so that this week 0 bleed served as antibody-induced controls. A second bleed was taken at week 5 (at which time the mice were sacrificed). Splenocytes were also isolated from sacrificed mice and used to examine T cell responses in the ELISpot assay described below.

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

Specifically, the ConWT1-L and ConWT-S antigens induced a T cell response that was about 400-fold higher than the T cell response induced by the optimized and non-optimized constructs. Thus, these data indicate that modification and removal of the zinc finger in WT1 significantly enhances the immunogenicity of WT1 immunogen, thereby providing a significant T cell response against WT1 immunogen.

Humoral immune responses to ConWT1-L and ConWT1-S antigens were examined by determining whether sera from week 5 bleeds contained antibodies specific for the antigen. Specifically, 293T cells were transfected with the vectors WT1-pVAX-L and WT 1-pVAX-S. After transfection, cells transfected with WT1-pVAX-L or WT1-pVAX-S were stained with DAPI to label the nuclei and serum taken at week 5 from mice immunized with WT1-pVAX-L or WT-pVAX-S. As shown in FIG. 13, sera from mice immunized with WT1-pVAX-L or WT1-pVAX-S detected the ConWT1-L or ConWT1-S antigen in transfected cells, respectively. Thus, these data indicate that immunization with constructs expressing the ConWT1-L and ConWT1-S antigens resulted in the production of antibodies immunoreactive with the ConWT1-L and ConWT1-S antigens.

The induction of humoral immune responses by constructs expressing ConWT1-L and ConWT1-S was further examined by immunoblotting. Specifically, lysates were obtained from the above transfected cells and probed with serum from mice immunized with WT1-pVAX-L or WT 1-pVAX-S. Figure 14 shows a representative immunoblot, in which lysates obtained from untransfected 293T cells were used as controls for background. The immunoblot in figure 14 was also probed with an anti-actin antibody (which shows that equal amounts of lysate loaded between 3 lanes). These data indicate that sera from immunized mice contained antibodies immunoreactive with ConWT1-L and ConWT1-S antigens, and further confirm the results of the above cell staining.

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

It should be understood that the foregoing detailed description and accompanying examples are illustrative only and are not intended to limit the scope of the invention, which is defined only by the appended claims and their equivalents.

Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications, including but not limited to those relating to the chemical structures, substituents, derivatives, intermediates, syntheses, compositions, formulations, or methods of use of the invention, may be made without departing from the spirit and scope thereof.

Claims (21)

1. An isolated nucleic acid molecule encoding at least one amino acid sequence selected from the group consisting of SEQ ID NO 2 and 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 and 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 set forth in claim 1.

5. A composition comprising one or more nucleic acid molecules set forth in claim 2.

6. Use of the isolated nucleic acid molecule of claim 1 or claim 2 in the manufacture of a medicament for inducing an immune response against WT1 in an individual.

7. Use of the composition of claim 4 or claim 5 in the preparation of a medicament for inducing an immune response against WT1 in an individual.

8. A protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO 2 and SEQ ID NO 4.

9. A nucleic acid molecule comprising a nucleic acid sequence encoding an antigen, wherein said nucleic acid sequence consists of SEQ ID No. 1.

10. A nucleic acid molecule comprising a nucleic acid sequence encoding an antigen, wherein said nucleic acid sequence consists of SEQ ID No. 3.

11. Use of the nucleic acid molecule of claim 9 or claim 10 in the preparation of a medicament for inducing an immune response against WT-1 in an individual.

12. A peptide comprising an antigen, wherein the antigen consists of the amino acid sequence shown in SEQ ID No. 2.

13. A peptide comprising an antigen, wherein the antigen consists of the amino acid sequence shown in SEQ ID No. 4.

14. Use of a peptide according to claim 12 or claim 13 in the manufacture of a medicament for inducing an immune response against WT-1 in an individual.

15. A vaccine comprising an antigen, wherein the antigen is encoded by SEQ ID No.1 or SEQ ID No. 3.

16. The vaccine of claim 15, wherein the antigen is encoded by SEQ ID No. 1.

17. The vaccine of claim 15, wherein the antigen is encoded by SEQ ID No. 3.

18. A vaccine comprising a peptide, wherein:

(a) the peptide comprises an antigen, wherein the antigen consists of the amino acid sequence shown in SEQ ID NO. 2; or

(b) The peptide comprises an antigen, wherein the antigen consists of the amino acid sequence shown in SEQ ID NO. 4.

19. The vaccine of claim 18, wherein the peptide comprises an antigen, wherein the antigen consists of the amino acid sequence set forth in SEQ ID NO 2.

20. The vaccine of claim 18, wherein the peptide comprises an antigen, wherein the antigen consists of the amino acid sequence set forth in SEQ ID No. 4.

21. Use of the vaccine of any one of claims 15-20 in the manufacture of a medicament for inducing an immune response against WT-1 in an individual.